Lactate mitochondrial oxidation drives stemness potential in metastatic breast cancer

Domain: neuroscience
Published: 2025-12-18
Editorial outcome: accept

Abstract
Metastatic cancer cells, originating from cancer stem cells with metastatic capacity, utilize nutrient flexibility to navigate the challenges of the metastatic cascade. However, the nutrient required to maintain the stemness potentials of metastatic cancer cells remains unclear. Here, we reveal that metastatic breast cancer cells sustain stemness and initiate metastasis upon detachment by taking up and oxidizing lactate. In detached metastasizing breast cancer cells, lactate is incorporated into the tricarboxylic acid cycle, boosting oxidative phosphorylation, and promoting the stemness potentials via α-KG-DNMT3B-mediated SOX2 hypomethylation. Moreover, lactate is taken up and oxidized in mitochondria by the CD147/MCT1/LDHB complex, which correlates with stemness potentials and tumor metastasis in patients with breast cancer. An intracellularly expressed single-chain variable fragment targeting mitochondrial CD147 (mito-CD147 scFv) effectively disrupts the mitochondrial CD147/MCT1/LDHB complex, inhibits lactate-induced stemness potential, depletes circulating breast cancer cells, and reduces metastatic burden, suggesting promising clinical applications in reducing lactate-fueled metastasis.

Introduction
Breast cancer is the most prevalent cancer and the leading cause of death in women worldwide, with 2.3 million diagnosed new cases and 670,000 deaths in 20221. In patients with breast cancer, metastasis contributes to over 90% of cancer-associated deaths2,3, although the process of metastasis is inefficient. During the metastatic cascade, millions of tumor cells detach from the primary tumor, but very few of them (less than 0.02%) can survive and effectively form growing metastatic foci4. This small proportion of cancer cells is a subgroup of cancer stem cells (CSCs) that are capable of initiating metastasis and setting up metastatic colonies in secondary organs, and are termed metastasis-initiating cells (MICs)5–7. Therefore, understanding the origin and maintenance mechanisms of MICs helps to specifically and effectively eradicate MICs and reduce metastasis.

Being different from CSCs that are identified with cancer-driving mutations, no metastasis-specific mutations occurred in MICs8. In fact, the origins of MICs are multifaceted. Besides originating from CSC subclones with pre-existing high metastatic potential and an elevated epithelial–mesenchymal transition (EMT) status9, MICs can also arise from non-CSCs that acquire stemness potentials during metastasis, such as detachment, manifesting phenotypic plasticity with dynamic changes in stemness potentials6. For example, in recurrent colorectal cancer, metastatic outgrowth is driven by Lgr5+ stem cells directly or by Lgr5+ cells that have transformed from Lgr5− non-stem cells via phenotypic conversion10,11. Similarly, our previous study demonstrated that detached ALDH− non-stem breast cancer cells gained the stemness potentials via resistance to detachment-induced anoikis12, the first major survival challenge for MICs and a critical barrier to forming metastasis13. Consequently, phenotypic plasticity ensures MICs bear the adaptability to counteract various stresses and survival pressures during the metastatic cascade and is considered an emerging hallmark of cancer stemness and tumor metastasis9,14,15.

Recently, metastatic cancer cells have been found to have metabolic adaptability and exhibit stage-dependent metabolic traits at every step of the metastatic cascade, which leads to phenotypic plasticity15. Evidence from single-cell RNA sequencing in human breast cancer showed that MICs switched their metabolic phenotype from glycolysis in primary tumors to oxidative phosphorylation (OXPHOS) in established micro-metastases16. Moreover, metabolites like pyruvate, lactate, glutamine, oleic acid, and cystine in the circulation all potentially help detached cancer cells defend against oxidative stress8 and further contribute to anoikis resistance.

These facts suggest that detached cancer cells possess metabolic flexibility, allowing them to adapt to various bioenergetic substrates according to their bioavailability. However, whether this metabolic flexibility contributes to the stemness potentials of MICs and the initiation of tumor metastasis in breast cancer remains unclear. It is well established that glucose uptake decreases upon detachment17–19, but the main energy substrates and corresponding metabolic pathways necessary for maintaining MIC stemness under stress and initiating metastasis still need to be determined.

In the present study, we investigated the energy substrates of MICs and their contributions to stemness properties in metastatic breast cancer from the perspective of metabolic adaptation. We found that lactate, taken up and oxidized by the mitochondrial cluster of differentiation 147 (CD147)/monocarboxylate transporter 1 (MCT1)/lactate dehydrogenase B (LDHB) complex, was converted into the tricarboxylic acid (TCA) cycle intermediate α-ketoglutaric acid (α-KG), which subsequently induced MIC stemness potentials via DNA methyltransferase 3 beta (DNMT3B)-mediated SOX2 hypomethylation. By developing an intracellularly expressed single-chain variable fragment (scFv) that specifically targets mitochondrial CD147, we demonstrated that mito-CD147 scFv effectively inhibited metastasis in breast cancer. Therefore, we propose that lactate serves as a fuel for the TCA cycle in detached metastatic breast cancer cells and promotes the stemness potentials of MICs, while blocking of lactate utilization could be used to eliminate such cells.

Results
Lactate serves as a fuel for the TCA cycle in detached breast cancer cells
To determine the energy substrates of detached breast cancer cells, we examined their utilization of major carbon sources. We noticed that the uptake of glucose and the expression of glucose transporter GLUT1 were significantly decreased in both ALDH− and total anoikis-resistant (AR) cells compared to adherent (Ad) cells (Fig. S1A–C). We also observed a significantly decreased uptake of fatty acid and glutamine in AR cells (Fig. S1D, E), but the reduction in fatty acid uptake (28.16±1.52%) and intracellular glutamine levels (19.71±0.71%) was significantly lower than that of glucose (50.62±3.79%). By analyzing the single-cell RNA sequencing data of breast cancer PDXs (GSE123837), however, we found significant enrichment of several metabolic pathways and organic acid metabolic processes in lung metastases compared to primary tumors (Fig. S1F). Given our previous findings showing that detached breast cancer cells exhibit significantly prolonged doubling times, these data suggest that their growth retardation may lead to the reduced utilization of three major nutrients while shifting toward the consumption of alternative energy sources.

To explore potential alternative energy sources for detached breast cancer cells, we conducted a non-targeted metabolomics analysis. The top five reduced metabolites in the supernatant of AR cells were valine, 2-Hydroxy-3-methylpentanoic acid, alanine, lactic acid (lactate), and phosphate (Fig. 1A, B). As glucose utilization was primarily decreased among the three major nutrients, we then focused on lactate, an organic acid and metabolic reservoir for glucose20. As expected, we found that lactate levels in AR cells were significantly decreased in the supernatant but increased inside the cells compared to those in Ad cells (Fig. 1C). Additionally, we observed that plasma lactate levels progressively declined during tumor progression and metastasis, while tumor tissue lactate concentrations increased in 4T1-bearing mice (Fig. 1D). These data suggested that lactate utilization is enhanced in AR cells and correlates with tumor metastasis.

To further investigate lactate utilization in AR cells with decreased glucose uptake, we cultured cells in glucose-free medium supplemented with lactate. We found that lactate levels were time-dependently and progressively decreased in the supernatant while increasing inside the cells in lactate-treated ALDH−, total, and ALDH+ cells (Fig. S1H). Lactate has been reported to be imported into cells, oxidized to pyruvate, and incorporated into the TCA cycle21,22. Thus, we analyzed the 13C -lactate-derived metabolites using stable isotope labeling techniques. Compared to Ad cells, significantly increased 13C -lactate-derived metabolites in AR cells included pyruvate, acetyl-CoA, TCA cycle intermediates (fold change from high to low: oxaloacetate, succinate, α-ketoglutarate, malate, fumarate, isocitrate, and citrate), and TCA cycle-derived metabolites (glutamate, aspartate, and ribose-5-phosphate), indicating enhanced lactate uptake and oxidation as well as increased TCA metabolism (Fig. 1E, F). Moreover, proteomics screening revealed that monocarboxylate transporter 1 (MCT1, protein for the SLC16A1 gene) and lactate dehydrogenase B (LDHB), responsible for lactate uptake and oxidation, respectively, were among the top three significantly upregulated proteins in AR cells (Fig. 1G). Their successively upregulation was also verified in AR ALDH−, total, and ALDH+ cells (Figs. 1H and S1I) as well as in lung metastases of breast cancer PDXs (Fig. S1G). Taken together, these results suggested that lactate is preferentially taken up by detached ER+ breast cancer cells (maybe dependent on ALDH status) and incorporated into the TCA cycle as a fuel.

Lactate induces stemness potentials in detached breast cancer cells
We previously demonstrated that detachment enhances the stemness potentials of AR breast cancer cells12. Given that lactate acts as a fuel for detached breast cancer cells, we wondered whether lactate contributes to their stemness potentials. Incubation of breast cancer cells in glucose-free medium supplemented with lactate induced a dose-dependent increase in ALDH+ and CD44highCD24−/low CSC subpopulations, as well as tumorsphere formation, with the most pronounced effects observed at 20 mM lactate (Fig. S2A, B). In contrast, these effects were attenuated in glucose-containing conditions. PCR array analysis revealed that 30 stem cell transcription factors were significantly altered in AR MCF7 ALDH− cells compared to those in their Ad counterparts, with SOX2 being the only factor changed in both AR and lactate-treated ALDH− and total breast cancer cells (Fig. S2C–E and Supplementary Data 1). Lactate also increased the expression of MCT1/LDHB (Fig. S2F, G), while the inhibition of lactate uptake or oxidation using MCT1/LDHB siRNAs or inhibitors significantly suppressed the stemness potential, specifically the ratios of ALDH+ and CD44highCD24−/low CSC subpopulation, expression of SOX2 protein, and tumorsphere numbers in both AR (Fig. S3A–D) and lactate-treated (Fig. S3E, F) cells. All these data indicate that lactate promotes the stemness potentials in detached ER+ breast cancer cells.

Moreover, lactate administration significantly increased the number of lung metastatic nodules, the ratios of ALDH+ CSC subpopulation, and the expression of SOX2/MCT1/LDHB in lung metastatic lesions from PDX1 (Figs. 2A–D and S3G), PDX2, and MDA-MB-231 human breast cancer cells (Fig. S3H–J), as well as in 4T1 mouse breast cancer cell-derived xenografts (Fig. 2B, C). In contrast, lactate administration had no obvious effect on the tumor growth (Fig. S3K, L) and stemness potentials of primary tumors in all the models mentioned above, although the median survival time in 4T1 xenograft-bearing mice was significantly shortened (Fig. 2E). Lactate administration also significantly increased the numbers (Fig. 2F) and the SOX2 expression (Fig. 2G–I) in CD45−CK18+ CTCs from PDX1 or 4T1 xenografts. However, inhibiting lactate uptake/oxidation by CHC/GNE significantly reduced the number of lung metastatic nodules (Fig. 2J, K), the ALDH+ and CD44highCD24−/low CSC ratios (Figs. 2L and S3M), the SOX2 expression in lung metastatic lesions (Fig. S3N) and in CK18+ CTCs (Fig. 2M, N). Taken together, these findings suggest that lactate increases the stemness potentials in detached triple-negative breast cancer (TNBC) cells, including lung metastatic cells and CTCs, and enhances the metastatic capacity of MICs. Interestingly, within tumor tissues of 4T1-bearing mice, we observed spatially coexistence of lactate-producing glycolytic tumor cells and lactate-importing oxidative tumor cells. Notably, oxidative tumor cells displayed significantly higher MCT1 and SOX2 expression (Fig. S3O, P), implying that these oxidative cells might enhance their stemness potentials through lactate consumption.

We previously showed that detachment induces the stemness potentials in non-stem breast cancer cells and reprograms non-CSCs into CSCs12. We then further analyzed the stemness potentials in ALDH− non-stem breast cancer cells cultivated with or without lactate treatment and found that lactate indeed increased the stemness potentials in these ER+ cells (Fig. 3A–C). Similarly, in 4T1 ALDH− cell-bearing mice, lactate administration significantly promoted lung metastasis (Fig. 3D) and augmented the stemness potentials in metastatic lesions (Fig. 3E, F), but did not alter primary tumor growth (Fig. 3G). By establishing tumor-bearing models with mCherry-labelled 4T1 ALDH− cells (Fig. 3H), we discovered that the administration of lactate significantly increased both the proportion of mCherry⁺ALDH+ cells in metastatic lung lesions and the expression level of SOX2 in mCherry⁺ cells isolated from these lesions (Fig. 3I, J). Besides, lactate increased the expression of MCT1/LDHB in ALDH− cells (Fig. 3C) and in lung metastatic lesions from ALDH− xenografts (Fig. 3F), while MCT1/LDHB siRNA or CHC/GNE significantly reduced the lactate- or AR-induced stemness potentials in ALDH− cells (Fig. 3K–N). CHC/GNE also significantly reduced the lactate-enhanced lung metastasis and stemness potentials in metastatic lesions from TNBC 4T1 ALDH− xenografts (Fig. 3O–Q). These results reveal that lactate induces the stemness potentials and facilitates metastatic capacity in non-stem breast cancer cells.

Lactate induces stemness potentials in detached breast cancer cells by fueling the TCA cycle and promoting OXPHOS
We next focused on how lactate induces stemness potentials in detached breast cancer cells. In addition to being a major circulating carbohydrate fuel, lactate can induce histone lactylation via the histone acetyltransferase P30023,24 or act as a ligand for the G-protein-coupled receptor GPR8125,26. By pre-incubating the lactate-treated cells with the P300 inhibitor A485 or GPR81 siRNA, our findings revealed that, except for the downregulation of SOX2 expression in T47D cells, neither P300 (Fig. S4A–D) nor GPR81 (Fig. S4E–I) significantly affected lactate-induced stemness potential.

To further explore the detailed role of lactate in stemness potential, we performed a proteomics analysis. In both AR and lactate-treated cells, the top two significantly enriched pathways were carbon metabolism and the citrate cycle (TCA cycle) (Figs. 4A and S4J). The TCA cycle is coupled to OXPHOS to provide cells with ATP. Indeed, we observed that ATP levels and oxygen consumption rates (OCRs) were significantly enhanced in both AR and lactate-treated ALDH− or total cells (Fig. 4B–D). Additionally, the activities of the electron transport chain complexes, especially mitochondrial complex I, were significantly increased in AR and lactate-treated ALDH− or total cells (Figs. 4E and S4K). Notably, while complex I assembly and mitochondrial mass remained unchanged, we observed significant upregulation of pan-Kla (lysine lactylation) on the complex I subunit NDUFS1 (Fig. S4L–N). In contrast, the suppression of lactate utilization by MCT1/LDHB inhibitors or siRNAs significantly weakened ATP production, OCR, and complex I activity in lactate-treated cells (Figs. 4F–H and S4O, P). Moreover, the ATP synthase inhibitor oligomycin and the complex I inhibitor rotenone both significantly decreased the ratios of ALDH+ or CD44highCD24−/low CSC subpopulations (Fig. 4I, J), tumorsphere numbers (Fig. 4K), and SOX2 expression in AR and lactate-treated ALDH− or total cells (Fig. 4L). Taken together, these data suggest that lactate induces stemness potentials in detached ER+ breast cancer cells by enhancing the Complex I activity and lactylation of its critical subunit NDUFS1.

Lactate induces the stemness potentials of detached breast cancer cells by promoting α-KG-DNMT3B-mediated hypomethylation of SOX2
To clarify the metabolic-epigenetic mechanism of lactate-induced stemness potentials in detached breast cancer cells via fueling the TCA cycle, we next focused on α-ketoglutarate (α-KG), a vital signaling molecule regulating stemness gene expression and one of the top three significantly increased TCA cycle intermediates (oxaloacetate, succinate, α-KG) in 13C -lactate treated AR cells (Fig. 1E, F). We found that the levels of α-KG were significantly increased in AR and lactate-treated cells (Fig. S5A), but were abolished by the MCT1/LDHB inhibitor CHC/GNE (Fig. S5B). Moreover, the AR- and lactate-boosted stemness potentials (Fig. 5A–C) were significantly facilitated by the α-KG analogue DM-αKG but were suppressed by the IDH inhibitor Enasidenib, which markedly decreased α-KG levels (Fig. S5C). These data indicated that lactate induces the stemness potentials of detached ER+ breast cancer cells through TCA cycle intermediates α-KG.

We then turned to determine whether the lactate-induced stemness potential was due to the α-KG-mediated methylation modification of DNA or histones, especially H3K27m3, a critical histone modification responsible for α-KG-regulated gene expression. We found that total DNA methylation levels were obviously decreased in AR (Fig. 5D–F) and lactate-treated (Fig. S5D, E) cells, while H3K27m3 protein levels remained unchanged (Fig. S5F). By predicting the possible regions in the SOX2 promoter where DNA methylation likely occurs via the Ensembl database (Fig. S5G) and designing corresponding primers, we found that the methylation levels in the SOX2 promoter region 181720250-181720500, identified by primer 2 and predicted to be a CpG island by MethPrimer 2.0, were significantly decreased in AR or lactate-treated cells (Fig. S5H). Moreover, DM-αKG significantly decreased, while Enasidenib increased, the methylation levels of the SOX2 promoter region 181720250-181720500 in both AR (Figs. 5G and S5J) and lactate-treated (Fig. S5I, J) cells. Notably, the DNMT inhibitor azacitidine (AZA) significantly counteracted the Enasidenib-induced hypermethylation of the SOX2 promoter region. These data demonstrated that α-KG upregulated SOX2 expression in detached ER+ cancer cells by reducing the methylation level in its promoter region 181720250-181720500.

Finally, we aimed to determine the enzyme involved in α-KG-suppressed methylation of the SOX2 promoter. As DNA methyltransferase DNMT3B was the top significantly downregulated protein upon detachment in our proteomics data (Fig. 1G), the expression and enzymatic activity of DNMT3B were downregulated in AR or lactate-treated cells (Fig. 5H, I), and further decreased by DM-αKG but abolished by Enasidenib (Fig. 5J). We then focused on DNMT3B. In AR and lactate-treated cells, we found that the binding of DNMT3B to the SOX2 promoter region (181720250-181720500) was negatively correlated with α-KG activities (Fig. 5K), and DNMT3B overexpression significantly abolished the α-KG-induced stemness potentials (Figs. 5L–N and S5K). These observations suggested that the lactate-converted α-KG induces stemness potentials in detached ER+ breast cancer cells by reducing DNMT3B-mediated SOX2 methylation.

Mitochondrial CD147/MCT1/LDHB complex responsible for lactate uptake and oxidation correlates with tumor metastasis and CTC stemness potentials in patients with breast cancer
Then, how was the uptake and oxidation of lactate enhanced in detached breast cancer cells? CD147, a chaperone protein that anchors MCTs to specific membrane locations and is listed as one of the top three significantly upregulated proteins (along with MCT1 and LDHB) in AR cells (Fig. 1G), has caught our attention. We noticed that CD147 expression was significantly upregulated in lactate-treated cells and lactate-administered metastatic tumors (Fig. S6A, B). Additionally, CD147 overexpression or knockdown12 correspondingly facilitated or suppressed lactate uptake (Fig. S6C), and the lactate-induced stemness potentials (Fig. S6D–G), as well as MCT1/LDHB expression (Fig. S6F) and lung metastatic nodules (Fig. S6H). These results indicated that CD147 promoted MCT1/LDHB-mediated lactate uptake and oxidation, facilitating lactate-induced stemness potentials in detached breast cancer cells.

The specific subcellular compartment for lactate uptake and oxidation, whether in the cytoplasm or mitochondria, remains controversial. By employing an isotope labeling scheme using 2-2H-lactate27,28, we found that more than 95% of lactate in the mitochondria of AR MCF7 cells was 2H-labeled, and that the 2H-labeled lactate in the mitochondria of AR MCF7 cells was significantly higher than that in Ad cells (Fig. 6A, B), indicating that lactate could be imported into the mitochondria of detached ER+ breast cancer cell.

As CD147 subcellular localization29 and lactate uptake/oxidation both occur in mitochondria, and PPI network analysis suggested a possible interaction between CD147 and MCT1 or LDHB (Fig. S6I), we presumed that CD147 might regulate lactate uptake/oxidation by interacting with MCT1 or LDHB in the mitochondria. By isolating high-purity mitochondria (Fig. S6J), we indeed found that the protein levels of CD147, MCT1, and LDHB were increased (Fig. S6K), their co-localization was enhanced (Fig. S6L), and the interactions between CD147 and MCT1 or LDHB were augmented (Figs. S6M and S7A) in AR or lactate-treated cells. By conducting protease K protection and mitochondria sub-fractionation isolation assays, we found that the increased expression of these three molecules occurred in the mitochondrial inner membrane and/or matrix of AR (Fig. 6C, D) or lactate-treated (Fig. S7B, C) cells. Excitingly, transmission electron microscopy (TEM) confirmed the existence of a CD147/MCT1/LDHB complex in the mitochondrial inner membrane of AR cells (Fig. 6E). Moreover, molecular docking simulations predicted the binding conformation of three molecules: CD147 structurally bridges between MCT1 and LDHB. Specifically, its transmembrane region near the C-terminus interacts with MCT1 while its N-terminus binds to LDHB (Fig. S7D). Building upon the well-established CD147-MCT1 interaction30, we further investigated the potential CD147-LDHB interaction. Using proximity ligation assay (PLA), we confirmed a direct interaction between CD147 and LDHB, suggesting their functional coupling. Notably, lactate treatment significantly enhanced this interaction (Fig. 6F). To further validate this finding, we conducted GST pull-down assays combined with site-directed mutagenesis using purified GST-tagged CD147 (N-terminal) and His-tagged LDHB proteins, supplemented by molecular docking analyses. These experiments confirmed a direct physical interaction between CD147 and LDHB, mediated by key residues: Glu168 and Arg106 in CD147, and Arg99 and Asp56 in LDHB (Figs. 6G and S7E, F). These results suggested that there is a CD147/MCT1/LDHB complex in the inner membrane, which facilitates mitochondrial lactate uptake and oxidation in ER+ breast cancer cells.

Consistently, CD147 was co-localized and interacted with MCT1 or LDHB in the mitochondria of lung metastases from lactate-administered TNBC PDX1/2 (Figs. 6H and S7G–I) and 4T1 ALDH− (Fig. S7J) bearing mice, unlike in primary tumors. In the primary breast cancer tissues of metastatic patients, the expression of CD147 showed a strong positive correlation with that of MCT1 or LDHB. In contrast, a moderate or low positive correlation was observed in non-metastatic patients (Figs. 6I, J and S7K). Patients with higher co-expression of CD147/MCT1/LDHB had significantly worse overall survival and higher tumor metastasis/recurrence, and their co-expression was an independent predictor of poor overall survival (Figs. 6K and S7L and Table S2–4). Moreover, by analyzing the CTCs in blood vessels of breast cancer patient tissues, we found that mitochondrial CD147/MCT1/LDHB complexes occurred in CTCs of 6 out of 10 patients (Fig. S7M), and patients whose CTCs bore the mitochondrial CD147/MCT1/LDHB complex exhibited high SOX2 expression (Fig. 6L–N and S7N, O) and more advanced stages (Fig. S7M). Altogether, these results uncovered a mitochondrial CD147/MCT1/LDHB complex for lactate uptake/oxidation, which positively correlated with tumor metastasis and CTC stemness potentials in patients with breast cancer.

Mitochondrial CD147 promotes the lactate-induced stemness potentials in detached breast cancer cells
Due to the key role of CD147 in the mitochondrial CD147/MCT1/LDHB complex, we investigated the influence of mitochondrial CD147 on lactate-induced stemness potentials and metastatic capacity by constructing cells with CD147 knocked out (KO) or expressed exclusively in mitochondria (mito147, Fig. S8A, B). In detached MCF7 ALDH− cells with different CD147 levels, the mitochondrial uptake of 2-2H-lactate was completely blocked in CD147 KO cells but was efficiently rescued in mito147 cells to a comparable level to wild type (WT) cells (Fig. 7A, B), which was verified by the corresponding alteration of lactate levels in the supernatant (Fig. 7C), further confirming the role of mitochondrial CD147 in lactate uptake. Upon detachment or lactate treatment, mitochondrial-expressed CD147 efficiently reversed the reduced mitochondrial respiration (Figs. 7D and S8C), stemness potentials (Figs. 7E and S8D), and MCT1/LDHB expression (Fig. S8D) in CD147 KO cells to a degree comparable to WT cells. Likewise, lactate-administered mito147 cell-bearing mice exhibited significantly recovered primary tumor growth, facilitated lung metastasis (Figs. 7F–G and S8E–G), enhanced stemness potentials in metastatic lesions (Figs. 7H, I and S8H), as well as increased CTC numbers and SOX2 expression in CTCs (Figs. 7J, K and S8I, J), compared to lactate-administered CD147 KO cell-bearing mice.

For the epigenetic mechanism by which mitochondrial CD147 promotes lactate-induced stemness potential, we found that mito147 cells had significantly higher α-KG accumulation (Fig. S8K), lower DNMT3B expression (Fig. 7L), and weaker binding of DNMT3B to the SOX2 promoter (Fig. S8L) than CD147 KO cells, whether under AR or lactate-treated conditions. Therefore, mitochondrial CD147 is essential for maintaining stemness potentials and promoting metastasis through enhanced lactate uptake/oxidation and the α-KG-DNMT3B mediated SOX2 hypomethylation.

Intracellularly expressed scFv targeting mitochondrial CD147 inhibits lactate-induced stemness potentials in detached breast cancer cells
To block the mitochondrial CD147-facilitated lactate uptake/oxidation and the induced stemness potentials in detached breast cancer cells, we intracellularly expressed a scFv specifically targeting mitochondrial CD147 (mito-CD147 scFv) by fusing a scFv sequence that targets CD147 to a matrix-targeting sequence that guides the scFv into mitochondria. The mito-CD147 scFv expressed in the pGV469 adenoviral vector specifically bound to the mitochondria localized CD147 in both mito147 and wild-type cells (Figs. 8A and S9A, B), and restrained the formation of the mitochondrial CD147/MCT1/LDHB complex (Figs. 8B and S9C); more importantly, it blocked the uptake of 2-2H-lactate into the mitochondria of MCF7 ALDH− cells (Fig. 7A, B). In lactate-treated WT or mito147 cells, mito-CD147 scFv significantly inhibited mitochondrial respiration (Fig. S9D), ALDH+ and CD44highCD24−/low ratios (Fig. S9E), tumorsphere formation (Fig. S9F up), and SOX2/MCT1/LDHB expression (Fig. S9G). Moreover, mito-CD147 scFv reduced the α-KG levels (Fig. S9F down) and recovered the binding of DNMT3B to the SOX2 promoter (Fig. S9H). These data indicated that mito-CD147 scFv functioned as a brake to the lactate-induced stemness potentials by inhibiting α-KG-DNMT3B mediated SOX2 hypomethylation.

Being injected into lactate-administered MCF7 mito147 cell xenografts (Fig. S10A), pGV469-mito-CD147 scFv adenovirus markedly abolished the lung metastasis facilitated by mitochondrial CD147 (Fig. S10B left), reduced CSC ratios (Fig. S10C), and decreased SOX2 expression in metastatic lesions (Fig. S10D), while not affecting stemness potentials or tumor growth in primary tumors (Fig. S10D, E). Meanwhile, mito-CD147 scFv significantly diminished the numbers of CTCs boosted by mitochondrial CD147 and its SOX2 levels in lactate-administered mice (Fig. S10B right, Fig. S10F, G).

To investigate the therapeutic potential on CTCs, mito-CD147 scFv adenovirus was administered via the tail vein to lactate-treated MMTV-PyMT spontaneous tumorigenic and PDX1-bearing mice that express native CD147 (Figs. 8C and S10H). We found that mito-CD147 scFv significantly decreased lactate-promoted lung metastasis (Fig. 8D and I), the CSC ratios, and SOX2 expression in lung metastatic lesions (Figs. 8E, F and J and S10I), as well as CTC numbers and stemness (Fig. 8G, H and K–M). Similar effects were also observed in MDA-MB-231 ALDH− cell-bearing mice (Fig. S10J–L). Altogether, mito-CD147 scFv effectively suppressed tumor metastasis and CTC stemness in breast cancer by blocking the formation of the mitochondrial CD147/MCT1/LDHB complex and inhibiting lactate uptake/oxidation.

Methods
Human tumor tissues
The human tissue study protocol was approved by the Ethics Committee of Fourth Military Medical University (Approval number KY20183305-l) and informed consents have been obtained from the participants. A tissue microarray (TMA) constructed from paraffin-embedded tissue blocks of 131 female patients with breast cancer was purchased from Shanghai Outdo Biotech Company. Clinicopathological data were collected from the patients’ medical records, which included age at diagnosis, tumor type and grade, TNM stage, AJCC stage, overall survival, recurrence, etc. (Tables S2–S4). Additionally, tissue slides from 10 female patients with breast cancer with CTCs in blood vessels were obtained from Bioaitech Co., Ltd (Fig. S7M).

Animals models and experiments
The animal study protocols were approved by the Institutional Animal Care and Use Committee of the National Translational Science Center for Molecular Medicine (2023-NTSCMM-ID003). Six-to eight-week-old female BALB/c mice were purchased from Vital River Laboratory Animal Technologies (Beijing, China). Six-to eight-week-old female BALB/c nude mice, NCG mice, and MMTV-PyMT mice were obtained from Gempharmatech (Nanjing, China). Mice were maintained in a specific pathogen-free (SPF) mouse facility (room temperature, 18–23°C; room humidity, 40–60%) with free access to food (XTI01ZJ-009, Xietong Shengwu, China) and water under a 12 h light/dark cycle.

The maximal permitted tumor burden was defined as a tumor volume not exceeding 1500 mm³. Tumorigenesis was monitored by measuring tumor dimensions every 3 days using a digital caliper. Tumor volume was calculated using the formula: Volume=(Length×Width²)/2. Mice were humanely euthanized before the tumor burden reached the maximum permitted size if they met any of the following predefined humane endpoint criteria: (1) tumor volume exceeding 1500 mm³; (2) ulceration, necrosis, or infection of the tumor that impaired the animal’s well-being; (3) significant weight loss (>20% of baseline body weight); (4) difficulty in breathing, locomotion, or feeding; (5) signs of prolonged pain or distress that were not alleviated by analgesia. These endpoints were strictly followed for all survival studies to minimize suffering and to generate the survival curves presented in this study. We confirm that the maximal tumor size/burden permitted by our ethics committee was never exceeded during this study. In the instances where animals reached the humane endpoint criteria described above, they were euthanized promptly.

For lactate administration experiments (Fig. 2A), patient-derived xenograft (PDX) 1/2 tumor masses (2×1×1 mm3), 1×106 MDA-MB-231 cells, 1×105 4T1-luciferase cells, or 1×107 4T1 ALDH− cells were inoculated into the mammary adipose tissue pad of mice. When tumor volume reached 100 mm3, mice were randomized into two groups and injected intraperitoneally (i.p.) with or without lactate (Lac 800 mg/kg: sodium lactate, 800 mg/kg, 100 μL; Lac 0 mg/kg: 0.9% sterile saline/NS, 100 μL) for five consecutive days. On day 25, mice with 4T1-luciferase xenografts were given 150 mg/kg D-Luciferin potassium salt (ST196, Beyotime) i.p. for bioluminescence imaging, and images were recorded using an IVIS Lumina II (PerkinElmer, Waltham, MA, USA). On day 28, whole-body blood from mice was obtained for circulating tumor cell (CTC) isolation, counting, and staining; then mice were euthanized, and lungs and tumors were collected for lung nodule counting and histological staining as well as for stemness potential analysis via flow cytometry and western blot.

For lactate uptake/oxidation inhibition experiments (Fig. 2J), mice bearing xenografts of 100 mm3 were randomly assigned into four groups and injected i.p. without or with inhibitors for two consecutive days; three days later, mice were treated without or with lactate for five consecutive days. On days 25 and 28, mice were imaged, and the lungs and tumors were collected for the same analysis as the above procedures. For mice bearing 1×105 4T1-luciferase xenografts, the three groups were: NS+Lac, CHC+Lac, and GNE+Lac; for mice bearing 1×107 4T1 ALDH− xenografts, the four groups were: NS, Lac, Lac+CHC, and Lac+GNE. CHC (5 mg/kg, 100 μL) and GNE (5 mg/kg, 100 μL) were used as inhibitors for MCT1 and LDHB, respectively, with 0.9% sterile saline (NS) as the vehicle control.

For experiment investigating the function of mitochondrial localized CD147 (Fig. S8E), nude mice of similar weights were inoculated with 1×106 MCF7 or 1×107 T47D derived WT, KO, and mito147 cells. On day 14, all mice received lactate for five consecutive days. On day 28, whole-body blood, as well as the lungs and tumors, were collected from the mice for further analysis.

For experiment involving therapeutic mito-CD147 scFv, we first adopted a mouse xenograft model inoculated with cells expressing CD147 exclusively in mitochondria (Fig. S10A). Mice of similar weights were inoculated with 1×106 MCF7-derived WT/KO/mito147 cells; on day 14, MCF7-mito147 cells were randomly assigned into two groups and intratumorally injected with pGV469-mito‑CD147 scFv or pGV469 adenovirus (109 pfu, 30 μL) at multiple sites for three consecutive days; two days later, all mice were treated with lactate and evaluated on day 28. For the mouse model with native CD147 expression, 10-week-old MMTV-PyMT mice and mice bearing 1×107 MDA-MB-231 ALDH− xenografts or PDX1 derived tumors (100 mm3) were first injected with Lac or NS for five consecutive days; then, two days later, they were treated with pGV469-mito‑CD147 scFv or pGV469 adenovirus (108 pfu, 100 μL, i.v.) for three consecutive days; at 13 weeks old or on day 28, mice were evaluated (Figs. 8C and S10H). In MMTV-PyMT mice or mice with MDA-MB-231 ALDH− xenografts, three groups were assigned: NS, Lac, and Lac+mito‑CD147 scFv. In mice bearing PDX1 xenografts, four groups were assigned: NS, mito‑CD147 scFv, Lac, and Lac+ mito-CD147 scFv.

Cell lines and treatments
MCF7, T47D, MDA-MB-231, and 4T1 cells were cultured in RPMI 1640 medium with 3.15 g/L-glucose. All cells were routinely supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 2% L-glutamine. The ER+ MCF7 and T47D cells were untreated with charcoal stripping and without exogenous E2 supplementation. They were regularly tested for mycoplasma contamination and authenticated by short tandem repeat (STR) DNA profiling to ensure their identity and purity.

As depicted in the Fig. S11, there are two distinct cellular models in our study. One is the Low-Attachment Model. Cells surviving for 7 days in glucose-containing medium (3.15 g/L) under detached suspension culture conditions in ultra-low attachment plates were considered anoikis-resistant (AR) cells, while cells cultured in uncoated common plates under attached conditions with glucose-containing medium (3.15 g/L) served as control adherent (Ad) cells12. The other is Glucose-Free Model with Lactate. Due to the reduced glucose uptake in detached cells, adherent cells cultured in glucose-free medium, with or without exogenous lactate incubation, were designated as “Lac 20 mM” and “Lac 0 mM”, which were designed to mimic the states of AR and Ad cells, respectively.

Cell lines stably overexpressing the luciferase gene (4T1-luciferase) were generated from 4T1 cells using the LV-17 luciferase lentivirus. DNMT3B overexpressing (DNMT3B) or control (VC) cells were established by transfecting pcDNA3.1(+)–DNMT3B or pcDNA3.1(+) vector control into MCF7/T47D cells.

CD147 knock-in (MCF7/T47D 147) or knockdown (MDA-MB-231 A6) cells were generated by transfecting pGV341 lentiviral CD147 cDNA or pGIPZ lentiviral CD147 shRNA (pGIPZ-A6) into MCF7/T47D or MDA-MB-231 cells, respectively12. The corresponding pGV341 lentiviral expression vector or pGIPZ lentiviral non-target control shRNA (pGIPZ-NTC) transfected cells, MCF7/T47D VC or MDA-MB-231 NTC, were used as control cells12.

CD147 knockout (MCF7/T47D KO) cells were generated by pGV392-U6-sgRNA-EF1a-Cas9-FLAG (pGV392-KO) lentivirus transfection. Cells with CD147 exclusively expressed in mitochondria (MCF7/T47D mito147) were obtained by introducing the mitochondrial CD147 overexpressing pGV371-mito147 lentivirus into MCF7/T47D KO cells. The mitochondrial CD147 exclusively expressed pGV371-U6-sgRNA-SV40 vector was constructed by cloning the mitochondrial targeting sequence (MTS, 5′-ATGTCCGTCCTGACGCCGCTGCTGCTGCGGGGCTTGACAGGCTCGGCCCGGCGGCTCCCAGTGCCGCGCGCCAAGATCCATTCGTTG-3′) of COX VIII and the DNA sequence encoding CD147 (NM_198589) into the pGV371 expression vector. pGV392 transfected wild-type cells (MCF7/T47D WT) were used as controls (Table S5).

Rationale for lactate concentration selection
To systematically evaluate the impact of lactate on the stemness potential of detached breast cancer cells, we employed an in vitro concentration range of 0–40 mM, with a primary experimental focus at 20 mM. This range was selected to encompass both physiological serum lactate levels (1.5–3 mM in healthy individuals)39,58 and pathological elevated concentrations observed in human serum (5–8 mM)59,60 and tumor tissues (10–50 mM)59.

For in vivo studies, we administered lactate via intraperitoneal injection at a dose of 800 mg/kg, a concentration previously demonstrated to be non-lethal, without inducing acidosis or severe inflammatory side effects61–63. These in vitro and in vivo conditions ensured biologically relevant lactate exposure while maintaining experimental feasibility and reproducibility.

Patient-derived xenograft (PDX) models and tumor cell suspension separation
Breast cancer PDX1/2 were obtained from Suling Liu’s lab64. Both PDX models were derived from triple-negative breast cancer patients. To ensure genetic fidelity and exclude contamination, the identities of the PDX models were rigorously monitored through the establishment a backup library, control of passaging number, and performing short tandem repeat (STR) profiling specificity. Tumor tissue was cut into small pieces (2×1×1 mm3), and inoculated into the mammary adipose tissue pad of NCG mice. The mice were euthanized when the maximum tumor diameter reached 1 cm. The tumor was removed, minced, and digested with 10 mL/g cell dissociation buffer (DMEM supplemented with 10% FBS, 1% mycillin, 200 U/mL collagenase type II, and 0.6 U/mL dispase) in a 50 mL conical tube at 37°C for 2 h with vertexing for 1 min every 20 min. The cells suspension was then filtered through a 70 µm strainer (Corning, San Jose, CA, USA) for subsequent experiments.

pGV469-mito-CD147 scFv adenoviral expression vector and virus
Adenoviral constructs targeting and antagonizing mitochondrial localized CD147 were generated by Genechem (Shanghai, China) using the pGV469 plasmid, wherein the gene of interest is expressed under an SV40 promoter. As detailed in Fig. S12, the pGV469-mito-CD147 scFv adenoviral expression vector was constructed by cloning the matrix-targeting sequence (MTS, 5′-ATGTCCGTCCTGACGCCGCTGCTGCTGCGGGGCTTGACAGGCTCGGCCCGGCGGCTCCCAGTGCCGCGCGCCAAGATCCATTCGTTG-3′) of COX VIII and the scFv sequence of HAb1857, a monoclonal antibody targeting CD147, into the GV469 plasmid using MluI/HindIII, with EGFP fused at the C terminus; the construct was applied for a Chinese invention patent (202410075009.2).

For in vitro cellular analysis, the pGV469-mito-CD147 scFv expression vector was transfected into cells (6×105) at 2.5 μg per well in a six-well plate. For in vivo animal experiments, the pGV469-mito-CD147 scFv expression vector was packaged into adenovirus and injected intratumorally at multiple sites (109 pfu, 30 μL) or intravenously into the tail of mice (108 pfu, 100 μL).

Metabolomics analysis
For metabolomics analysis, supernatants from 1×106 MCF7 AR and Ad cells (n=6 biologically independent samples) were collected and centrifuged at 3000×g for 10 min at 4°C to remove cell debris and the lipid layer, followed by untargeted metabolic profiling by MetaboProfile Inc (Shanghai, China). Briefly, each aliquot of 50 μL of sample was mixed with 10 μL of the internal standard 4-chlorophenylalanine, and then 175 μL of pre-chilled methanol/chloroform (v/v=3/1) was added. After 20 min of centrifugation at 14,000×g and 4°C, 200 μL of supernatant from each sample was evaporated and lyophilized using a FreeZone freeze dryer (Labconco, Kansas City, MI, USA), while the remaining supernatant from each sample was pooled as quality control. A time-of-flight mass spectrometry (GC-TOF/MS) system (Pegasus HT, Kennett Square, PA, USA) with an Agilent 7890B gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) and a Gerstel multipurpose sample MPS2 with dual heads (Gerstel, Germany) was used to investigate the metabolite profiling, with an Rxi-5ms capillary column (30 m×250 μm i.d., 0.25-μm film thickness; Restek Corporation, Bellefonte, PA, USA) employed for the separation of metabolites. Metabolite annotation was performed by comparing the retention indices and mass spectral data with those previously generated from reference standards of known structures using ChromaTOF software. The resulting datasets were analyzed using the SIMCA-P 14.1 software package or MetaboAnalyst 5.0 online software (https://www.metaboanalyst.ca/) at the univariate level with an unpaired two-sided nonparametric t-test65. The cut-off for significantly different metabolites between AR and Ad was set to VIP 1.0 and FDR 0.05.

Glucose and fatty acid uptake assay
The uptake of glucose or fatty acids in AR and Ad cells was measured using the fluorescent deoxyglucose analog 2-NBDG (ab287845, Abcam, Cambridge, UK) or TF2-C12 fatty acid (36385, AAT Bioquest, Pleasanton, CA, USA), respectively. Briefly, cells seeded in six-well plates at a density of 8×105/2 mL per well were cultivated overnight and then transferred into serum-free medium for 1 h. Subsequently, cells were incubated with 100 μg/mL 2-NBDG in glucose-free medium or with 100 μL/well of the fatty acid dye-loading solution for 1 h. Then, 1×106 cells were collected, and the fluorescence signal was measured using a flow cytometer (BD Fortessa, BD Biosciences, Franklin Lakes, NJ, USA) at excitation/emission (Ex/Em) wavelengths of 485/535 nm or 485/515 nm, respectively. The data (mean fluorescence intensity, MFI) were standardized with respect to a reference of 1×106 cells.

Extracellular or intracellular glutamine and lactate measurement
Glutamine levels in culture supernatants and cell lysates were detected using a glutamine assay kit (G0429F, Grace Biotechnology, Bend, OR, USA) according to the manufacturer’s protocols. Briefly, AR and Ad cells were cultivated overnight at a density of 8×105/2 mL per well, and the corresponding culture supernatants were collected. The cells were disrupted by ultrasound in 200 μL of extraction solution, with the supernatant after centrifugation used for detection, while the culture supernatant was taken for direct detection after centrifugation. After reacting with glutaminase, glutamate dehydrogenase, and color-developing reagents, the absorbance at 450 nm was measured, and the glutamine content was calculated according to the standard curve. The glutamine levels in culture supernatants were normalized to 1×106 cells, while those in cell lysates were standardized against total cell protein levels.

Lactate levels in mouse serum and culture supernatants were tested using a lactic acid assay kit (A019-2-1, Jiancheng Bio, Nanjing, China), with lactate levels in cellular supernatants normalized to 1×106 cells. In cell lysates, lactate levels were assayed using an L-lactate assay kit (ab65331, Abcam), with total cell protein levels used for data normalization. Serum from 1×105 4T1-luciferase cell-bearing Balb/c mice was collected from 100 μL of blood every 4 days starting from the 5th day after inoculation. Cells and the corresponding culture supernatants from AR/Ad cells were collected similarly to the procedures for glutamine measurement, while samples from lactate-treated cells were collected by culturing cells in glucose-free medium with 20 mM lactate incubation for 1, 2, 4, 8, 12, 24, 36, and 48 h.

Proteomics analysis by bottom-up mass spectrometry
1×106 AR/Ad or with/without lactate-treated MCF7/T47D cells (n=3 biologically independent samples) were lysed with RIPA lysis buffer, and the protein lysates were electrophoresed on 10% SDS-PAGE gels. After Coomassie Brilliant Blue staining, five equal parts of the gels, divided by molecular weight, were evaporated using acetonitrile and reconstituted in 1% (v/v) formic acid, then digested with trypsin and injected into the mass spectrometer (Model LTQ, Thermo Fisher Scientific, Waltham, MA, USA). The mass spectrometer (MS) was calibrated using standard compounds and operated in data-dependent mode, cycling between full MS scans (m/z 300–2000), while the MS data were collected by targeting MS scans on the ten most abundant ions detected in the MS scan.

MS raw data were processed using Proteome Discoverer software (version 2.5.0.4) for database searching, quantification, and false discovery rate (FDR) estimation. Spectra were queried against the UniProt Homo sapiens reference proteome database (release 2023_08, containing 20349 sequences). The search parameters were set as follows: Trypsin/P was specified as the protease with up to 2 missed cleavages permitted. Precursor mass tolerance was set to 10 ppm, while the fragment mass tolerance was defined as 0.02 Da. The minimum required peptide length was set to 6 amino acids. Methionine oxidation and protein N-terminal acetylation and lysine methylation were specified as the variable modifications, while cysteine carbamidomethylation was set as the stable modification.

Site possibility threshold for peptide-to-spectrum matching (PSM) was set to minimum 75 for PTM site localization. To ensure high-confidence identifications, the false discovery rate (FDR) was controlled at <1% at both the peptide-spectrum match level and the protein level. This was determined by searching against a reversed decoy database. Finally, protein identification required at least two unique peptides per protein group. Three biologically independent samples were conducted for each cell, and proteins with variable importance in projection (VIP) of ≥1 and P<0.05 were considered differentially expressed proteins.

Western blot
Tumor tissues and cells were lysed in RIPA lysis buffer supplemented with protease inhibitors (4693159001, Roche, Basel, Switzerland). Protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific), and 20 μg of protein per sample (the default amount if not specified otherwise) were subjected to SDS-PAGE, transferred onto PVDF membranes, and incubated with primary antibodies summarized in the key resources table, including GLUT1 (1:1000), MCT1 (1:1000), LDHB (1:1000), SOX2 (1:1000), GPR81 (1:1000), DNMT3B (1:1000), H3 (1:1000), H3K27m3 (1:1000), COXIV (1:1000), TOMM20 (1:1000), Na+-K+ ATPase (1:1000), LAMP1 (1:1000), GM130 (1:1000), GRP78 (1:1000), α-Tubulin (1:5000), and anti-CD147 (HAb18, 1:3000) produced in our laboratory54,66. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Thermo Fisher Scientific, 1:5000), the proteins were visualized using chemiluminescence reagents (29050, Engreen, San Jose, CA, USA) with a Gel Doc EZ Imager (Bio-Rad Laboratories, Hercules, CA, USA) and analyzed using the Image Lab software (Bio-Rad Laboratories).

RT-PCR and PCR array
Total RNA was isolated using an E.Z.N.A. Total RNA Kit II (OMEGA, BioTek, Norcross, GA, USA), and 1 μg of RNA was subjected to reverse transcription with the PrimerScriptTM RT Master Mix kit (RR036A, TaKaRa, Otsu, Japan). Subsequently, RT-PCR was performed with a SYBR Green PCR kit (TaKaRa) on the QuantStudioTM 7 Flex Real-Time PCR system (Life Technologies, Thermo Fisher Scientific) to detect the expression levels of the target genes. Gene expression was quantified using the ΔCt method, normalized to 18s RNA. All primers were synthesized by Tsingke Biotech (Beijing, China), and the primer sequences are shown in Table S1.

For the human stem cell transcription factors PCR array (PAHS-501Z, Qiagen, Duesseldorf, Germany), the gene expression of interest was normalized relative to housekeeping genes (ACTB, B2M, HPRT1, RPLP0; Supplementary data 1). Fold changes in gene expression, scatterplots, and heatmaps were analyzed and generated according to the RT2 PCR array data analysis website (https://geneglobe.qiagen.com/us/analyze).

13C -lactate metabolic flux analysis
1×107 MCF7 AR/Ad cells (n=6 biologically independent samples) cultivated in regular complete RPMI 1640 medium containing 10 mM 13C -lactate for 12 h were pelleted, stored at −80°C, and then sent to Metabo-Profile Inc (Shanghai, China) for metabolic flux assay using ultra-performance liquid chromatography triple-quadrupole mass spectrometry (UPLC-TQ-MS), with unlabeled AR and Ad cells collected at 0 h being used to correct for the natural isotopic abundance that could potentially contribute to false positives.

For the disruption of cell pellets, 400 μL of 80% methanol solution was added, followed by ultrasonic disruption and centrifugation at 4°C at 18,000×g for 15 min. The supernatant was concentrated (Labconco), dissolved in 100 μL of 80% methanol solution, and then prepared for injection and analysis. Metabolites were separated using an Acquity amide and an Acquity HSS C18 column (2.1 mm×100 mm, 1.7 μm, Waters Corp) equipped with an ACQUITY UPLC VanGuard Pre-Column (Waters Corp, Milford, MA, USA). A 5 μL aliquot of the sample was injected into the column, with the temperature maintained at 40°C and the flow rate set constantly at 0.4 mL/min. The raw UPLC-MS data were analyzed using TargetLynx Application Manager software version 4.1 (Waters Corp). Quantification of each metabolite was performed using linear regression analysis of the peak area of the metabolite versus concentration.

The mass distribution vector (MDV) of 13C-labeled metabolites, which describes the fractional abundance of each isotopologue normalized to the sum of all possible isotopologues, was measured by correcting for naturally occurring isotopes according to a previous publication67. First, the proportion (P0) of natural 13C-labeled metabolite relative to non-13C-labeled metabolite in MCF7 cells collected at 0 h was calculated as: P0 = abundance of natural 13C-labeled metabolite / abundance of non-13C-labeled metabolite; while the abundance of the natural 13C-labeled metabolite in MCF7 cells cultured with 13C -lactate for 12 h was calculated using the following formula: abundance of natural 13C-labeled metabolite = abundance of non-13C-labeled metabolite × P0. Then, the abundance of the natural 13C-labeled metabolite was subtracted from the total 13C-labeled metabolite in MCF7 cells cultured with 13C -lactate. Finally, the MDVs of 13C-labeled metabolites were obtained using the following formula: MDVs = (abundances of 13C-labeled metabolites—abundances of natural 13C-labeled metabolites)/(abundances of total metabolites—abundances of natural 13C-labeled metabolites).

Flow cytometry
The analysis of cancer stem cell (CSC) ratios was performed using the ALDEFLUOR assay and CD44highCD24–/low staining. In the ALDEFLUOR assay (01700, STEMCELL Technologies, Vancouver, CA), functional stem cells were analyzed and isolated based on the fact that aldehyde dehydrogenase (ALDH) is a widely recognized key marker for breast CSCs68. Specifically, cells with ALDH enzymatic activity (ALDH+) are considered CSCs, while those without ALDH enzymatic activity (ALDH−) are non-CSCs. The detailed procedures for sorting ALDH− non-CSCs are described in our paper12. Immediately following sorting, these ALDH− cells were directly utilized in the subsequent experiments without undergoing any additional passaging.

For in vivo analysis of CSC ratios in tumors, PE anti-mouse EPCAM and APC anti-mouse CD45 staining were used to distinguish tumor cells from other cell populations. The CSC ratio analysis was performed on a BD LSRFortessaTM cell analyzer, and cell sorting was conducted on a BD FACSAriaTM III. Data collection and analyses were carried out using BD FACSDiVaTM and FlowJo 7.6 software. Corresponding flow cytometry gating plots were provided in the supplementary information file.

siRNA knockdown
The siRNAs targeting MCT1, LDHB, and GPR81, along with the universal negative control siRNA (Table S1), were synthesized by Sangon Biotech (Shanghai, China) and transfected into cells using Lipofectamine 2000™ reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). After 48 h, total RNA and whole cell lysates were extracted to assess the efficiency of siRNA interference using RT-PCR and western blot, respectively.

Tumorsphere formation assay
The tumorsphere formation assay was performed as described in our previous paper12. Briefly, 2×105 cells were cultured in ultra-low attachment six-well plates using serum-free DMEM/F12 medium supplemented with 1% N2, 2% B27, 15% glucose, 1 µg/mL hydrocortisone, 5 µg/mL insulin, 10 ng/mL epidermal growth factor (EGF), 10 ng/mL basic fibroblast growth factor (bFGF), and 0.2% heparin (Table S5). The number of spheres with a diameter greater than 50 μm was monitored and quantified under an inverted microscope (Olympus, Tokyo, Japan) on days 10–14. In lactate-treated cells, the DMEM/F12 medium was replaced with glucose-free DMEM/F12 medium (Procell, Canton, MA, USA), and the addition of 15% glucose was omitted.

Immunofluorescence (IF) staining
Ad cells cultured on glass-bottom dishes (Cellvis, Mountain View, CA, USA) or AR suspended cells mounted on adhesive slide were fixed with 4% paraformaldehyde, permeabilized for 5 min with 0.2% Triton X-100, blocked with 10% goat serum for 30 min, and then incubated overnight at 4°C with primary antibodies against 5-mC (1:1000; 28692, Cell Signaling Technology, Danvers, MA, USA) for methylation detection, or against CD147 (1:200) and COXIV (a mitochondrial marker; 1:200; 11242-1-AP, Proteintech, Rosemont, IL, USA) to observe the mitochondrial localization of CD147 and the binding of mito-CD147 scFv to mitochondrial CD147. After incubation with fluorescently labeled secondary antibodies, nuclei were counterstained with DAPI (Invitrogen). All images were captured using laser scanning confocal microscopy (LSCM, Olympus FV3000) and collected using FV31S-SW 2.4.1 with consistent settings across all samples, and fluorescence intensity was quantified using ImageJ 1.50i (NIH, Bethesda, MD, USA).

For cellular co-localization analysis among CD147, MCT1, LDHB, and COX IV, cells were initially stained with primary antibodies against CD147 (1:200), MCT1 (1:200; 20139-1-AP, Proteintech), and LDHB (1:100; ab195964, Abcam) overnight at 4°C, followed by incubation with Alexa Fluor 555-, 647- or 405-conjugated antibodies, respectively, at room temperature for 1 h. This was followed by incubation with an Alexa Fluor 488-conjugated COX IV antibody (1:200; CL488-60251, Proteintech) at room temperature for 3–4 h. Finally, nuclei were counterstained, and images were captured following the same procedures as described above. For human or mouse tumor samples, tissues were fixed, embedded, sectioned, and antigen retrieval was performed using the same procedures as outlined in the Histological Staining and Analysis section below, and stained following the same protocols as for cells.

Histological staining and analysis
Human or mouse breast primary or lung metastatic tumor tissues were fixed, embedded in paraffin, and cut into 4-µm-thick sections. Sections were deparaffinized, dehydrated, and cleared in xylene and gradient alcohol, then used for hematoxylin-eosin (H&E) or immunohistochemical (IHC) staining.

For H&E staining, the slides were counterstained with hematoxylin for 2 min and eosin for 3 min.

For IHC staining, the slides underwent antigen retrieval with 10 μM citrate buffer (pH 6.0) in pressure cookers at 100°C for 2 min, followed by blocking with 5% goat serum and staining with anti-SOX2 antibody (1:100; 11064-1-AP, Proteintech) for 2 h at room temperature. Next, the slides were stained with immunoperoxidase using a General SP kit (SP-9000, ZSGB-BIO, Beijing, China), developed with 3,3′-diaminobenzidine (ZLI-9019, ZSGB-BIO), and counterstained with hematoxylin. To evaluate the expression of SOX2, IHC scores were calculated by two independent pathologists according to the following equation: score = 3 (strong positive) × percentage value + 2 (moderate positive × percentage value + 1 (weak positive × percentage value + 0 (negative) × percentage value. The percentage index 0–4 was defined using the following criteria: 0 (<10% positive cells), 1 (10–25% positive cells), 2 (25–50% positive cells), 3 (50–75% positive cells), and 4 (75–100% positive cells).

For multiplex immunohistochemical (mIHC) analysis, the deparaffinized, dehydrated, and antigen-retrieved tissues were permeabilized with 0.5% Triton X-100, stained with primary antibodies, and detected using a TSA-indirect kit (PerkinElmer) according to the manufacturer’s instructions. Nuclei were stained with DAPI. The image was scored using HALO software (Indica Labs, USA). During data analysis, the immunostained sections were divided into high- and low-expression groups, with the mean score value serving as the cut-off. High expression was defined as scores of CD147≥29.5, scores of MCT1≥17.5, and scores of LDHB≥4.5; scores below these thresholds were considered as low expression.

Circulating tumor cells isolation, counting, and IF staining
Circulating tumor cells (CTCs) were isolated using EPCAM+ magnetic beads (130-105-958, Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, 800 μL of blood collected from mice was lysed to remove red blood cells and incubated with EPCAM+ magnetic beads at 4°C for 15 min. Subsequently, EPCAM+ cells that adhere to the LS column in a magnetic field were eluted. The identification and counting of CK18+CD45− CTCs, as well as the examination of SOX2 levels in CTCs, were conducted through IF staining using the following primary antibodies: anti-CD45 (1:200, 60287-1-Ig, Proteintech), CoraLite® Plus 488-conjugated anti-CK18 (1:200, CL488-66187, Proteintech), and CoraLite® Plus 647-conjugated anti-SOX2 (1:200, CL647-66411, Proteintech). Additionally, Alexa Fluor 555 donkey anti-mouse IgG (H+L) (1:200, A31570, Invitrogen) was used to visualize CD45 expression. Finally, IF signals were quantified using Image J 1.50i (NIH).

ATP measurement
A quantity of 1×106 cells was lysed to detect cellular ATP levels using an ATP bioluminescence assay kit (S0026, Beyotime) according to the manufacturer’s instructions. The bioluminescence signal generated by luciferase was measured as relative light units (RLU) in a luminometer (Promega, USA Gloma), and the ATP levels were calculated using a standard curve.

Oxygen consumption rate measurement
Cellular oxygen consumption rate (OCR) was measured using the XF96 Flux Analyzer (Agilent Technologies). Briefly, Ad cells were seeded into pre-hydrated 96-well plates (1.5×104 cells/well) and cultivated overnight, while AR cells at the same density were seeded directly into poly-L-lysine precoated wells just before the measurement. For lactate-treated cells, they were first cultured in glucose-free media without or with 20 mM lactate for 48 h. Subsequently, an equal number of cells (1.5×104 cells per well) were seeded into pre-hydrated 96-well plates and allowed to adhere for 6 h. Then, AR/Ad cells were incubated at 37°C for 45 min without CO2 in seahorse assay medium containing 10 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate, while the lactate-treated cells were incubated at 37°C for 45 min without CO2 in a substrate-limited medium containing 0.5 mM glucose, 0.5 mM L-glutamine, and 0.2 mM sodium pyruvate. OCR was measured by adding inhibitors at the following times: oligomycin (14 min, 1 μM), FCCP (33 min, 1 μM), and rotenone/antimycin A (53 min, 0.5 μM). Three readings were taken after each injection. Cells in each well were subsequently trypsinized and counted, and OCR values were normalized to the corresponding cell number per well. Data were collected using WAVE 2.6.1 (Alignment) and final results were presented as pmoles/min for OCR (y-axis) versus time (x-axis).

Additionally, OCR was detected using a commercial kit (600800, Cayman Chemical, Ann Arbor, MI, USA) following the manufacturer’s protocols. Briefly, cells at a density of 5×105/2 mL per well were cultivated in a six-well plate for 48 h. Subsequently, MitoXpress-Xtra solution was added, and the plate was read immediately at 650 nm for 2 h using a spectrophotometer (BioTek Instruments, Winooski, VT, USA). The OCR was calculated as (mOD1-mOD2)/T, where mOD1 is the initial absorbance, mOD2 is the final absorbance, and T is time in minutes.

Mitochondrial complex activity assay
The activities of mitochondrial complexes were measured using the enzyme activity microplate assay kit (Abcam). Briefly, 1×107 cells were homogenized, and total protein concentrations were adjusted to 5.5 mg/mL with PBS. To extract the proteins, 1/10 volume of detergent solution was added; then the supernatant from lysed cells was incubated at room temperature for 2–3 h in a 96-well microplate precoated with monoclonal antibodies to complexes I, II, or IV. After adding the specific substrates, absorbances were measured continuously using a spectrophotometer (BioTek Instruments) as follows: complex I (450 nm, 30 min), complex II (600 nm, 2 h), and complex IV (550 nm, 2 h). The enzyme activity of each complex (mOD/min), expressed as the reaction rate, was calculated by (mOD1-mOD2)/T, similar to the calculation in the OCR assay mentioned above.

α-ketoglutarate (α-KG) measurement
α-KG levels were measured using an α-ketoglutarate assay kit (ab83431, Abcam). Briefly, lysates of 1×106 cells were deproteinized and incubated with the enzyme reaction mixture for 30 min. The α-KG level was calculated by measuring optical density at 570 nm in a spectrophotometer (Bio-Rad Laboratories) using a standard curve.

5-methelcytosine (5-mC) DNA dot blot
To detect global DNA methylation levels, genomic DNA was extracted and purified from cells using a DNA extraction kit (DP304, TIANGEN BIOTECH, Beijing, China), followed by quantification, sonication, and denaturation. Next, 1 μg of DNA was transferred to a PVDF membrane and incubated successively with a 5-mC primary antibody (1:1000; 28692, Cell Signaling Technology) and an HRP-conjugated secondary antibody. The chemiluminescence signals were then visualized with a Gel Doc EZ Imager (Bio-Rad Laboratories) and analyzed using Image Lab 5.2.1 (Bio-Rad Laboratories).

Mitochondrial isolation
The mitochondria were isolated from 2×107 cells using the Minute™ Mitochondria Isolation Kit (MP-007, Invent Biotechnologies, Plymouth, MN, USA), which employs protein extraction filter cartridges and differential centrifugation. The isolated mitochondria were stored in Minute™ denatured protein solution for western blot analysis or in Minute™ nondenatured protein solution for co-immunoprecipitation. The purity of the mitochondrial fractions was assessed by performing western blot analysis in a concentration-dependent manner (2, 10, 20, and 50 μg) using antibodies against COXIV, α-tubulin, Na+/K+-ATPase, LAMP1, GM130, and GRP78 to indicate the fractions of mitochondria, cytoplasm, plasma membrane, lysosome, Golgi apparatus, and endoplasmic reticulum, respectively.

Mitochondrial sub-fractionation isolation and protease protection assay
The sub-fractionation of mitochondria was performed according to Huang’s report69. Briefly, 1 mg of mitochondria (measured by protein content) suspended in 40 μL of T10E20 buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.6) was incubated with 0.1 mg of digitonin for 20 min. After adding 120 μL of 250 mM sucrose, the mitochondria were centrifuged at 10,000×g for 15 min. The fraction of the mitochondrial inner membrane and matrix (I/M) was collected from the pellet, while the outer membrane and intermembrane (O/I) were obtained from the supernatant.

For further mitochondrial fractionation experiments, protease protection assays were conducted by incubating intact mitochondria with 0.5 μg/mL of proteinase K (K) for 10 min, either alone or following a 5 min pretreatment with 0.2% Triton X-100, and then subjected to western blotting. TOMM20 or COXIV were used to indicate proteins located in the outer or inner mitochondrial membrane, respectively.

Blue native PAGE analysis
Blue native PAGE analysis was performed as described previously62,70. In brief, mitochondrial proteins were isolated using the procedures detailed in the Mitochondrial Isolation section. After isolation, 30 μg of mitochondrial proteins were loaded onto a 4–13% Bis-Tris Native PAGE gel (P0545S, Beyotime) for electrophoresis-based separation. Then the gel was stained with Coomassie Brilliant Blue to visualize the protein bands.

Mitochondrial mass analysis
Mitochondrial mass was assessed via flow cytometric analysis of cells stained with MitoTracker Green FM (M46750, Thermo Fisher Scientific). Briefly, cells were incubated with 100 nM Mitotracker Green FM for 30 min at 37°C. Subsequently, the cells were rinsed with PBS, detached using trypsin, and then centrifuged at 130×g for 4 min. The resulting cell pellets were resuspended in PBS supplemented with 1% FBS for FACS. Given that MitoTracker Green FM has an excitation wavelength of 490 nm and an emission wavelength of 516 nm, the FITC channel, with an excitation at 494 nm and emission at 520 nm, was used to detect the signal. Data analyses were performed using FlowJo 7.6 software (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

Pyruvate assay
Pyruvate levels were measured using a pyruvate colorimetric assay kit (E-BC-K-130-S, Elabscience). Briefly, 20 µL of cytoplasm or mitochondrial subfraction, along with standard and distilled water (blank control), were incubated with 100 µL of chromogenic agent at 37°C for 10 min. Subsequently, 500 µL of alkali reagents were added to develop color for 5 min at room temperature. The absorbance values for the cytoplasm or mitochondrial subfraction and the standard at 505 nm were obtained by subtracting the absorbance value of the blank control. Pyruvate content was then calculated based on the standard curve and normalized to the protein concentration.

Methylation assay of the SOX2 promoter region
The methylation level in the SOX2 promoter region was assessed using a fast bisulfite conversion kit (ab117127, Abcam) according to the manufacturer’s instructions. Briefly, genomic DNA from 1×106 cells was extracted (DP304, TIANGEN), purified, and quantified. Two hundred ng of DNA was denatured and bisulfite converted at 95°C for 20 min, followed by methylation-specific real-time PCR using three pairs of primers designed with MethPrimer that targeted the CpG islands in the SOX2 promoter region (Table S1). The relative methylation level was calculated as the ratio of the Ct value for methylated DNA to the sum of the Ct values for both methylated and unmethylated DNA.

DNMT3B enzyme activity
The enzyme activity of DNMT3B was measured using a DNMT3B direct activity assay kit (ab113471, Abcam, Cambridge, UK). Briefly, lysates of 1×106 cells were incubated with S-adenosylmethionine donor at 37°C for 2 h in a plate pre-coated with DNA substrate. This was followed by successive incubations with anti-5-mC primary antibody, HRP-labeled secondary antibody, and chemiluminescent substrate. Relative DNMT3B enzyme activity was calculated as the ratio of luminescence values measured using a Bio-Tek fluorescent microplate reader.

Chromatin immunoprecipitation (ChIP)
ChIP assays were performed using a SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads, 9005s, Cell Signaling Technology). Briefly, 4×106 cells were crosslinked in 1% formaldehyde at room temperature for 10 min, with the reaction quenched by 2.5 M glycine. Next, cells were lysed, and chromatin was extracted by incubating with micrococcal nuclease at 37°C for 20 min, followed by brief sonication. The resulting cell lysates (input) were immunoprecipitated at 4°C for 12–16 h with 3 µg of anti-5-mC (28692, Cell Signaling Technology) and anti-DNMT3B (57868, Cell Signaling Technology). The immunocomplexes were then rotationally incubated with 30 µL of ChIP-Grade Protein G Magnetic Beads at 4°C for 2 h, and the ChIP DNA was eluted using ChIP elution buffer at 65°C for 30 min. After reverse cross-linking and proteinase K/RNase treatment, ChIP DNA was purified, amplified, and quantified by RT-PCR. The PCR results for DNA methylation were presented as the percentage of ChIP DNA relative to total input DNA (2(Ctinput–Ctsample)), with the Ct values detected by the indicated antibodies normalized to a Ct value obtained from control ChIP performed with nonspecific rabbit IgGs. The specific primer pair for the SOX2 promoter region is listed in Table S1.

2-2H-lactate metabolic flux analysis
Overall, 2×107 MCF7 AR and Ad cells (n=3 biologically independent samples) were cultured in medium containing 10 mM 2-2H-lactate (693987, ≥98% (CP), Sigma) for 6 h. The mitochondria were isolated, verified for purity, and then sent to Applied Protein Technology (Shanghai, China) for targeted LC/MS analysis of 2H-labeled metabolites.

2H-labeled metabolites in the mitochondria were extracted by adding 500 μL of cold extraction buffer (methanol: acetonitrile: water = 2:2:1, v/v/v), followed by sonication for 2 min and centrifugation at 14,000×g for 5 min at 4°C. The supernatants were thoroughly lyophilized and reconstituted in 50 μL of methanol-water (1:1, v/v) just prior to measurement. A mixture of equal amounts of supernatant from individual samples, analyzed using the same procedure for experimental samples, was used as quality control.

2H-labeled metabolites in the mitochondria were analyzed with LC-HRMS using Thermo Fisher Q-Exactive mass spectrometry, equipped with a Vanquish UPLC system (Thermo Fisher, MA, US), employing a SeQuant ZIC-HILIC column (100 mm×2.1 mm, i.d. 3.5 μm, Merck, Germany). A 1 μL aliquot of supernatant extracted from the mitochondria was injected into the column, with the temperature maintained at 35°C and the flow rate set at 0.4 mL/min. The mass spectrometry operated in both negative and positive modes with a capillary voltage of 2.0 kV. The source temperature and desolvation gas temperature were set at 120 and 500°C, respectively.

Raw peak data were processed using TraceFinder 5.0 and Xcalibur 4.0 software (Thermo Fisher Scientific), with peak alignment and detection performed according to the manufacturer’s protocol. The method “peak alignment and frame extraction” was applied for targeted metabolite analysis. An input file containing theoretical m/z values and detected retention times was used for targeted metabolite analysis, with the m/z width set to 5 ppm. An output file was generated after data processing, which included detected m/z values and relative intensities across different samples. Metabolite mass isotopomer distribution (MID, also known as MDV) was determined based on the ratio of the integrated peak areas of the chosen isotopomer to the sum of all integrated peak areas of the possible isotopomers for the given metabolites. Correction for natural abundance is necessary for meaningful interpretation. The natural isotopic correction was performed by treating a control group of unlabeled samples (mitochondria isolated from cells collected at 0 h) using both a generally applicable correction matrix and an isotopic correction matrix, as previously reported71,72.

Co-immunoprecipitation (Co-IP) assay
Mitochondria were isolated following the protocol described in the Methods Section under Mitochondrial Isolation. Approximately 500 μg of the mitochondrial pellet was resuspended in 200 μL of nondenatured lysis buffer (WA-010, Invent Biotechnologies), a mild formulation designed to preserve native protein-protein interactions within the mitochondria while solubilizing mitochondrial membranes. The suspension was gently pipetted to ensure homogeneity, followed by centrifugation at 12,000×g for 2 min to remove insoluble components. The resulting supernatant was then subjected to a Co-IP experiment according to standard procedures. Briefly, the supernatant was incubated with 30 μg of primary antibodies to CD147, MCT1 (20139-1-AP, Proteintech), or LDHB (14824-1-AP, Proteintech) at 4°C overnight, with irrelevant mouse IgG (P5000-Y, GENIA) or rabbit IgG (I8140, Sigma) used as negative controls. The following day, Protein A&G beads (Invitrogen) were added and incubated at 4°C for 4 h. The immuno-precipitates were then eluted and identified by western blot, with the isolated mitochondria used as an input control.

Transmission electron microscope
For colloidal gold staining, 2×106 cells were fixed with PLP Fixing Solution (G2220, Solarbio, Beijing, China) at 4°C for 24 h and then subjected to dehydration and embedding. Ultrathin sections of the cells were transferred onto grids, blocked with goat serum for 30 min at room temperature, and incubated overnight at 4°C with primary antibodies against CD147 (mouse, 1:100), MCT1 (rabbit, 20139-1-AP, Proteintech, 1:100), and LDHB (goat, ab195964, Abcam, 1:100). This was followed by a 1-h incubation at room temperature with 10 nm gold-conjugated anti-mouse IgG (ab39593, Abcam, 1:200), 20 nm gold-conjugated anti-rabbit IgG (ab270563, Abcam, 1:200), and 35 nm gold-conjugated anti-goat IgG (K0038D-Gold, Solarbio, 1:200). Finally, the sections were consecutively counterstained with 5% uranium acetate and lead acetate and observed under an EM-1230 transmission electron microscope (JEOL Ltd., Tokyo, Japan).

Proximity ligation assay (PLA)
The Duolink® in situ assay kit (DUO92008, Sigma) was utilized according to the manufacturer’s instructions. In Brief, MCF7 cells were treated with either 0 or 20 mM lactate and then cultured on small round cover glasses. Subsequently, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. After being blocked with the Duolink® blocking solution, the cells were incubated overnight at 4°C with mouse monoclonal anti-CD147 (produced in our laboratory, dilution 1:200) and rabbit polyclonal anti-LDHB (14824-1-AP, Proteintech, dilution 1:100). Next, the PLA probe solution was added, and the cells were incubated in a pre-heated humidity chamber at 37°C for 1 h. After thorough washing, the ligation solution was applied for 30 min at 37°C, followed by the addition of the amplification solution for 100 min at 37°C. Afterward, DAPI was used to stain the cell nuclei for 15 min at RT. Images were captured via laser scanning confocal microscopy (LSCM, Olympus FV3000) and analyzed using the ImageJ software.

Molecular docking simulations
The molecular docking simulations were performed using AutoDock 4.273. Briefly, PDBQT files were generated for CD147, MCT1, and LDHB with Gasteiger partial charge assigned, and the AutoGrid algorithm was employed to precalculate the atomic affinity grid for docking calculations. Candidate complexes were selected based on binding energy scores and geometric complementarity, the latter evaluated through cluster analysis. The detailed molecular interactions were analyzed using LIGPLOT version 4.4.274, with hydrophobic interactions defined as ligand-protein residue hydrophobic atom distances <3.9 Å, while hydrogen bonds were identified when (i) the donor-acceptor pair matched predefined criteria and (ii) bond angles and distances met default thresholds.

Expression plasmids Construction, protein expression, and purification
The cDNA sequences encoding the extracellular domain of CD147 (1–205 aa) and the full-length LDHB were cloned into the pGEX-6P-1 and pET-28a plasmids, respectively. Subsequently, GST-CD147 N-terminal and His-LDHB proteins were expressed using BL21 (DE3) bacteria cells. Specifically, bacteria cells carrying the plasmid encoding these proteins were initially incubated at 37°C for 6 h, then the expression was induced with 0.5 mmol/L isopropyl-β-d-thiogalactopyranoside (IPTG) at 16°C overnight. After that, cell lysates were collected by centrifugation at 13,500×g for 30 min at 4°C. Prior to use, appropriate beads were pre-washed with PBS and equilibrated in lysis buffer. Glutathione Sepharose Beads (SMART Lifesciences, Changzhou, China) and His60 Ni Superflow Resin (TaKaRa, Japan) were incubated with cell lysates at 4°C for 4 h with continuous rotation. Following incubation, the beads were washed with lysis buffer, and the proteins were eluted with GST elution buffer (75 mmol/L Tris-HCl pH 8.0, 20 mmol/L-Glutathione) and His elution buffer (50 mmol/L NaH2PO4, 300 mmol/L NaCl, 250 mmol/L imidazole, adjust pH to 8.0), respectively.

For the expression of proteins with specific-sites mutations, the codons in the cDNA sequence corresponding to the target sites were replaced with alanine-encoding codons. Subsequently, the same experimental procedure as described above was followed.

GST pull-down assay
GST pull-down assays were performed following the instructions provided by the manufacturer of the Pierce GST Protein Interaction Pull-Down Kit (21516, Thermo Fisher Scientific). First, the glutathione agarose was equilibrated. Then, 100 μg of the purified GST-CD147 N-terminal protein or 100 μg of the GST protein alone, each in a 300 μL volume, were incubated with glutathione-agarose beads at 4°C for 1 h. After the incubation, the beads were washed with the wash buffer. Subsequently, 100 μg of the purified His-LDHB were added to the column and incubated at 4°C for 2 h. After another round of washing with the wash buffer, the eluted protein was subjected to immunoblotting analysis using HRP-conjugated rabbit polyclonal anti-GST (1006-3, Huabio, dilution 1:500) or mouse monoclonal anti-His (66005-I-Ig, Proteintech, dilution 1:1000) antibodies, respectively.

Statistics and reproducibility
All data were presented as mean ± SEM from at least three independent experiments, and statistical analyses were performed using GraphPad Prism 8.0 software. To determine statistical significance, Student’s t-test or one-way ANOVA was conducted to compare continuous variables between two groups or more than two groups, while the χ2 test was used for categorical variables. Spearman correlation analysis was employed to investigate the correlation between the expressions of CD147 and MCT1 or LDHB. The Kaplan–Meier method and the log-rank tests were applied to compare survival and recurrence times between patients with high or low CD147/MCT1/LDHB co-expression. The multivariate Cox proportional hazards model was used to explore independent prognostic predictors for the overall survival of patients with breast cancer. For all statistical analyses, p-values less than 0.05 were considered significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the authors investigate increased lactate uptake and utilization during anchorage independence. Intraperitoneal lactate injections are shown to increase tumor metastasis and decrease survival in mouse models. The authors noted increased SOX2 expression in metastatic lesions of mice treated with lactate. They hypothesized the increased SOX2 expression was due to a change in the methylation state of the SOX2 promoter and attempted to tie this change in promoter methylation to increased α-KG levels in anchorage resistant or lactate treated cells. A number of issues need to be addressed.\n\nMajor Comments\n1. ALDH- cells are not well defined, and it is not clear why they are chosen as an in vitro model. The authors refer to their own papers that isolate a cell population with low ALDH1 activity as determined by a commercially available kit. This should be explained in the introduction or methods section. Are these ALDH1- cells sorted from the bulk population immediately before experiments? Or is this a population that is sorted and then cultured for several passages? Do the authors periodically verify the population continues to be ALDH1- after several passages? Why are ALDH- cells used for experiments and not the total adherent population which per the authors prior work are composed of 95-99% ALDH- cells?\n2. AR cells are also poorly defined. The following statement is concerning and confusing- “Besides, based on the decreased glucose uptake in detached cells, adherent cells cultured in glucose-free medium with or without exogenous lactate incubation were considered as a simulation for AR or Ad cells, respectively.” Separate nomenclature should be used throughout the manuscript for cells that were treated under these very different conditions since they are not equivalent. This has significant implications for interpretation of the entire paper. For instance, it is shown that “AR” cells and cells treated with lactate have increased abundance of TCA cycle intermediates including α-KG. Are these true AR cells or simply adherent cells grown in the absence of glucose and lactate? If the latter, the difference between TCA cycle intermediates would be expected, as the cells they term AD are being grown without adequate fuel for the TCA cycle while the so-called AR cells are simply adherent cells grown without glucose but with lactate. A better comparison would be true anchorage independent cells grown in a low attachment dish with media containing a physiologic amount of glucose compared to adherent cells grown under the same conditions, since CTCs in-vivo are exposed to far more glucose than lactate in circulation. The premise of the manuscript is preferential uptake and utilization of lactate by CTCs when glucose is available as a carbon source. The in-vitro experiments (aside from the seahorse experiment as detailed below) should be performed under similar conditions, with media containing glucose and lactate.\n3. Of importance, both in vitro and in vivo experiments rely heavily on adding high levels of exogenous lactate to see any effects, biasing results strongly towards finding a role for lactate metabolism. The question above of whether an “anoikis resistance” state is actually growth in low glucose/high lactate makes this effect more concerning. For instance, in Figure 2 JK, CHC/GNE inhibit tumor growth in the presence of lactate given IP—do they work without extra lactate? Most experiments in Figures 2-8 require added lactate to demonstrate the effects of intervention. In Figure 4FG and S4 L,M, do inhibitors decrease ATP levels and OCR if lactate is not added? In Figures 8 and S10, the effect of scFv to all endpoints is dependent on adding exogenous lactate as well. Figure 8I-K, which are the only experiments in which scFv is added without extra lactate, show that scFv has no effect. In in vivo systems (PDX1/2, 4T1, MCF, MMTV-PyMT), does CHC/GNE treatment inhibit metastasis and stemness in the absence of extra lactate, which is the clinically translatable experiment?\n4. Related to this, do the authors propose that lactate uptake and oxidation an intrinsic and stable property of anoikis resistance or does the state of detachment induce this metabolic switch? That is, if you replate floating AR cells on solid matrix or plastic, will their lactate utilization revert to match the adherent state?\n5. Figure 1F. Was this tracing experiment performed in glucose and pyruvate free media? While a lower fractional abundance of M+3 pyruvate than the fractional abundance of M+2 TCA cycle intermediates is possible with separate pyruvate pools, a fractional enrichment of only 2% for pyruvate and 25% for TCA cycle intermediates would be very unusual as lactate carbons must go through pyruvate to enter the TCA cycle. The data presented suggests the total pyruvate pool outside the mitochondria is very large compared to the mitochondrial pool, with minimal import of pyruvate into the mitochondria, and the majority of the mitochondrial pyruvate derived from lactate imported into the mitochondria. This degree of compartmentalization of the pyruvate pools is usually not seen. The data also implies a minimal conversion of lactate into pyruvate in the cytoplasm by LDHA.\n6. Figures S1A-C, S1D-E. The decrease in nutrient utilization could also be explained by lower growth rates when detached, which the authors’ prior paper (Meng 2020) demonstrates. Comparative doubling times between AR and attached cells should be provided here again.\n7. Figure 1D. A decrease in plasma lactate levels with tumor progression is an unexpected result. Plasma lactate levels are usually maintained in a tight range, with increasing tumor burden leading to increased plasma lactate levels. This does not fit with the hypothesis as the vast majority of tumor cells are anchored and not CSCs or detached CTCs and should have lower lactate consumption as demonstrated in the authors’ experiments. How do the authors reconcile this?\n8. Figure 4C-E. Seahorse assays should be normalized to cell count. These differences in respiration could be due to a different number of cells per well.\n9. Figure4D-E. A dramatic increase in OCR with addition of a metabolite is unlikely in normal seahorse media. Substrate limited media with significantly lower levels of glucose and glutamine are required to investigate the contribution of individual metabolites to respiration. The materials and methods section does not specify the use of substrate limited media. This should be clarified.\n10. Figure 4E. The authors claim lactate is utilized as a fuel for the TCA cycle and increased TCA cycle turnover is promoting oxidative phosphorylation and increased respiration in the seahorse assay. However, Isolated Complex 1 activity is very different than Complex 1 activity in an intact cell. Respiration in the seahorse assay, including ATP uncoupled respiration, is dependent on the availability of NADH mostly provided by the TCA cycle. In the isolated Complex 1 activity kit from Abcam a fixed amount of NADH substrate is provided and thus differences in TCA turnover in the intact cell have no bearing on isolated Complex 1 activity. The dramatic differences in isolated Complex 1 activity suggest either differences in mitochondrial mass or abundance, defects in Complex 1 assembly, or PTMs of Complex 1 subunits. The authors should perform blue native page for complex assembly and a western blot for mitochondrial proteins such as citrate synthase, and flow cytometry to estimate mitochondrial mass.\n11. In Figure 5, D-2HG is used as an α-KG “inhibitor.” However, D-2HG has protean effects which include inhibition of LDH (Science 2022;377:1519), which means its effects are not specific for -KG.\n12. In Figure 5 and 7L, α-KG and CD147 seem to dramatically decrease the expression of DNMT3B. The effect is much greater than the modest loss of DNMT3B activity (Figure 5I). What explains this discrepancy, and how does -KG decrease protein levels of DNMT3B?\n13. Figure 6B does not imply that 100% of the labelled lactate exists in the mitochondria as stated in the results section, rather this simply indicates 100% labelling of mitochondrial lactate.\n14. Figures S6C-H shows overexpression of CD147 in two cell lines and knockdown in a third cell line. It would be more convincing to do paired experiments in the same cell line background. Is there a major difference in baseline expression of CD147 between the cell lines (ie low in MCF7 and T47D and high in MDA-MB-231) that prompted this experimental design? If so, is the role of endogenous CD147 cell type specific?\n15. The method of co-IP experiments in Figures 6 and S7 is not clear. Without mild detergent, mitochondrial or submitochondrial particles will be isolated, especially with centrifugation (vs magnetic) techniques. Thus, intramolecular associations may not exist if conditions are not appropriate and controls are not included. The TEMs are not convincing for more than random colocalization on organelles. Perhaps a more convincing method would be a proximity ligation assay. The human tissue IF shown in Figure 6H,L does not demonstrate colocalization of CD137, MCT1, and LDHB, although magnification may be limiting. Overall, evidence for a macromolecular complex between these 3 proteins is unconvincing and seems overstated. However, functional cooperation with LDHA would not require physical association.\n16. Figures 6 and S7 show that is there a correlation between CD137 and MCT1/LDHB only in metastatic but not nonmetastatic patients. They are not necessarily higher in metastatic patients, only co-correlated. These data suggest that the actual level of expression of these proteins is unlinked to metastasis, while it is linked to survival. What accounts for this discrepancy?\n17. Figures 7B-D. It is well established CD147 is required for proper trafficking and membrane insertion of MCT1 and MCT4, the predominant plasma membrane lactate transporters. Consistent with this the authors demonstrate a dramatic reduction in MCT1 protein abundance when CD147 is knocked out (Fig S8D). Yet when a mitochondrial localized version is introduced to cells, the authors demonstrate a complete restoration of MCT1 protein levels, restoration of mitochondrial lactate levels, and lactate induced increase in cellular respiration. This begs the question – how is lactate entering the cytoplasm to be taken up by the mitochondria as presumably cytoplasmic MCT1 localization is impaired when CD147 with an MLS is introduced into cells? And with the majority of MCT1 located in the plasma membrane and not the mitochondria, how are MCT1 protein levels restored on a total cell level with introduction of mito147?\n18. There are a very large number of grammatical errors which make the paper difficult to follow.\n\nMinor Comments\n1. Figure 1B. The Y axis is missing a label.\n2. Western blots should be provided for all siRNA experiments to demonstrate on target knockdown.\n3. Figure 4K. No bars are provided to indicate the comparisons for which significance is being reported.\n4. Total pool sizes should be provided for all labelling experiments.\n5. Figure S4D,I show that A485 and siGPR81 both decrease SOX2 expression, in contrast to what the authors state.\n6. Figure S6J should have controls from whole cells or another fraction to assess enrichment. In Figure S6M, CD147 does not co-IP with MCT1 in T47D cells. Have the lanes been mislabeled?\n7. Figure S8B—the amount of CD147 in non-mitochondrial fractions should be shown to confirm that mito 147 is limited to the mitochondrion.", "reviewer_2": "In this manuscript by Zhang et al., the authors describe a new role for the metabolite lactate in the regulation of breast cancer metastasis-initiating cells (MICs). They show that MICs uptake extracellular lactate that they import into the mitochondria and oxidize it through a protein complex formed by CD147/MCT1/LDHB. Lactate-derived pyruvate feeds the TCA cycle and increases the levels of a-KG, which in turn inhibits the DNA methyltransferase DNMT3 leading to a decrease in the levels of DNA methylation on the promoter of SOX2 and to an increase of its expression. By performing both in vitro and in vivo functional experiments, the authors show that lactate increases the number and metastatic properties of breast cancer stem cells (CSCs), phenotypes that are abolished upon inhibition of either MCT1 or LDHB. Lastly, and to prove the involvement of CD147, they develop an adenoviral system to express a svFc to target mitochondrial CD147 and show that it efficiently inhibits lactate-dependent metastatic potential of CSCs.\n\nThis manuscript provides novel and important insights into the metabolic regulation of metastasis-initiating cells involving a metabolic-epigenetic axis linking lactate metabolism to gene expression. It also establishes a new role for the chaperone CD147 on mitochondrial lactate import and metabolism, while providing the rationale to target this chaperone to reduce breast cancer metastasis. In this context, the findings reported in this study are of interest and relevant for the scientific community. The manuscript contains an impressive amount of data, which, overall, is well presented and support most of the authors´ conclusions. However, there are some issues that need to be addressed, as specified below.\n\nMajor issues\n- There is no information about why the authors use the indicated concentrations of lactate (mM range). I understand that this range might be the one observed in physiological conditions, but they did not provide any explanation.\n- Throughout the manuscript, the authors use the expression “lactate feeding” referring to their in vivo studies, but they are injecting lactate intraperitoneally. Related to this, there is no experimental data on the levels of lactate upon i.p. injection in mice. The authors should check the levels of lactate in the serum and within tumors to prove that the lactate injected is reaching cancer cells, especially considering that lactate is the primary source of carbon feeding the TCA cycle in most tissues (PMID: 29045397).\n- The effect of lactate treatment clearly inhibits metastasis formation. But does it affect primary tumor growth?\n- Some of the immunostaining experiments are not very clear and they do not support the authors ‘claims. For instance, in Fig.2I, the staining for SOX2 is not nuclear, in Fig.2M it seems that SOX2 is expressed n CK18- cells, in Fig.6K there is no staining for SOX2, in Fig 6H there is no overlay of CD147, MCT1 and LDHB in CTCs and in Fig.6F it looks like the signal in the lactate treated samples is just background (it is exactly the same non-specific signal in all channels).\n- Another important issue of this manuscript is the authors ‘claim that lactate can induce stem cell properties in non-stem cells based on the results presented in Figure 3. It could be possible that ALDH- cells can become ALDH+ cells in vivo, which are the ones targeted by lactate, as they prove in Figure 2. This is supported by the fact that the number of ALDH+ cells is basically the same in primary tumors and metastasis derived from ALDH+ or ALDH- cells (compare Fig. 2C and Fig. 3F), indicating that the effect of lactate could be on the newly formed ALDH+ cells and is not driving the acquisition of stem cell properties in ALDH- cells. To fully prove this hypothesis, they authors should do tracing experiments.\n- In their in vivo experiments, they demonstrate that lactate uptake by CSCs is important for metastasis formation by injecting lactate into mice. In these conditions, they show an increase in the number of metastatic nodules. However, is this mechanism also occurring in more “physiological” conditions? For instance, is lactate uptake by CSCs in non-treated mice involved in metastasis formation? Are there other cancer cells releasing lactate under normal conditions (for instance glycolytic cancer cells) and feeding this CSCs to become more metastatic? This could be easily assessed by staining tumor sections with specific markers.\n- The authors claim that the effect of lactate is independent of histone lactoylation and the activation of the lactate receptor GPR81. However, Figure S4D and S4I show that, in T47D cells, both the inhibitor of P300 and the silencing of GPR81 decrease SOX2 expression. Interestingly, the number of spheres and CSCs is not decreased, suggesting an uncoupling of these two phenotypes.\n- The co-IP experiment in Fig. S6M (upper panel) is confusing. The blot for CD147 in the MCT1 IP shows that either CD147 is being IPed by non-specific IgGs in T47D cells or, if the labelling of the lanes is wrong, then CD147 is being IPed by non- specific IgGs in MCF7 cells.\n- Lastly, the authors did a very elegant approach to target CD147, which provided new information about the role of this chaperone in controlling lactate metabolism in the mitochondria. Importantly, this approach also implicates CD147 as a potential target to inhibit breast cancer metastasis. However, it is not clear what will it be the advantage of targeting CD147 instead of using MCT1 or LDHb inhibition, which, as the authors proved in this manuscript, efficiently blocks lactate-driven metastasis formation. I think that the authors could discuss this further to contextualize their findings.\n\nMinor issues\n- Figure 5k: how is the data represented? The figure legend states that n=3 experiments have been done, but one representative experiment is shown. If so, how has the statistics been done?\n- Why CD147-KD reduces the number of ALDH+ cells in primary tumors from MDA-MB-231 cells (Fig.S6G)?\n- In figure S6J, it would be nice to include a lane with protein form the other fractions (membrane, cytosol, nuclei, lysosomes) as a positive control for the antibodies used to claim that mitochondrial fractions are pure.\n- A writing revision/editing of the manuscript is encouraged to improve its quality.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "Zhang, Tian, and Song and colleagues investigate lactate catabolism role in breast cancer stem like cell features. Understanding tumor metabolism, cancer stem cells, and metastasis are highly relevant areas of clinical importance that will be of interest to a very broad readership. The study is fascinating and presents several novel findings, with important biological and translational implications.\n\nThe ample discussion of CSC vs MIC properties, phenotypic plasticity, and context dependence of their properties is a strength. Numerous exciting and novel data are presented in this study. The finding that lactate can essentiall transform non CSC into CSC is provocative. Mechanistic experiments demonstrating the lactate stemness effect is due to its role in oxphos, and probably not p300 etc, are robust. The manuscript includes copious data, most of it complementary and thorough, and a wealth of novel information that will be important for the field. Enthusiasm for this study is high.\n\nThe paper is well written, but grammatical editing is needed.\n\nSpecific comments\n1. There are no methods in the main paper. Methods should be readily visible to the readership and not relegated to the supplement as they are required to be able to accurately interpret the data.\n2. First results section- cell line models are inadequately described, as is the generation of Ad vs AR cells. A diagram would be beneficial\n3. Fig S1 , ALDH+ population data should also be provided, as they may contribute to met initiation and/or may also have a similar metabolic fuel adaptation. It is important to denote whether these phenotypes are dependent on ALDH status or not. I would expect that ALDH+ compared to ALDH- population inherently has higher lactate uptake and MCT1 protein levels. This should be experimentally tested.\n4. [lactate] in tumors as they progress would support the circulating lactate data in Fig 1\n5. The first few figures data (besides the 4T1 expt) are all from t47d and MCF7 cells and it is very important to make this clear in the text and to state that the findings are from two ER+ brca cell lines. Stating ‘breast cancer’ throughout the paper is misleading and it is certainly not true that all breast cancers can be lumped together metabolically speaking. Even within TNBC there are major metabolic differences between tumors and cell lines (Gong Cell Metabolism 2021)\na. -OR- congruent results need to be provided in two TNBC cell lines and then it can be stated in text the findings are from ER+ and TNBC cell lines\nb. Also, results section needs to mention whether ER+ cells were cultured in presence of E2 and if charcoal stripped media was used\n6. Fig 1- the invitro lactate data is from MCF7 but mouse data from 4T1. Congruent datasets need to be added to be able to compare in vitro and in vivo\n7. Throughout, I would avoid stressing xyz result ‘in metastatic cells’ when you have not compared them to non-metastatic cells. I would only mention metastasis when you are specifically assaying metastasis or direct metastasis phenotypes.\n8. Paragraph lines 175-188 stemness potential needs to be changed to stemness ‘markers’ or ‘proteins’ as not functional assay is conducted (mammosphere formation primary and secondary would be ideal)\n9. No information about properties of the PDXs is provided- is there a citation in which they’ve been described before? If not, this publication must do the work of presenting basic characteristics (patient properties of donor biopsies ; hormone receptor status ; treatment status and met status from patient; basic histology needs to be shown). How the PDX identities were tracked and validated needs to be described (STR?)\n10. Primary tumor growth for Fig 2 needs to be reported. It is not clear if the affects of lactate would equally impact primary tumor\n11. Fig 4- I would expect that adding any carbon fuel to cell culture media would have potential to elevate oxphos, despite whatever preference the cells/mitochondria may have. Fig 1 results indicate selectivity for lactate in these MICs, so authors should test glucose / glutamine / lipid as negative controls to see if OCR is elevated in the relevant cell populations under investigation. Same goes for checking SOX2 protein level.\n12. Results suggest that both oxphos and aKG-mediated histone methylation are important for the lactate driven stemness phenotype. Authors need to clarify- when they inhibit ETC proteins, is aKG also reduced? Experiments to determine if both branches are necessary to support stemness are needed. Alternatively perhaps only the methylation aspect is important and the elevated oxphos is a ‘side effect’ of increased TCA cycling but the redox balance/ATP generation from oxphos is not actually important for the CSC phenotype.\n13. Fig6 TEM and IF indicate drastic change in mitochondrial structure and perhaps subcellular localization upon lactate treatment. This should be quantified and reported.\nMinor comments\n1. Line 70- the statement ‘90% of tumor cells detached from the primary tumor’ is confusing and is certainly not a generalizable fact. The cited review does not prove this.\n2. Especially since this is a metabolism study, cells should be cultured in physiologic glucose concentration. The [gluc] is not reported in the methods. 2.5-5 nM is standard to achieve near physiologic glucose level.\n3. Incorporation of lactate into TCA cycle has been reported in TNBC cells and should be mentioned, in the context of therapeutic stress. Potential similarities between stress of therapy and of metastasis should be discussed (Baek et al Oncogene 2023)."}, "author_response": "Questions and answers:\nReviewer#1 (Remarks totheAuthor)\nIn this manuscript, the authors investigate increased lactate uptake and utilization\nduring anchorage independence. Intraperitoneal lactate injections are shown to\nincrease tumor metastasis and decrease survival in mouse models. The authors noted\nincreased SOX2 expression in metastatic lesions of mice treated with lactate. They\nhypothesized the increased SOX2 expression was due to a change in the methylation\nstate of the SOX2 promoter and attempted to tie this change in promoter methylation\nto increased α-KG levels in anchorage resistant or lactate treated cells. A number of\nissues need tobe addressed.\nWe sincerely thank you for your thoughtful and constructive feedback on our\nmanuscript. Your insights are truly invaluable for strengthening the quality of our\nwork. In the revised version of the manuscript, we have meticulously addressed all\nraised issues through additional experimental evidence, expanded methodological\nclarifications, and refined data interpretations. These comprehensive revisions\nsignificantly enhanced the study's robustness and clarity. Thank you again for your\ntime and supportin helping us improve ourresearch. Below,youwill find ourdetailed,\npoint-by-point responses to yourquestions.\nMajorComments\n1. ALDH- cells are not well defined, and it is not clear why they are chosen as an in\nvitro model. The authors refer to their own papers that isolate a cell population with\nlow ALDH1 activity as determined by a commercially available kit. This should be\nexplained in the introduction or methods section. Are these ALDH1-cells sorted from\nthe bulk population immediately before experiments? Or is this a population that is\nsorted and then cultured for several passages? Do the authors periodically verify the\npopulation continues to be ALDH1- after several passages? Why are ALDH- cells\nused forexperiments and not the total adherent population which per the authors prior\nworkare composed of95-99%ALDH-cells?\nResponse:\nWe sincerely apologize for the confusion that have caused by the definition and\napplicationofALDH-cells in ourstudy.\nAldehyde dehydrogenase (ALDH)is widely recognized as a keymarker forCSCs,\nespecially for breast CSCs[1, 2]. Consequently, we have utilized this characteristic to\nanalysis and isolate functional stem cells by the ALDEFLUOR assay (Cat#01700,\nSTEMCELL Technologies). In this assay, cells with ALDH enzymatic activity\n(ALDH+) are considered CSCs, while those without ALDH enzymatic activity\n(ALDH-) are non-CSCs. To clarify, we have added a detailed description of the\ndefinition and rationale for using ALDH- cells in yellow highlight in the Methods\nSection under “Flow cytometry and cell sorting” as following: In the ALDEFLUOR\nassay (Cat#01700, STEMCELL Technologies, Vancouver, CA), functional stem cells\nwere analyzed and isolated based on the fact that aldehyde dehydrogenase (ALDH) is\na widely recognized key marker for breast CSCs[1]. Specifically, cells with ALDH\nenzymatic activity (ALDH+) are considered CSCs, while those without ALDH\nenzymatic activity (ALDH-)arenon-CSCs.\nAs stated in the Introduction Section (Page 4, Paragraph 2, Line 89-91,\n“Similarly, our previous study demonstrated that detached ALDH- non-stem breast\ncancer cells gained the stemness potentials via resistance to detachment-induced\nanoikis (Meng et al., 2020),…”), our previous work indicated that detachment\nreprogramed ALDH-non-CSCs intoALDH⁺ CSCs[3]. In the present study, we aimed\nto further explore whether lactate, an important energy substrate for detached breast\ncancer cells, can also reprogramALDH- non-CSCs intoALDH⁺ CSCs. Therefore, we\nused ALDH- cells as an in vitro model, which have been also stated in the Result\nSection (Page 7, Paragraph 3, Line 204-208, “We previously showed that\ndetachment induces the stemness potentials in non-stem breast cancer cells and\nreprograms non-CSCs into CSCs. We then further analyzed the stemness potentials in\nALDH- non-stem breast cancer cells cultivated with or without lactate treatment and\nfoundthat lactateindeed increased the stemness potentials inthese cells”).\nFor the sortedALDH⁻ cells, we directly utilized them in subsequent experiments.\nAs such, we do not cultureALDH- cells for several passages, thereby minimizing any\npotential changes over time and ensuring the non-stemness status of ALDH-\nsubpopulation. To clarify, we have included more detailed description for using\nALDH- cells in yellow highlight in the Method Section under “Flow cytometry and\ncell sorting” as following: Immediately following sorting, these ALDH⁻ cells are\ndirectly utilized in the subsequent experiments without undergoing any additional\npassaging.\nIt is true that 95-99% of the adherent cells are ALDH- non-CSCs, however, the\nrare (1-5%) but potent ALDH⁺ CSCs dominate the stemness potentials. When we\ncarried out experiments to investigate whether lactate could induce stemness\npotentials inALDH- non-CSCs and reprogram them intoALDH⁺ CSCs, it was critical\nto eliminate the confounding influence of pre-existing ALDH⁺ CSCs. Thus, we used\nthe sorted ALDH- subpopulation but not the total adherent population for\nreprogramming-focused experiments(e.g.,Figure 3 andFigure S3).\n2. AR cells are also poorly defined. The following statement is concerning and\nconfusing- “Besides, based on the decreased glucose uptake in detached cells,\nadherent cells cultured in glucose-free medium with or without exogenous lactate\nincubationwere considered as a simulation forAR orAd cells, respectively.” Separate\nnomenclature should be used throughout the manuscript for cells that were treated\nunder these very different conditions since they are not equivalent. This has\nsignificant implications for interpretation of the entire paper. For instance, it is shown\nthat “AR” cells and cells treated with lactate have increased abundance of TCAcycle\nintermediates including α-KG.Are these trueAR cells or simply adherent cells grown\nin the absence of glucose and lactate? If the latter, the difference between TCA cycle\nintermediates would be expected, as the cells they termAD are being grown without\nadequate fuel forthe TCAcycle while the so-calledAR cells are simply adherent cells\ngrown without glucose but with lactate.Abetter comparison would be true anchorage\nindependent cells grownin a low attachment dishwith media containing a physiologic\namount of glucose compared to adherent cells grown under the same conditions, since\nCTCs in-vivo are exposed to far more glucose than lactate in circulation. The premise\nof the manuscript is preferential uptake and utilization of lactate by CTCs when\nglucose is available as a carbon source. The in-vitro experiments (aside from the\nseahorse experiment as detailed below) should be performed under similar conditions,\nwithmedia containing glucose and lactate.\nResponse:\nWe sincerely apologize for any confusion that may have arisen from imprecise\ndescriptions in the original text and would like to provide a clear and detailed\nclarificationofourexperimental design as follows:\nIn our study, we investigated the impact of lactate in promoting stemness\npotential in detached cancer cells using two complementary experimental models\n(see diagram below).1) Low-Attachment Model: This model replicates the detached\nstate of metastatic cells. Parental adherent cells (Ad cells) underwent anoikis when\ncultured in low-attachment plates, whereas cells surviving 7-days of continuous\nsuspension culture (AR cells) acquired robust anoikis resistance. Both Ad and AR\ncells were maintained in glucose-containing RPMI 1640 complete medium to ensure\nphenotypic comparability. 2) Glucose-Free Model with Lactate: To mimic the\nmetabolic trait of detached cancer cells that characterized by nutrient-deprived, we\nemployed a glucose-free system supplemented with lactate. This approach was based\non our findings that detached cells exhibit reduced glucose uptake (via GLUT1\ndownregulation[4]) and enhanced lactate uptake (Figure 1 and Figure S1). Notably,\nexogenous lactate (20 mM) suppressed stemness under glucose-replete conditions\n(Response Figure 1), further supporting the rationale for this model. In this setup,\ncells were cultured in glucose-free medium supplemented with 20 mM lactate (Lac\n20mM) were designated as lactate-treated cells, while in glucose-free medium alone\n(Lac 0mM)were controlcells.\nCreated in BioRender. Song, C. (2025) https://BioRender.com/pzpeq9s\nTwo cellular models were used in the study: the low-attachment model for replicating the\ndetached state of metastatic cells, and the glucose-free model for mimicking the nutrient-deprived\nmetabolic characteristic of detachment.\nResponse Figure 1. Lactate inhibits the stemness potential of breast cancer cells under\nglucose-containing condition. (A-B) ALDH+ (A) and CD44highCD24-/low (B) subpopulations in\nbreast cancer cells cultured under glucose-containing condition. Data are represented as mean ±\nSEM.Statistical analysis: one-wayANOVA(Lac5,10,20,40mM vs.Lac 0mM).*p<0.05,**p<0.01,***p<0.001,****p<0.0001.\nAdditionally, we have supplemented the diagram above and revised the relevant\nstatement in yellow highlight in the Method Section under “Cell lines and\ntreatments” as follows: “As depicted in the following diagram, there are two distinct\ncellular models in our study.One is the Low-Attachment Model. Cells surviving for 7\ndays in glucose-containing medium (3.15g/L) under detached suspension culture\nconditions inultra-low attachmentplates wereconsideredanoikis-resistant (AR)cells,\nwhile cells cultured in uncoated common plates under attached conditions with\nglucose-containing medium (3.15g/L) served as control adherent (Ad) cells[3]. The\nother is Glucose-Free Model with Lactate. Due to the reduced glucose uptake in\ndetached cells, adherent cells cultured in glucose-free medium, with or without\nexogenous lactate incubation, were designated as “Lac 20mM” and “Lac 0mM”,\nwhich weredesigned tomimic the states ofAR andAdcells,respectively.”\nIn our study design based on the aforementioned models, we first utilized the\nlow-attachment model to investigate the detachment-induced changes in multiple\naspects, including metabolomics (Figure 1A), 13C -lactate (Figure 1F) /2-2H-lactate\n3\nmetabolic flux (Figure 6A-B), proteomics (Figure 1G, 4A), screening of stem cell\ntranscription factors (Figure S2C), OXPHOS metabolism (Figure 4C), complex I\nactivity (Figure 4E), α-KG-DNMT3B mediated SOX2 hypomethylation (Figure\n5E-G), the mitochondrial CD147/MCT1/LDHB lactate oxidation complex (Figure\n6C-E), and mitochondrial CD147-mediated OXPHOS and stemness potential (Figure\n7C-E). Subsequently, we applied the glucose-free model with lactate to further\nvalidate the observed alterations. Considering our previous research[3] that\ndemonstratedthe inductionofstemness potential invitro and in vivo upondetachment,\nthe current study specifically focuses on exploring the metabolic adaption mechanism\nunderlying the inductionofstemness potential during the metastasis process.\nWe agree with your point. The core premise of our manuscript posits that when\nglucose is available as a carbon source, CTCs preferential uptake and utilize lactate.\nIn the in-vivo environment, CTCs in the circulation are indeed exposed to a much\nhigher concentration of glucose than lactate. However, as previously reported by Uen\net al.[4] and visually depicted in Figure 1 and Figure S1, the uptake of glucose in\ndetached cells like CTCs is significantly reduced due to the downregulation of\nGLUT1. Notably, under glucose-replete conditions, the addition of 20 mM exogenous\nlactate was found to suppress stemness potential of these cells (Response Figure 1).\nThese significant findings have guided our subsequent validation experiments on cells\nthat cultured in glucose-free conditions supplemented with lactate (hereafter referred\ntoas lactatetreated cells).\n3. Of importance, both in vitro and in vivo experiments rely heavily on adding high\nlevels of exogenous lactate to see any effects, biasing results strongly towards finding\na role for lactate metabolism. The question above of whether an “anoikis resistance”\nstateis actually growth in low glucose/high lactatemakes this effect more concerning.\nFor instance, in Figure 2 JK, CHC/GNE inhibit tumor growth in the presence of\nlactate given IP—do they work without extra lactate? Most experiments in\nFigures 2-8 require added lactate to demonstrate the effects of intervention. In Figure\n4FG and S4 L, M, do inhibitors decreaseATPlevels and OCR if lactate is not added?\nIn Figures 8 and S10 (SCFV), the effect of scFv to all endpoints is dependent on\nadding exogenous lactate as well. Figure 8I-K, which are the only experiments in\nwhich scFv is added without extra lactate, show that scFv has no effect. In in vivo\nsystems (PDX1/2, 4T1, MCF, MMTV-PyMT), does CHC/GNE treatment inhibit\nmetastasis and stemness in the absence of extra lactate, which is the clinically\ntranslatable experiment?\nResponse:\nWe acknowledge your point that in vivo experiments were carried out by adding\nexogenous lactate, which may have biased the results toward emphasizing lactate\nmetabolism. For in vitro experiments, however, we first utilized the low-attachment\nmodel to investigate the detachment-induced changes in multiple aspects, including\nmetabolomics (Figure 1A), 13C -lactate (Figure 1F) /2-2H-lactate metabolic flux\n3\n(Figure 6A-B), proteomics (Figure 1G, 4A), screening of stem cell transcription\nfactors (Figure S2C), OXPHOS metabolism (Figure 4C), complex I activity (Figure\n4E), α-KG-DNMT3B mediated SOX2 hypomethylation (Figure 5E-G), the\nmitochondrial CD147/MCT1/LDHB lactate oxidation complex (Figure 6C-E), and\nmitochondrial CD147-mediated OXPHOS and stemness potential (Figure 7C-E).\nAfterward, we applied a glucose-free model supplemented with lactate to further\nvalidatethe observed alterations.\nAs established in prior studies, physiological lactate concentration in healthy\nhuman serum typically ranges between 1.5-3 mM[5, 6], whereas tumor tissues exhibit\nsignificantly higher levels, reaching 10-30 mM in intratumoral regions and up to 50\nmM in necrotic cores[6, 7]. Notably, in in vivo experimental models like mice, lactate\ndoses below 1.5g/kg via intraperitoneal injection have been shown to be non-lethal\nand devoid ofacidosis orsevere inflammatoryside effects[8-10].\nTo comprehensively evaluate the impact of lactate on the stemness potential of\ndetached breast cancer cells in vitro, we selected a concentration range of 0-40 mM,\nwith a primary experimental concentration of 20 mM, encompassing both\nphysiological and pathological benchmarks. For in vivo studies, we administered\nlactate via intraperitoneal injection at 800 mg/kg, which elevated serum lactate levels\nto approximately 5 mM (consistent with Cerda-Kohler et al.[10]), while tumoral\nlactate concentrations reached 6-8 mM (as shown in Response Figure 2 below). This\ndosing regimen ensured biologically and clinically relevant lactate exposure while\nmaintaining experimental robustness.\nResponse Figure 2. Lactate levels in plasma (left) and tumors (right)of PDX1(A), 4T1 (B),and\nMCF7 (C)-bearing mice (n=4 mice for each group) with 800 mg/kg lactate. Data are represented\nasmean±SEM.Statisticalanalysis:ttest.**p<0.01,***p<0.001.\nMoreover, we sincerely apologize for the lack of clarity in the original text\nregarding the culture medium for AR cells, which should have clearly specified that\nwe used the regular glucose-containing RPMI 1640 medium.\nIn response to your constructive suggestion, we have conducted additional\nexperiments to clarify the role ofCHC/GNE ormito147 scFv in inhibitingATPlevels,\nComplex I and OCR (Updated Figure 4F-G, S4L-M), as well as lung metastatic\nnodules or stemness properties in 4T1 (Updated Figure 2J-K), PDX1 (Response\nFigure 5) or MCF7 WT/KO/mito147 (Updated Figure S10A-D) tumor-bearing mice\nwithoutorwith exogenouslactate.\nBelow is a summary ofthe experimental design:\nFigures Experiments Originalgroups Newgroups\n4F-G/S4L-M ATP,Complex Lac20mM 1) Lac0mM NS/CHC/GNE\n(MCF/T47D) IandOCR NS/CHC/GNE 2) Lac20mMNS/CHC/GNE\n2J-K metastasisand Lac800mg/kg 1) Lac0mg/kg NS/CHC/GNE\n(4T1) stemness NS/CHC/GNE 2) Lac800mg/kg NS/CHC/GNE\nNewRF5 metastasisand Lac800mg/kg 1) Lac0mg/kg NS/CHC/GNE\n(PDX1) stemness NS/CHC/GNE 2) Lac800mg/kg NS/CHC/GNE\n1) Lac0mg/kgWT/KO/\nS10A-D Lac800mg/kg\nmetastasisand mito147/mito147+scFv\n(MCF WT/KO/mito147/\nstemness 2) Lac800mg/kgWT/KO/\nWT/KO/mito147) mito147+scFv\nmito147/mito147+scFv\nNote: New RF5,new Response Figure5.\nResponse Figure 3 (Updated Figure 4F-G and S4L-M): CHC or GNE (A-B) or\nMCT1/LDHB siRNAs (C-E) reduced ATP levels, complex I activity, and OCR in\nlactate- treated MCF7/T47D cells, while showing no significant effects in cells\nculturedwithoutlactatesupplementation.\nResponse Figure 3 (Updated Figure 4F-G and S4L-M). (A-B) ATP levels (A) and complex I\nactivities(B)incellsincubatedwith0mMor20mMlactatefor48hoursandpretreatedwithPBS\n(NC)or 1 mM CHC/GNEfor 2 hours.(C-E) ATPlevels (C), complex I activities (D),and OCRs\n(E) in cells incubated with 0 mM or 20 mM lactate for 48 hours and pretreated with siNC or\nMCT1/LDHB siRNA for 48 hours. Data are represented as mean ± SEM. Statistical analysis:\none-wayANOVA.**p<0.01,***p<0.001.\nResponse Figure 4 (Updated Figure 2J-K): CHC or GNE effectively inhibited tumor\nmetastasis (A) and stemness potential (B-E) in 4T1-bearing mice subjected to lactate\nadministration. In contrast, this treatment exhibited minimal impacts on mice in the\nabsence ofintraperitoneal administrationofexogenous lactate.\nResponse Figure 4 (Updated Figure 2J-K). (A-E) Quantitation of lung metastatic nodules (A,\nn=4 mice for each group), ratios of ALDH+ or CD44highCD24-/lowsubpopulation (B-C, n=4 mice\nfor each group), SOX2 protein levels and quantification (D-E, n=3 mice for each group) in\nprimary and lung metastatic tumor from CHC/GNE treated 4T1-bearing mice with or without\nlactateadministration.Dataarerepresentedasmean±SEM.Statisticalanalysis:one-wayANOVA.\n*p<0.05,***p<0.001,****p<0.0001,ns:notsignificant.\nSimilarly, CHC or GNE effectively inhibited tumor metastasis (Response Figure\n5A) and stemness potential (Response Figure 5B-E) in PDX1-bearing mice subjected\nto lactate administration. In contrast, this treatment exhibited minimal impacts on\nmice in the absence ofintraperitoneal administrationofexogenouslactate.\nResponse Figure 5.(A-E) Quantitation of lung metastatic nodules (A, n=4mice for each group),\nratios of ALDH+ or CD44highCD24-/low subpopulation (B-C, n=4 mice for each group), SOX2\nprotein levels and quantification (D-E, n=3 mice for each group) in primary and lung metastatic\ntumor from CHC/GNE treated PDX1-bearing mice with or without lactate administration. Data\narerepresented asmean±SEM.Statistical analysis: one-wayANOVA.**p<0.01,***p <0.001,\n****p<0.0001,ns:notsignificant.\nResponse Figure 6 (Updated Figure S10A-D): mito147 scFv effectively inhibited\ntumor metastasis (A) and stemness potential (B-E) in MCF7-bearing mice subjected\nto lactate administration. In contrast, this treatment exhibited minimal impacts on\nmice in the absence ofintraperitoneal administrationofexogenouslactate.\nResponseFigure6(UpdatedFigureS10A-D).(A-E)Quantitationoflungmetastaticnodules(A,\nn=5 mice for each group), ratios of ALDH+ or CD44highCD24-/lowsubpopulation (B-C, n=5 mice\nfor each group), SOX2 protein levels and quantification (D-E, n=3 mice for each group) in\nprimary and lung metastatic tumor from scFv treated MCF7 WT/KO/mito147 cells bearing mice\nwith or without lactate administration. Data are represented as mean ± SEM. Statistical analysis:\none-wayANOVA.***p<0.001,****p<0.0001,ns:notsignificant.\nIn summary,we observed that CHC/GNE ormito-CD147scFv has minimal effect\non tumor metastasis or stemness potential in mice without exogenous lactate\nsupplementation. This finding underscores the potential clinical relevance of\nCHC/GNE or mito-CD147 scFv for inhibiting tumor metastasis in patients with\nelevated circulating lactate levels. We propose that this targeted approach is\nwell-suited to precision medicine strategies, potentially offering optimized clinical\noutcomes throughbiomarker-guided treatment selection.\n4. Related to this, do the authors propose that lactate uptake and oxidation an intrinsic\nand stable property of anoikis resistance or does the state of detachment induce this\nmetabolic switch? That is, if you replate floating AR cells on solid matrix or plastic,\nwill their lactateutilizationrevertto match the adherent state?\nResponse:\nWe extend our heartfelt gratitude for your insightful and constructive suggestion.\nIn addressing your question, we replated the floatingAR cells onto uncoated common\nplates and allowed them to adhere and grow for 48 hours (hereafter referred to as\nAR-Ad cells). Upon examination, we observed that the expression levels of MCT1\nand LDHB, two key regulators governing lactate uptake and oxidation, reduced to\nlevels comparable to those observed in Ad cells (Response Figure 7A). Furthermore,\nthe extracellular lactate levels increased, while the intracellular lactate levels\ndecreased, both approaching the levels comparable to those in Ad cells (Response\nFigure 7B). These findings indicate that lactate utilization in AR-Ad cells has been\nrestored to a state comparable to that in the adherent state. In summary, our\ncomprehensive data support the conclusion that it is the detached state that triggers\nthis metabolic switch in breast cancer cells, leading to an enhancement in lactate\nuptake and oxidation.\nResponse Figure 7. Replating floating AR cells restores lactate utilization to Ad cell levels.\n(A-B) MCT1 and LDHB protein levels (A), extracellular and intracellular lactate concentrations\n(B) in adherent (Ad), anoikis resistance (AR) and replating floatingAR (AR-Ad) cells. Data are\nrepresentedasmean±SEM.Statisticalanalysis: (B)one-wayANOVA.**p<0.01,***p<0.001,\n****p<0.0001.\n5. Figure 1F. Was this tracing experiment performed in glucose and pyruvate free\nmedia? While a lower fractional abundance of M+3 pyruvate than the fractional\nabundance of M+2 TCAcycle intermediates is possible with separate pyruvate pools,\na fractional enrichment of only 2%for pyruvateand 25% forTCAcycle intermediates\nwould be very unusual as lactate carbons must go through pyruvate to enter the TCA\ncycle. The data presented suggests the total pyruvate pool outside the mitochondria is\nvery large compared to the mitochondrial pool, with minimal import of pyruvate into\nthe mitochondria, and the majority of the mitochondrial pyruvate derived from lactate\nimported into the mitochondria. This degree of compartmentalization of the pyruvate\npools is usually not seen. The data also implies a minimal conversion of lactate into\npyruvatein the cytoplasm by LDHA.\nResponse:\nWe sincerely apologize for the oversight in omitting the details of the culture\nmedium used for13C -lactate metabolic flux analysis.Toclarify,bothAR andAd cells\n3\nwere maintained in regular RPMI 1640 complete medium, which contained glucose\n(3.15 g/L) and pyruvate (110 mg/L). Prior to sample collection, the cells were treated\nwith10 mM 13C -lactate for12hours understandard cultureconditions.\nfractional enrichment of M+3 pyruvate (2%) compared to the higher enrichment of\nM+2 TCAcycle intermediates (25%).We fully agree with your interpretation that the\nmajority of labeled lactate was not converted to pyruvate in the cytosol (as evidenced\nby the low M+3 pyruvate levels) but rather directly entered the mitochondria, where it\nwas metabolized into pyruvate (leading to the observed high enrichment of M+2 TCA\ncycle intermediates). This apparent discrepancy challenges conventional models in\nwhich cytosolic lactate-derived pyruvate serves as the primary mitochondrial\nsubstrate. Instead, our findings support a model of compartmentalized pyruvate\nmetabolism, wherein lactate is preferentially oxidized within mitochondria,\nbypassing canonical cytosolic conversion. We propose that this phenomenon can be\nexplained in two ways:\n1) Pool Dilution Effects: The large cytosolic pyruvate pool, derived from both\nglycolytic flux (unlabeled glucose in RPMI 1640 medium) and medium’s endogenous\n12C -pyruvate (110 mg/L sodium pyruvate), dilutes the 13C -pyruvate generated from\n3 3\nthe 13C -lactate tracer, and thus substantially reduces the observed M+3 pyruvate\n3\nenrichment(2%).\n2) Lactate Oxidation Pathway: While LDHB-mediated cytoplasmic lactate to\npyruvate conversion is the canonical pathway, recent studies[11-13] align with our\n13C -lactate tracing (Figures 1F) and subsequent 2-2H-lactate metabolic flux (Figures\n3\n6A-B) experiments in detached breast cancer cells all support direct mitochondrial\nlactate uptake and oxidation, which resulting in higher labeled carbon entry into the\nTCA cycle (yielding 25% M+2 intermediates). Moreover, our study identifies a\nCD147/MCT1/LDHB complex that is specifically localized on the mitochondrial\ninner membrane (Figures S6-S7). This complex enables a mitochondrial lactate\n\noxidation, directly converting lactate to pyruvate within mitochondria and bypassing\ncytosolic oxidationbeforemitochondrial uptake.\nIn summary, the observed compartmentalized pyruvate metabolism and\nmitochondrial lactate oxidation highlights an adaptive metabolic reprogramming in\ndetached breast cancer cells. This reprogramming is characterized by the preferential\ndirect utilization of lactate as a direct energy source via its uptake and oxidation\nwithin the mitochondria. By decoupling lactate oxidation from cytosolic pyruvate\npools, detached breast cancer cells maintain redox balance andATPproduction during\ndetachment—a critical survival mechanism for metastatic dissemination. Targeting\nthis mitochondrial lactate oxidation complex would disrupt metastasis-specific energy\nmetabolism, thereby presenting a novel therapeutic vulnerability.\n6. Figures S1A-C, S1D-E.The decrease in nutrient utilization could also be explained\nby lower growth rates when detached, which the authors’ prior paper (Meng 2020)\ndemonstrates. Comparative doubling times between AR and attached cells should be\nprovided here again.\nResponse:\nWecompletely agree with yourinsightful observationthat the decrease in nutrient\nutilization could also be explained by lower growth rates upon detachment, as\nevidenced by the significantly prolonged Td (doubling times) ofAR cells. In response\nto your valuable and constructive suggestion, we have incorporated comparative Td\ndata into the manuscript, as follows: “Given our previous findings showing that\ndetached breast cancer cells exhibit significantly prolonged doubling times, these\ndata suggest that their growth retardation may lead to the reduced utilization of three\nmajor nutrients while shifting toward the consumption of alternative energy sources”.\nAdditionally, we have included these comparative Td data in Response Figure 8A(as\nshownbelow)to furthersupportourconclusions.\nIn contrast to the reduced utilization of three major nutrients upon detachment,\nlactate consumption is markedly increased in detached cells (Figures 1C, 1E-F). To\ninvestigate the lactate’s functional role, we assessed its effects on cell growth, cell\ncycle progression, apoptosis, and stemness potentials. Strikingly, when cells were\ncultured in glucose-free medium supplemented with 20 mM lactate for 48 hours,\nstemness potentials was significantly enhanced (i.e., Figures S2.1A, S2.1C-G),\nwhereas cell growth, cell cycle distribution, and apoptosis remained unaffected\n(Response Figure 8B-E). These findings suggest that lactate serves as a preferred\nsubstrateforsustaining stemness rather than proliferationin detached cells.\nResponse Figure 8. Enhanced lactate utilization promotes cellular growth without altering\nthe cellcycle and apoptosis ofdetached breastcancercells. (A)Cell growth wasmonitored by\nmeasuringthe absorbance ofWST-8dye at450nm inARorAd breastcancercells replated under\nadherent condition for up to 12 days. Td, doubling time. These data were adopted from the study\nby Meng et al. [3]. (B-C) CCK8 analysis was performed in MCF7/T47D cells cultured in\nglucose-free or glucose-containing medium supplemented with varying concentrations of lactate\n(0, 5, 10, 20, 40 mM) for 48 hours (B) or supplemented with lactate (0 or 20 mM) for 24, 48, 72\nhours (C). (D-E) Cell cycle analysis (D) and apoptosis assay (E) was performed in MCF7/T47D\ncells cultured in glucose-free or/and glucose-containing medium supplemented with lactate (0 or\n20 mM) for 48 hours. Data are represented as mean ± SEM. Statistical analysis: (A, C-E) t test\n(AR vs.Ad; Lac 20 mM vs. Lac 0 mM); (B) one-wayANOVA(Lac 5, 10, 20, 40 mM vs. Lac 0\nmM).*p<0.05,**p<0.01,***p<0.001.\n7. Figure 1D. A decrease in plasma lactate levels with tumor progression is an\nunexpected result. Plasma lactate levels are usually maintained in a tight range, with\nincreasing tumor burden leading to increased plasma lactate levels. This does not fit\nwith the hypothesis as the vast majority of tumor cells are anchored and not CSCs or\ndetached CTCs and should have lower lactate consumption as demonstrated in the\nauthors’experiments. How dothe authorsreconcile this?\nResponse:\nWe acknowledge the unexpected decrease in plasma lactate levels during tumor\nprogression, which appears to contradict with conventional expectations. This\ndiscrepancy can be explained by the dynamic balance between lactate production and\nconsumption,particularly as tumors evolve into metastatic states.\nAs depicted in Response Figure 9 (Updated Figure 1D) below, primary breast\ncancer cells primarily function as lactate producers before Day 21, maintaining\nplasma lactate levels within a stable range (5.580.41 to 5.500.49 mM).However, as\nthe tumor progression advances, marked by increased intratumoral lactate levels (Day\n21-29), primary tumor cells detached as CTCs, reprogrammed into CSCs, and\nultimately established lung metastasis. Notably, this transition coincides with a sharp\ndecline in plasma lactate levels, reaching its lowest point (2.970.21 mM) by Day 29.\nThese findings suggest that lactate may support the establishment of pre-metastatic\nniches in distant organs (e.g., lungs) partly by fueling metastatic cells or CTCs in\ncirculation, resulting in its active consumption and subsequent reduction in plasma\nlactate concentrations, consistent with prior reports[14]. The metabolic shift—from\nlactate production in primary tumors to its utilization by metastatic cells—likely\nexplains the observed reductionin systemic lactatedespite increasing tumorburden.\nResponse Figure 9 (Updated Figure 1D). Representative images of tumor burden (left) and\nlactate levels of plasma (middle) and tumor (right) in 4T1 luciferase-bearing mice (n=7 mice).\nDataarerepresentedasmean±SEM.\nAdditionally, other cells within the tumor microenvironment—such as CD8+ T\ncells[15], tumor-infiltrating regulatory T cells[16], or tumor-associated macrophages\n(TAMs)[17]—may also metabolize lactate, further reducing its presence in the\nsystemic circulation.\n8. Figure 4C-E.Seahorse assays should be normalized to cell count.These differences\nin respiration could be due toa different number ofcells perwell.\nResponse:\nWe fully agree with your insightful advice concerning the seahorse assays data\npresented in Figure 4C-D. To preclude any potential confounding impacts of cell\nnumber variations on OCR measurements, we carefully controlled seeding densities\nacrossall experimentalconditions.\nSpecifically, in Figure 4C, adherent (Ad) cells were seeded at a density of\n1.5×104 cells per well into pre-hydrated 96-well plates one day prior to the assay to\nensure complete attachment, while anoikis resistant (AR) cells of the same density\nwere plated using an adhesion reagent right before the measurement. DespiteAd cells\nmay have slightly high cell numbers after overnight incubation, AR cells still\nexhibited significantly higher OCR, confirming their inherently elevated oxygen\nconsumption. In Figure 4D, MCF7/T47D cells were firstly cultured in glucose-free\nmedia, either without or with 20 mM lactate, for 48 hours before OCR assessment.\nSubsequently, an equal number of cells (1.5×104 cells per well) were seeded into\npre-hydrated 96-well plates and allowed 6 hours for adhesion. Since our prior data\nconfirmed that 20 mM 48-hour lactate exposure does not alter cellular proliferation\nrate, the comparable final cell number between lactate treated and untreated groups\nvalidate that the observed OCR differences reflect genuine metabolic alterations\nrather thandisparities in cell number.\nToenhance clarity, we have revised the relevant statement in yellow highlight in\nthe Method Section under “Oxygen consumption rate measurement” as follows:\n“Briefly, Ad cells were seeded into pre-hydrated 96-well plates (1.5×104 cells/well)\nand cultivated overnight, while AR cells at the same density were seeded directly into\npoly-L-lysine precoated wells just before the measurement. For lactate-treated cells,\nthey were first cultured in glucose-free media without or with 20 mM lactate for 48\nhours. Subsequently, an equal number of cells (1.5×104 cells per well) were seeded\nintopre-hydrated 96-wellplates and allowed toadherefor 6 hours.”\n9. Figure4D-E. A dramatic increase in OCR with addition of a metabolite is unlikely\nin normal seahorse media. Substrate limited media with significantly lower levels of\nglucose and glutamine are required to investigate the contribution of individual\nmetabolites to respiration. The materials and methods section does not specify the use\nofsubstratelimited media. This should be clarified.\nResponse:\nWe sincerely appreciate your insightful suggestions and apologize for the initial\nomission of methodological details in the initial submission. Following your valuable\nadvice, we have added the details regarding the use of substrate limited media in\nyellow highlight in the Methods Section under “Oxygen consumption rate\nmeasurement” as follows: “…, while the lactate-treatedcells wereincubated at 37°C\nfor 45minutes without CO in asubstrate-limitedmedium containing 0.5mMglucose,\n2\n0.5mML-glutamine,and0.2mMsodium pyruvate.”\n10. Figure 4E. The authors claim lactate is utilized as a fuel for the TCA cycle and\nincreased TCA cycle turnover is promoting oxidative phosphorylation and increased\nrespiration in the seahorse assay. However, Isolated Complex 1 activity is very\ndifferent than Complex 1 activity in an intact cell. Respiration in the seahorse assay,\nincluding ATP uncoupled respiration, is dependent on the availability of NADH\nmostly provided by the TCAcycle. Inthe isolated Complex 1 activity kit fromAbcam\na fixed amount of NADH substrate is provided and thus differences in TCA turnover\nin the intact cell have no bearing on isolated Complex 1 activity. The dramatic\ndifferences in isolated Complex 1 activity suggest either differences in mitochondrial\nmass or abundance, defects in Complex 1 assembly, or PTMs of Complex 1 subunits.\nThe authors should performblue native page forcomplex assembly and awesternblot\nfor mitochondrial proteins such as citrate synthase, and flow cytometry to estimate\nmitochondrial mass.\nResponse:\nWe fully agree with your opinion that “The isolated Complex I activity is very\ndifferent than Complex I activity in an intact cell”, and that“The dramatic differences\nin isolated Complex 1 activity suggest either differences in mitochondrial mass or\nabundance, defects in Complex 1 assembly, or PTMs of Complex 1 subunits”.\nFollowing your insightful suggestions, we have performed additional experiments,\nincluding blue native page to assess complex assembly, western blot to measure the\nexpression of mitochondrial citrate synthase, and flow cytometry to evaluate\nmitochondrial mass.\nAs illustrated below, lactate treatment had minimal impact on both the assembly\nof mitochondrial Complex I (Response Figure 10A) and the mitochondrial number\n(Response Figure 10B) in breast cancer cells. However, it increased the expression of\ncitrate synthase, the first and a key rate-limiting enzyme in the TCAcycle (Response\nFigure 10C). Collectively, these findings indicate that the significant increase in the\nactivity of isolated Complex I might be associated with the detachment or lactate\nenhancedTCAturnover,Complex IelectrontransportandATPsynthesis.\nResponse Figure 10. Lactate treatment increased the protein levels of citrate synthase\nwithout affecting the complex I assembly or mitochondrial mass. (A) Blue native PAGE\nanalysis of complex I-V in the isolated mitochondria from lactate treated MCF7/T47D cells.\n(B) Mitochondrial mass of MCF7/T47D cells cultured in glucose-free media supplemented with\n0, 5, 10, 20, 40 mM lactate. (C) Citrate synthase (CS) protein levels in AR or lactate treated\nMCF7/T47D cells. Data are represented as mean ± SEM. Statistical analysis: (B) one-way\nANOVA(Lac5,10,20,40mMvs.Lac0mM).\n11.In Figure 5, D-2HG is used as an α-KG “inhibitor.” However, D-2HG has protean\neffects which include inhibition of LDH (Science 2022;377:1519), which means its\neffects arenot specific for-KG.\nResponse:\nWe sincerely appreciate your thoughtful comments. We fully acknowledge that\nD-2HG, a well-characterized competitive inhibitor of α-KG, has been shown to\npotently suppress α-KG-dependent enzymatic activities in numerous metabolic\nstudies[18-20]. As you astutely pointed out, however, D-2HG has also been reported\ntoinhibit LDH activity (Science 2022;377:1519).\nTo ensure the rigor of our findings, we conducted an additional experiment using\nan isocitrate dehydrogenase (IDH) inhibitor (Enasidenib; Cat#HY-18690, MCE) to\ndirectly block α-KG biosynthesis. As shown in Response Figure 11, Enasidenib\nconsistently generated effects similar to those of D-2HG, further reinforcing our\nconclusion that α-KG availability is critical for regulating stemness induction during\ndetachment throughits mediationofSOX2 hypomethylation.\nResponse Figure 11. (A-C) ALDH+ ratios (A), tumorsphere numbers (B), and SOX2 protein\nlevels(C)inAR/lactatetreated MCF7/T47Dcells combinedwith PBS (NC),0.5μMDM-αKGor\n1 μM Enasidenib for 48 hours. (D) The methylation levels for the 181720250-181720500 region\nof SOX2 promoter detected by real-time qPCR in AR cells cultured with PBS (NC), 0.5 μM\nDM-αKG or 1 μM Enasidenib for 48 hours. (E) DNMT3B protein levels in lactate treated cells\ncombined with PBS (NC), 0.5 μM DM-αKG or 1 μM Enasidenib for 48 hours. (F) The binding\nactivity of DNMT3B to the 181720250-181720500 region of SOX2 promoter detected by\nChIP-qPCR in AR or lactate treated cells combined with PBS (NC), 0.5 μM DM-αKG or 1 μM\nEnasidenib for 48 hours. H3, positive control; IgG, negative control. (G-I) ALDH+ ratios (G),\ntumorsphere numbers (H) and SOX2 protein levels (I) in 20 mM lactate incubated DNMT3B\noverexpressed cells combined with PBS (NC), 0.5 μM DM-αKG or 1 μM Enasidenib treatment\nfor 48 hours. Data are represented as mean ± SEM. Statistical analysis: one-wayANOVA. *p <\n0.05,**p<0.01,***p<0.001,****p<0.0001.\n12. In Figure 5 and 7L, α-KG and CD147 seem to dramatically decrease the\nexpression ofDNMT3B. The effect is much greaterthan the modest loss of DNMT3B\nactivity (Figure 5I). What explains this discrepancy, and how does -KG decrease\nprotein levels ofDNMT3B?\nResponse:\nWe appreciate your insightful question regarding the apparent discrepancy\nmentioned above, where the expression of DNMT3B was significantly reduced (55%)\n(Figure 5H) but its enzymatic activity showed only a modest decline (15%) in the\noriginal Figure 5I. To investigate whether this inconsistency stemmed from the\nrelatively low sensitivity of the initial enzymic activity detection kit (Cat#52034,BPS\nBioscience), we purchased an alternative kit from a different supplier (Cat#ab113471,\nAbcam). Repeating the assay with this new kit revealed a more pronounced decrease\n(34%) in DNMT3B enzymatic activity. To ensure clarity and avoid confusion, we\nhave updatedFigure 5Iaccordingly with these new results.\nResponse Figure 12 (Updated Figure 5I). DNMT3B enzyme activities were significantly\ndecreased in AR or lactate treated cells. DNMT3B enzyme activities in AR or lactate treated\ncells were detected by commercial kits fromAbcam (Cat#ab113471). Data are shown as mean ±\nSEM.Student’sttestswereusedforstatisticalanalysis.**p<0.01,***p<0.001.\nBeyond the limitations of detection sensitivity, the observed decoupling between\nreduced DNMT3B protein expression and sustained enzymatic activity can be\nattributed to the following established regulatory mechanisms: 1)\nDNMT3L-Dependent Stabilization: DNMT3B’s catalytic activity depends on its\ninteraction with the catalytically inactive cofactor DNMT3L. High DNMT3L\nexpression stabilizes the catalytic conformation of DNMT3B, effectively maintaining\nits enzymatic activity even when total DNMT3B levels decline[21]. 2)\nCompensatory Role of DNMT3B3: The splicing variant DNMT3B3, which is catalytically inactive,\nmimics DNMT3L’s scaffolding function, acting as an accessory factor to maintain\nenzymatic activity despite reduced total DNMT3B expression[22, 23]. 3)\nMethodological Discrepancy: Western blot quantifies total amount of DNMT3B\n(including inactive isoforms like DNMT3B3), whereas enzymatic assays only\nmeasure the activity of functional DNMT3B molecules. This can create an apparent\ndisparity where total protein levels appear reduced while enzymatic activity show\nminimal change.\nIn response to your question regarding how α-KG decreases protein levels of\nDNMT3B, we have conducted a thorough literature review and propose the following\nthree possible mechanisms with sincere consideration: 1) α-KG serves as an essential\ncofactorfor the JmjC family of histone demethylases (such as JMJD3), catalyzing the\ndemethylation of inhibitory histone marks like H3K27me3, thereby activating gene\nexpression[24]. This process may indirectly affect the transcriptional activity of the\nDNMT3B promoter, leading to reduced mRNA synthesis. 2) DNMT3B deficiency\ncan enhance the activity of isocitrate dehydrogenase (IDH), promoting the\naccumulation of α-KG. Conversely, α-KG treatment downregulates DNMT3B\nexpression[25].This may be attributedtothe feedback inhibition ofIDHactivity upon\nα-KG supplementation, further contributing to the reduction of DNMT3B. 3) In\ncolorectal cancer, proteasome inhibitors inactivate mTOR, suppressing the translation\ninitiation of DNMT3B and significantly downregulating its expression[26]. It is\nplausible that α-KG may exert similar effects on DNMT3B synthesis through\ncomparable mechanisms.\n13. Figure 6B does not imply that 100% of the labelled lactate exists in the\nmitochondria as stated in the results section, rather this simply indicates 100%\nlabelling ofmitochondrial lactate.\nResponse:\nWe sincerely apologize for the imprecise description of the data. As correctly\nnoted, Figure 6B only demonstrates that mitochondrial lactate is fully (100%) labeled,\nnotthat “all labeled lactateis exclusively mitochondrial”.Following yourconstructive\nsuggestion, we have revised the original text (Page 10, Paragraph 2, Line 300) to\nclarify this point. The original sentence: “we found that more than 95% 2H-labled\nlactate were occurred in mitochondria of AR MCF7 cells” has been corrected to: “we\nfound that more than 95% of lactate in the mitochondria of AR MCF7 cells were\n2H-labeled”. This revision more accurately reflects our experimental observation that\nthe majorityof mitochondriallactateinAR MCF7 cells carried the 2H label.\n14. Figures S6C-H shows overexpression of CD147 in two cell lines and knockdown\nin a third cell line. It would be more convincing to do paired experiments in the same\ncell line background. Is there a major difference in baseline expression of CD147\nbetween the cell lines (ie low in MCF7 and T47D and high in MDA-MB-231) that\nprompted this experimental design? If so, is the role of endogenous CD147 cell type\nspecific?\nResponse:\nWe sincerely appreciate your insightful comment regarding experimental design.\nWe fully agree that performing CD147 overexpression or knockdown experiments\nwithin the same cell line background would further strengthen the conclusions, as it\nwould better control for intrinsic cellular variability. As you astutely noted, however,\nour experimental design was guided by the baseline CD147 expression levels across\ndifferent breast cancer cell lines. Our preliminary research revealed that\nanoikis-sensitive T47D and MCF7 cells exhibit low endogenous CD147 expression\n(making them suitable for overexpression studies), whereas anoikis-resistant\nMDA-MB-231 cells intrinsically express high CD147 levels (supporting knockdown\nstudies),as demonstratedin Response Figure 13A-B[3].\nMoreover, as shown in Response Figure 13C-D, our team’s findings further\nvalidated that metastatic MDA-MB-231 cells with strong migration and invasion\npotential exhibit a higher expression level of CD147 compared to non-metastatic\nMCF7 cells bearing with weak migration and invasion potential, underscoring the\nstriking correlation between CD147 expression and the enhanced invasive and\nmetastatic capabilities of tumor cells[27]. In human breast carcinoma tissues,\nHAb18G/CD147, a protein sharing identical DNA and amino acid sequences with\nCD147, was positively associated with local recurrence and distant metastasis while\nnegatively correlated with estrogen receptor (ER) and progesterone receptor (PR)\nexpression. Notably, multivariate analysis identified HAb18G and lymph node\nmetastasis as independent prognostic factors for both reduced progression-free\nsurvival (PFS) and overall survival (OS)[28].\nCollectively, these data demonstrated that CD147 expression levels may serve as\na clinically relevant biomarker for malignant potential across distinct breast cancer\nsubtypes. Importantly, our results support the conclusion that CD147's functional role\ndepends primarily onits expression level rather than onthe specific cellular context.\nResponse Figure 13. CD147 is highly expressed in metastatic breast cancer cell. (A) Flow\ncytometry analysis of membranous CD147 levels in T47D, MCF7, and MDA-MB-231 cells\ncultured under an attached or detached condition for 0, 1, and 3 days. (B) Immunoblot assay of\nCD147 protein levels in T47D, MCF7, and MDA-MB-231 cultured under an attached (Ad) or\nanoikis resistant (AR) condition for 7 days. (C) Real-time RT-PCR analysis of CD147expression\nin a series of human breastcancer cell lines and normal tissues. (D)Western blot detection of the\nexpression of CD147, Sp1 and c-Myc in a series of human breast cancer cell lines and normal\ntissues. Dataare represented asmean± SEM.Statistical analysis: (A)one-wayANOVA(1,3days\nvs. 0 day); (C) t test. *p < 0.05, **p < 0.01, ***p < 0.001. Notes: FigureA/B were adopted from\nour previous paper meng et al.[3], while Figure C/Dwere adopted from our team’s paper kong et\nal.[27].\n15. The method of co-IP experiments in Figures 6 and S7 is not clear. Without mild\ndetergent, mitochondrial or submitochondrial particles will be isolated, especially\nwith centrifugation (vs magnetic) techniques. Thus, intramolecular associations may\nnotexist if conditions are notappropriateandcontrols are notincluded.The TEMs are\nnot convincing for more than random colocalization on organelles. Perhaps a more\nconvincing method would be a proximity ligation assay. The human tissue IF shown\nin Figure 6H, L does not demonstrate colocalization of CD137, MCT1, and LDHB,\nalthough magnification may be limiting. Overall, evidence for a macromolecular\ncomplex between these 3 proteins is unconvincing and seems overstated. However,\nfunctional cooperationwithLDHAwould notrequire physical association.\nResponse:\nWe sincerely apologize for your insightful comments regarding the co-IP\nexperiments in Figures 6 and S7. Below, we provide clarifications and additional\nmethodological details to address these concerns. Following mitochondria isolation,\nthe mitochondrial pellets were lysed using MinuteTM Non-Denatured Protein\nSolubilization Reagent (WA-010, Invent Biotechnologies). This mild lysis buffer is\nspecially formulated to preserve protein-protein interactions while yielding native\nproteins compatible with subsequent co-IPanalyses[29, 30]. To minimize nonspecific\nbinding, we included negative controls (IgG) to rule out potential false-positive\ninteractions. To address your concern, we have thoughtfully incorporated these co-IP\nmethodological details into the Methods Section under “Co-immunoprecipitation\n(Co-IP) assay” as follows: “Mitochondria were isolated following the protocol\ndescribed in the Methods Section under \"Mitochondrial Isolation\". Approximately\n500 μg of the mitochondrial pellet was resuspended in 200 μL of nondenatured lysis\nbuffer (WA-010, Invent Biotechnologies), a mild formulation designed to preserve\nnative protein-protein interactions within the mitochondria while solubilizing\nmitochondrial membranes.The suspension was gently pipetted toensurehomogeneity,\nfollowed by centrifugation at 12000×g for 2 minutes to remove insoluble components.\nThe resulting supernatant was then subjected to a Co-IP experiment according to\nstandard procedures.” We hope these revisions can enhance clarity and ensure\ncomprehensive understanding ofthe experimentalprocedures.\nWe wholeheartedly agree with your view that TEM images alone are insufficient\ntodistinguish specific protein interactionfromrandom colocalizationon mitochondria.\nNevertheless, our TEM observations have revealed a potential existence of protein\ncomplexes. Moreover, molecular docking simulations have predicted the binding\nconformation of three molecules: CD147 structurally bridges between MCT1 and\nLDHB. Specifically, its transmembrane region near the C-terminus interacts with\nMCT1 while its N-terminus binds to LDHB (Response Figure 14A). Considering that\nthe CD147-MCT1 interaction has been previously confirmed[31], we then employed\nPLA to verify that lactate significantly enhances the CD147-LDHB interactions in\nbreast cancer cells, which supporting their functional coupling (Response Figure 14B).\nFurther supporting this, we conducted pull-down assays and site-mutation analysis by\nusing purified GST-CD147 N-terminal and His-LDHB protein, along with molecular\ndocking techniques. The results of these experiments demonstrated an interaction\nbetween the two proteins, involving in Glu168 and Arg106 in CD147, as well as\nArg99andAsp56 in LDHB (Response Figure 14C-F).\nIn Figure 6H, our objective was to explore the potential correlation between the\nco-expression of CD147, MCT1, and LDHB and the metastatic potential of breast\ncancer patients, differentiating between those with or without metastasis, rather than\nfocusing on revealing the formation of the CD147/MCT1/LDHB complex through\nco-localization analysis of these three molecules. Conversely, in Figure 6L, our goal\nwas to verify the spatial assembly of this ternary complex, which achieving by\nspecifically addressing the formation of the CD147/MCT1/LDHB complex on the\nmitochondrial membranes of CTC during metastasis. To address the concern you\nraised that “the human tissue IF images failed to clearly demonstrate the\ncolocalization of CD147, MCT1, and LDHB”, we have now provided and updated\nhigh magnification IF images in the updated Figure 6L (Response Figure 15), which\nclearly display typical colocalization.\nTaken together, we agree that functional cooperation among proteins may not\nnecessarily require direct physical binding. However, a comprehensive array of our\nexperimental and computational evidence, including co-IP, TEM, PLA, protein\npull-down, specific site mutation, human tissue IF, and computational analyses,\nconsistently demonstrates that the mitochondrial colocalization ofCD147, MCT1,and\nLDHB represents a specific, functionally relevant macromolecular complex rather\nthan stochastic proximity.\nResponse Figure 14. Conformational binding characteristics of the CD147/MCT1/LDHB\ncomplex and the corresponding experimental validation. (A) The molecular docking\ntechnology simulated binding conformation of the CD147/MCT1/LDHBcomplex,elucidating the\nstructural basis of their interactions. Yellow: CD147; Purple: MCT1; Red: LDHB. (B) Proximity\nligation assay signal and the corresponding quantification of CD147 and LDHB in lactate treated\nMCF7 cells. Scale bars, 5 μm. (C) Coomassie Brilliant Blue staining showed the successful\nexpression and purification of GST-CD147 N-terminal protein (left, 46 kDa) and His-LDHB\nprotein (right, 36 kDa). (D) Pull-down assay confirmed the interaction between GST-CD147\nN-terminal protein and His-LDHB protein. (E) Site mutation analysis in pull-down assay\nidentified critical residues involved in the interaction between GST-CD147 N-terminal protein\n(left) and His-LDHB protein (right). (F) Molecular docking simulations revealed that the 168th\nglutamic acid of CD147 interacts with the 99th arginine of LDHB (left), and the 106th arginine of\nCD147 interacts with the 56th aspartic acid of LDHB (right), suggesting a\nhydrogen-bond-mediatedinteractionthatstabilizesthecomplex structure. Residuesare colored by\nprotein: CD147 in blue, LDHB in yellow. Data are represented as mean ± SEM. Statistical\nanalysis:(B)ttest.****p<0.0001.\nResponse Figure 15 (Updated Figure 6L). LSCM observed co-localization of CD147 (red),\nLDHB (blue) and MCT1 (purple) in the COX IV (green) indicated mitochondria of CTCs from\nbreast cancer patient. Merge1: COX IV,CD147 and LDHB. Merge2: merge1 and MCT1. Orange\narrowsshowtheco-localizationcell.Scalebars,20μm.\n16. Figures 6 and S7 show that is there a correlation between CD137 and\nMCT1/LDHB only in metastatic but not nonmetastatic patients. They are not\nnecessarily higher in metastatic patients, only co-correlated. These data suggest that\nthe actual level of expression of these proteins is unlinked to metastasis, while it is\nlinked to survival. What accountsforthis discrepancy?\nResponse:\nWe sincerely apologize for the confusion caused by the description of the\ncorrelation between CD147 and MCT1/LDHB in metastatic and non-metastatic\npatients, as well as the relationship between their co-expression and metastasis in\nFigure 6and S7.\nIn Figure 6I, a significantly strong correlation was observed between the\nexpression of CD147 and MCT1/LDHB in metastatic breast cancer patients (with R²\nvalues of 0.9295 and 0.9071 for MCT1 and LDHB, respectively). In contrast, only a\nmoderately or weakly significant correlation were found between them in\nnon-metastatic patients (R² values of 0.6621 and 0.3540 for MCT1 and LDHB\nrespectively, as shown in Figure S7G). To enhance clarity, we have revised the\noriginal description in Page 10, Paragraph 4, Line 322 of the results section. The\noriginal text “In breast cancer patient tissues, the expression of CD147 had strongly\npositive correlations withthat of MCT1or LDHB,with thecorrelationcoefficients (R2)\nmuch higher in metastatic patients than that in non-metastatic patients (Fig 6H-I, Fig\nS7G).” has been changed to “In breast cancer tissues, the expression of CD147 was\nstrongly positively correlated with that of MCT1 or LDHB in metastatic patients,\nwhile it showed amoderate or low positive correlationin non-metastatic patients (Fig\n6H-I,Fig S7G).”\nRegarding the relationship between the co-expression of CD147, MCT1, and\nLDHB and metastasis, Figure S7H reveals that the individual expression levels of\nCD147, MCT1, or LDHB are significantly higher in metastatic and recurrent breast\ncancer patients compared to non-metastatic and non-recurrent patients. Moreover, as\nshown in the Response Figure 16 (Updated Figure S7H) below, the ratios of\nco-expression of CD147, MCT1, or LDHB in metastatic and recurrent breast cancer\npatients are both significantly higher than those in non-metastatic and non-recurrent\npatients.\nResponse Figure 16 (Updated Figure S7H). Quantification of CD147/MCT1/LDHB protein\nco-expression in CK18+ breast cancer cells from 131 patients without or with metastasis (no,\nn=120 patients without metastasis; yes, n=11 patients with metastasis) / recurrence (no, n=96\npatients without recurrence; yes, n=35 patients with recurrence). Data are represented as mean ±\nSEM.Statisticalanalysis:ttest.****p<0.0001.\nTaken together, these findings not only suggest a strong correlation among\nCD147, MCT1, or LDHB in patients with metastatic and recurrent breast cancer but\nalso unequivocally demonstrate a link between their co-expression levels, metastasis,\nand patient survival, thereby resolving any potentialdiscrepancies.\n17. Figures 7B-D. It is well established CD147 is required for proper trafficking and\nmembrane insertion of MCT1 and MCT4, the predominant plasma membrane lactate\ntransporters. Consistent with this the authors demonstrate a dramatic reduction in\nMCT1 protein abundance when CD147 is knocked out (Fig S8D). Yet when a\nmitochondrial localized version is introduced to cells, the authors demonstrate a\ncomplete restoration of MCT1 protein levels, restoration of mitochondrial lactate\nlevels, and lactate induced increase in cellular respiration. This begs the question –\nhow is lactate entering the cytoplasm to be taken up by the mitochondria as\npresumably cytoplasmic MCT1 localization is impaired when CD147 with an MLS is\nintroduced into cells? And with the majority of MCT1 located in the plasma\nmembrane and not the mitochondria, how are MCT1 protein levels restored on a total\ncell level with introductionof mito147?\nResponse:\nWe sincerely appreciate your thoughtful question and fully acknowledge that\nCD147 serves as a critical chaperone protein for MCT1, playing a pivotal role in its\nmembrane trafficking and localization. However, ourdata in Figure S8D revealed that\ntotal MCT1 protein levels were slightly reduced in Ad CD147 KO cells compared to\nAd WT/mito147 cells, significantly increased in AR WT/mito147 cells, and unchanged\nin AR CD147 KO cells. Fractionation analysis of lysates from isolated mitochondria\nand non-mitochondria (primarily plasma membrane complements) further\ndemonstrate that the MCT1 levels in mito147 cells were increased within\nmitochondria, reduced on the plasma membrane (but not disappear), and unchanged\nin total cells,as compared to that in WTcells (as shownin Response Figure 17below).\nThis residual plasma membrane MCT1 localization supports efficient cytoplasmic\nlactate uptake; meanwhile, the increased mitochondrial MCT1 localization enables\neffective mitochondrial lactateuptake and oxidation forrespiration. The persistence of\nplasma membrane MCT1 localization despite CD147 KO suggests the existence of\ncompensatory mechanisms. Embigin, an alternative chaperone, may partially\nsubstitute for CD147’s trafficking function during its deficiency, thereby sustaining\nplasma membrane MCT1[32,33].\nThe CD147-N-terminal mitochondrial localization signal (MLS) construct in\nmito147 cells shares the same sequence as WT CD147, including the critical Glu218\nresidue within the transmembrane region essential for MCT1 membrane\nlocalization[31, 34, 35], differing only by the addition of the MLS. Given the\nestablished role of membrane-localized CD147 in trafficking MCT1 to the plasma\nmembrane and considering the natural colocalization of CD147 and MCT1 within\nmitochondria[12], we propose that it is mitochondrial-localized CD147, rather than\nplasma membrane-localized CD147, that facilitates MCT1 trafficking and\nstabilization to the mitochondrial membrane, and lactate utilization in the\nmitochondria in response to detachment (Figure S8D). This hypothesis, however,\nnecessitatesfurther experimentalvalidation.\nResponse Figure 17(Updated Figure S8B). Mito147cells expressing CD147in mitochondria\nexhibit reduced plasma membrane MCT1 but elevated mitochondrial MCT1 compared to\nWT cells. CD147 and MCT1 expression was detected by western blot in 30 μg total cell lysate\n(total, left), isolated mitochondria (mito, middle) or non-mitochondria (non-mito, right) lysates\nfrom MCF7/T47D derived WT/KO/mito147 cells. α-Tubulin, maker of cytoplasm; COX IV,\nmakerofmitochondrialinnermembrane;Na+-K+ATPase,makerofplasmamembrane.\n18. There are a very large number of grammatical errors which make the paper\ndifficult to follow.\nResponse:\nWe sincerely appreciate your valuable feedback and apologize for the\ngrammatical errors in the manuscript, which may have hindered readability. To\naddress this issue, we have conducted a comprehensive, line-by-liner review of the\nmanuscript to correct all grammatical, syntactical, and typographical errors. In\naddition, we also sought assistance from a native English speaker to further polish the\nlanguage, ensuring natural phrasing and improved clarity. The corresponding editing\ncertificate is provided below. Please note that the article title has also been revised.\nThe updated title, \"Lactate oxidation drive stemness potentials via mitochondrial\nCD147/MCT1/LDHB in metastatic breast cancer\", has now been incorporated into\nthe revised manuscript.\nWe are confident that these revisions have significantly improve the linguistic\nquality and readability of the manuscript. Thank you again for your time and\nconstructive criticism—it has greatly strengthened our work.\nMinorComments\n1. Figure 1B.TheY axis is missing a label.\nResponse:\nWesincerely apologize forthe oversight in omitting theY-axis label in Figure 1B.\nWehave nowcorrected this by clearly labeling the axis (\"Normalized Intensity (a.u.)\")\nand have updatedthe figure accordingly.\n2. Western blots should be provided for all siRNA experiments to demonstrate on\ntargetknockdown.\nResponse:\nWe sincerely appreciate your insightful suggestion regarding the inclusion of\nwestern blot (WB) data to validate siRNA-mediated protein knockdown.Although we\noriginally performed WB analysis for all siRNA experiments to confirm target\nspecificity and knockdown efficacy, space limitations prevented their inclusion in the\nmanuscript.\nTo fully address your concern, we now provided the omitted WB data for Figure\n3H-I, S3C-F, S4F-I, S4L-M and S6C-H in the following. These results consistently\ndemonstrate effective protein knockdown across all experimental conditions, thereby\nconfirming the reliability of our siRNA approach and further supporting the\nconclusions ofourstudy.\nResponse Figure 18. Western blot validation of siRNA or shRNA-mediated knockdown of\nMCT1, LDHB, GPR81 and CD147 protein expression under various conditions. (Fig 3H-I)\nMCT1 and LDHB protein levels in Ad/AR MCF7/T47D ALDH- cells treated with siRNA\ntargetingMCT1/LDHBfor48hours.(FigS3C-F)MCT1andLDHBproteinlevelsinAd/AR(Fig\nS3C-D)or0/20mMlactateincubated(FigS3E-F)MCF7/T47DcellstreatedwithsiRNAtargeting\nMCT1/LDHB for 48 hours. (Fig S4F-I) GPR81 protein levels in 0/20 mM lactate incubated\nMCF7/T47D cells treated with siRNA targeting GPR81 for 48 hours. (Fig S4L-M) MCT1 and\nLDHB protein levels in 20 mM lactate incubated MCF7/T47D cells treated with siRNAtargeting\nMCT1/LDHB for 48 hours. (Fig S6C-H) CD147 protein levels in MCF7/T47D CD147 knockin\n(147) or vector control (VC) cells as well as in MDA-MB-231 CD147 knockdown (A6) or\nnon-targetcontrol(NTC)cells.siNC,negativesiRNAcontrol.\n3. Figure 4K. No bars are provided to indicate the comparisons forwhich significance\nis being reported.\nResponse:\nWe sincerely apologize for the omission of significance bars in the original\nversion of Figure 4K, which prevented clear interpretation of the statistical\ncomparisons.The updated Figure 4Know incorporates the necessarybars forclarity.\nUpdated Figure 4K. Tumorsphere numbers in AR or lactate incubated ALDH- or total cells\npre-incubated with PBS (NC), 1 mM rotenone (Rot) or 1 mM oligomycin for 2 hours. Data are\nrepresentedasmean±SEM.Statisticalanalysis: one-wayANOVA.*p<0.05,**p<0.01,***p<\n0.001.\n4.Totalpoolsizes should be provided forall labelling experiments.\nResponse:\nWe sincerely appreciate your incisive question. The \"total pool size\" you referred\nto represents the total intracellular concentration orquantity ofa particular metabolite.\nIt encompasses the sum of all its isotopologues, including unlabeled, partially labeled,\nand fully labeled forms.\nWe fully agree that absolute quantification of pool sizes is crucial for accurate\nflux interpretation. Unfortunately, in the current study, our metabolic flux analysis\nwas unable to measure the absolute quantities of metabolite. Instead, as an alternative\nsemi-quantitative approach, we employed relative quantification. Specifically, we\nused relative peak areas (normalized to an internal standard) to represent the relative\nabundance ofmetabolites.\nTo assist you in the review process, we have summarized the normalized peak\nareas ofkey metabolites in the table below foryourreference.\nFig 1F\nPeakarea(mean±SD)\nMetabolitename\nAd AR\nLactate 8.61×105±6.66×103 1.17×106±3.19×105\nPyruvate 1.66×106±3.18×105 1.44×106±1.72×104\nRibose-5-phosphate 1.20×105±3.96×104 1.52×105±2.72×104\nAcetyl-CoA 2.98×104±1.25×104 2.76×104±8.96×103\nCitrate 3.34×106±6.26×105 2.26×106±4.33×105\nIsocitrate 1.48×104±2.92×103 1.17×104±2.30×103\nα-ketoglutarate 6.19×105±2.44×105 5.03×105±5.60×104\nSuccinate 4.98×105±9.86×104 5.79×105±2.25×104\nFumarate 5.49×105±3.44×104 6.19×105±1.67×104\nMalate 2.04×106±1.45×105 1.91×106±1.66×104\nOxaloacetate 3.42×104±3.83×103 2.87×104±4.00×103\nGlutamate 1.22×106±1.63×105 1.24×106±2.41×104\nAspartate 5.17×106±9.67×105 5.30×106±2.13×104\nFig 6B\nPeakarea(mean±SD)\nMetabolitename\nAd AR\nLactate 6.20×107±7.26×106 5.85×1010±1.10×108\nPyruvate 2.60×106±8.86×103 7.64×107±2.04×107\nAlanine 9.49×105±2.93×105 6.66×105±2.15×105\nMalate 1.80×106±3.51×105 1.16×107±6.96×106\nFig 7B\nMetabolite Peakarea(mean±SD)\nname WT KO mito147 mito147+scFv\n5.04×1010±7.55 0.93×107±1.06 5.71×1010±3.13× 4.60×106±2.01×\nLactate\n×109 ×107 109 106\n5.09×107±7.86 1.69×106±4.83 5.79×107±6.70 1.12×106±3.82\nPyruvate\n×106 ×105 ×106 ×105\n6.93×106±2.39 6.80×106±2.22 8.96×106±3.88 5.54×106±1.09\nAlanine\n×106 ×106 ×106 ×105\nWe understand that the notation “(% of total pool)” on the Y-axis of Figure\n1F may have given rise to your concerns. We sincerely apologize for any confusion\ncaused by this phrasing. To address this issue and ensure greater clarity, we have\npromptly removed itfrom the figure.\n5. Figure S4D,I show that A485 and siGPR81 both decrease SOX2 expression, in\ncontrast towhat the authors state.\nResponse:\nWe fully acknowledge your observation that both A485 and siGPR81 decrease\nSOX2 expression in 20 mM lactate-treated T47D cells, but do not in MCF7 cells\n(Figure S4D and S4I). This cell line-specific discrepancy may arise from the inherent\nsusceptibility ofT47Dcells todetachment condition[3].\nTo resolve this ambiguity, we have revised the relevant section in the Results\n(Page 8,Paragraph 2,Line 224-226).The original statement,“Bypre-incubatingthe\nlactate-treated cells with P300 inhibitor A485 or GPR81 siRNA, however, we found\nthat neither P300 (Fig S4A-D) nor GPR81 (Fig S4E-I) altered the lactate induced\nstemness potentials.” has been replaced with the following updated description: “By\npre-incubating the lactate-treated cells with P300 inhibitor A485 or GPR81 siRNA,\nour findings revealed that,except for the downregulation of SOX2expression in T47D\ncells, neither P300 (Fig S4A-D) nor GPR81 (Fig S4E-I) significantly affected\nlactate-induced stemness potentials.” We believe this revision provides a more\nprecise descriptionofourresults and appropriately addresses the concern you raised.\n6. Figure S6J should have controls from whole cells or another fraction to assess\nenrichment. In Figure S6M, CD147 does not co-IP with MCT1 in T47D cells. Have\nthe lanes been mislabeled?\nResponse:\nWe sincerely appreciate your insightful suggestion. In response, we have\nperformed additional western blot analyses for assessing non-mitochondrial\ncomponent in the updated Figure S6J below. This data confirms that the absence of\nnon-mitochondrial signals in the mitochondrial fraction reflects high purity of the\nisolatedmitochondria,rather than inadequateenrichment.\nIn addition, we sincerely apologize for the labeling error in Figure S6M. To\nensure accuracy and clarity, we have corrected the mistake by adding explicit\nlabeling forCD147 co-IPwithMCT1 orLDHB inT47Dcells.\nResponse Figure 19 (Updated Figure S6J). The isolated mitochondrial or non-mitochondrial\nfractions from MCF7 cells at different contents (2 g, 10 g, 20 g, 50 g) were analyzed by\nimmunoblotting using antibodies to COX IV, α-Tubulin, Na+-K+ ATPase, LAMP1, GM130,\nGRP78 and Lamin B1 for indicating the fraction of mitochondria, cytoplasm, plasma membrane,\nlysosome,Golgiapparatus,endoplasmicreticulumandnucleus,respectively.\nUpdated Figure S6M. Interaction of CD147 with MCT1 or LDHB via Co-IP assay in\nmitochondriaisolatedfromARMCF7/T47Dcells.IgG,captureantibodycontrol.\n7. Figure S8B—the amount of CD147 in non-mitochondrial fractions should be\nshowntoconfirm that mito 147is limited to the mitochondrion.\nResponse:\nWesincerely appreciate your insightful critique, which significantly enhanced the\nrigor of our conclusions. Following your advice, we performed additional western\nblot analyses assessing CD147 expression in non-mitochondrial fractions (updated\nFigure S8B below). The negligible CD147 signal observed in non-mitochondrial\nfractions robustly confirms its exclusive mitochondrial localizationin mito147cells.\nResponse Figure 20 (Updated Figure S8B). CD147 expression detected by immunoblot in total\ncell (total), isolated mitochondria (mito) or non-mitochondria (non-mito) lysates from\nMCF7/T47D derived WT/KO/mito147 cells. α-Tubulin, maker of cytoplasm; COX IV, maker of\nmitochondrialinnermembrane;Na+-K+ATPase,makerofplasmamembrane.\nReviewer#2 (Remarks totheAuthor)\nIn this manuscript by Zhang et al., the authors describe a new role for the metabolite\nlactate in the regulation ofbreast cancer metastasis-initiating cells (MICs). They show\nthat MICs uptake extracellular lactate that they import into the mitochondria and\noxidize it through a protein complex formed by CD147/MCT1/LDHB.\nLactate-derived pyruvate feeds the TCAcycle and increases the levels of a-KG, which\nin turninhibits the DNAmethyltransferase DNMT3 leading to a decrease in the levels\nofDNAmethylation onthe promoterof SOX2and toan increase ofits expression. By\nperforming both in vitro and in vivo functional experiments, the authors show that\nlactate increases the number and metastatic properties of breast cancer stem cells\n(CSCs), phenotypes that are abolished upon inhibition of either MCT1 or LDHB.\nLastly, and to prove the involvement of CD147, they develop an adenoviral system to\nexpress a svFc to target mitochondrial CD147 and show that it efficiently inhibits\nlactate-dependent metastatic potentialof CSCs.\nThis manuscript provides novel and important insights into the metabolic regulation\nof metastasis-initiating cells involving a metabolic-epigenetic axis linking lactate\nmetabolism to gene expression. It also establishes a new role for the chaperone\nCD147 on mitochondrial lactate import and metabolism, while providing the rationale\nto target this chaperone to reduce breast cancer metastasis. In this context, the\nfindings reported in this studyare ofinterest and relevant forthe scientific community.\nThe manuscript contains an impressive amount of data, which, overall, is well\npresented and support most of the authors´ conclusions. However, there are some\nissues that need to be addressed,as specified below.\nWesincerely thank you for the time and expertise you dedicated to reviewing our\nmanuscript.Your acknowledgement of the scientific significance of our study and the\nrobustness of our data is profoundly encouraging. Wehave meticulously addressed all\npoints raised and implemented comprehensive revisions. These efforts aim to further\nstrengthen the methodological rigor and conceptual clarity of the work. We are\nconfident that the revised manuscript now meets the high quality you advocate. Thank\nyou again for your insightful feedback. Detailed point-by-point responses to your\ncomments areprovided below.\nMajorissues\n1. There is no information about why the authors use the indicated concentrations of\nlactate (mM range). I understand that this range might be the one observed in\nphysiological conditions,but they did not provide any explanation.\nResponse:\nThanks for your question. We acknowledge the need to clarify the rationale\nbehind the lactateconcentrationranges selected in our study.\nAs established in prior studies, physiological lactate concentration in healthy\nhuman serum typically ranges between 1.5-3 mM[5, 6], whereas tumor tissues exhibit\nsignificantly higher levels, reaching 10-30 mM in intratumoral regions and up to 50\nmM in necrotic cores[6, 7]. Notably, in in vivo experimental models like mice, lactate\ndoses below 1.5g/kg via intraperitoneal injection have been shown to be non-lethal\nand devoid ofacidosis orsevere inflammatoryside effects[8-10].\nTo comprehensively evaluate the impact of lactate on the stemness potential of\ndetached breast cancer cells in vitro, we selected a concentration range of 0-40 mM,\nwith a primary experimental concentration of 20 mM, encompassing both\nphysiological and pathological benchmarks. For in vivo studies, we administered\nlactate via intraperitoneal injection at 800 mg/kg, which elevated serum lactate levels\nto approximately 5 mM (consistent with Cerda-Kohler et al.[10]), while tumoral\nlactate concentrations reached 6-8 mM (as shown in Response Figure 21 below).This\ndosing regimen ensured biologically and clinically relevant lactate exposure while\nmaintaining experimental robustness.\n2. Throughout the manuscript, the authors use the expression “lactate feeding”\nreferring to their in vivo studies, but they are injecting lactate intraperitoneally.\nRelatedtothis, there is no experimentaldataonthe levels of lactateupon i.p.injection\nin mice.The authors should check the levels of lactate in the serum and within tumors\nto prove that the lactate injected is reaching cancer cells, especially considering that\nlactate is the primary source of carbon feeding the TCAcycle in most tissues (PMID:\n29045397).\nResponse:\nWe sincerely express our gratitude for your insightful questions and invaluable\nsuggestions. As you correctly noted, lactate in the in vivo experiments was\nadministered via intraperitoneal injection. To ensure accuracy, we have\ncomprehensively revised the term \"lactate feeding\" to \"lactate administration\"\nthroughoutthe manuscript.\nTo verify whether the administered lactate effectively reaches cancer cells, we\nfollowed your recommendation and measured lactate levels in both serum and tumors\nof PDX1, 4T1, and MCF7 tumor-bearing mice following intraperitoneal lactate\nadministration. Our data in Response Figure 21 demonstrate a significant increase in\nlactate concentrations in treated mice compared to untreated controls both in serum\nand tumors. Notably, lactate levels were higher in tumor tissues (7.23±0.37) than in\nserum (5.15±0.40), confirming effective lactate uptake within tumors relative to\nnormal tissues.\nResponseFigure21.Lactatelevelsinplasma (left)andtumors(right)ofPDX1(A),4T1(B),and\nMCF7 (C)-bearing mice (n=4 mice for each group) with 800 mg/kg lactate. Data are represented\nasmean±SEM.Statisticalanalysis:ttest.**p<0.01,***p<0.001.\n3. The effect of lactate treatment clearly inhibits metastasis formation. But does it\naffect primary tumorgrowth?\nResponse:\nThank you for raising this question. We have carried out a comprehensive\ninvestigation into how lactate impacts both primary tumor growth and metastasis.\nRegrettably, due to the space constraint, these data were not included in the submitted\nmanuscript. As shown in Response Figure 22 below, our data showed that\nadministering lactate to PDX1, 4T1, or 4T1 ALDH- tumor-bearing mice did not lead\ntoany significant alterations in the growthrate ofprimary tumors.\nFor the reason behind this phenomenon, we hypothesize that tumor cells within\nprimary tumors have sufficient glucose supply and thus predominantly utilize glucose\nas their energysource.In contrast,metastatic breast cancer cells have been reportedto\nhave the reduced glucose uptake[4]and rely mainly on lactate, a metabolic byproduct,\nto sustain their survival and maintenance their stemness. Therefore, it can be\nreasonably concluded that lactate primarily exerts its influence on the formation of\nmetastatic tumor,while having minimal effect on the growthof primarytumor.\nResponse Figure 22. Representative tumor images and tumor growth curve of PDX1 (A, n=5\nmice),4T1 (B, n=5 mice)and 4T1ALDH- (C, n=8mice) tumor-bearing mice with or without 800\nmg/kg lactate administration. Data are represented as mean ± SEM. Statistical analysis: repeated\nmeasurestwo-wayANOVA.ns:notsignificant.\n4. Some of the immunostaining experiments are not very clear and they do not\nsupport the authors’ claims. For instance, in Fig.2I, the staining for SOX2 is not\nnuclear, in Fig.2M it seems that SOX2 is expressed n CK18- cells, in Fig.6K there is\nno staining for SOX2, in Fig 6H there is no overlay of CD147, MCT1 and LDHB in\nCTCs and in Fig.6F it looks like the signal in the lactate treated samples is just\nbackground(it is exactly the same non-specific signal in all channels).\nResponse:\nWe sincerely apologize for the confusion arising from the unclear and improper\npresentation of the immunostaining images. To rectify these issues, we have\nmeticulously updated Figures 2I, 2M, 6K, and 6F. We appreciate your patience as we\narecommitted topresenting ourdatawiththe utmostclarity and accuracy.\nIn the original Figure 2I, the nuclear localization of SOX2 staining was unclear.\nTo address this, we have carefully selected and replaced the image. The updated\nFigure 2I (shown below) now better illustrate the specific SOX2 staining within\nnucleus.\nIn original Figure 2M, we aimed to illustrate SOX2 immunostaining in nucleated\nDAPI+CK18+ CTCs within the vascular lumen. Compared to mild to strong\nnon-specific CK18 staining in anuclear erythrocytes, the CK18 signal in CTCs was\nrelatively weak, potentially causing misinterpretation. To address this concern, we\ndirectly isolated CTCs from murine blood using magnetic bead separation, followed\nby rigorous CTC identification and SOX2 staining. As clearly shown in the updated\nFigure 2M presented below, the non-specific CK18 signal in CTCs has been\nsuccessfully eliminated. Furthermore, the data reveals that both the number of CTCs\nand the levels of SOX2 expression in CHC- and GNE-treated groups are significantly\nreduced compared to the control(NS)group,consistent with ouroriginal findings.\nFor the weak SOX2 staining in original Figure 6K, we have conducted a\nrepetition of the immunohistochemical (IHC) staining procedure using consecutive\nsections from the same patient.As vividly presented in the newly updated Figure 6K\nbelow, distinct brown staining now be clearly discerned within the nuclei of CTCs,\nindicating a positive SOX2staining in CTCs.\nIn Figure 6H, tumor tissue sections obtained from metastatic and non-metastatic\nbreast cancer patients were applied to investigate the potential correlationbetween the\nindividual expression levels of CD147, MCT1, and LDHB and the metastatic\npropensity of breast cancer patients. Notably, this analysis did not focus on the\nformationofthe CD147/MCT1/LDHB complex on circulating tumorcells (CTCs) nor\ndid it involve co-localization analysis (e.g., fluorescence overlay) of these three\nmolecules. Conversely, Figure 6L specifically demonstrates the co-localization of\nCD147, MCT1, LDHB on the mitochondrial membranes of CTCs from NO.9 breast\ncancer patient in Figure S7I, directly supporting the spatial assembly of\nCD147/MCT1/LDHB ternary complex in CTCs during the metastatic process.\nIn the original Figure 6F, the co-localization signal in the lactate treated samples\nseemed non-specific. To address this concern, we have meticulously replaced these\nimages with high-quality one, which clearly showing strong co-localization signals in\nthe lung metastatic tumors from PDX1-bearing mice with 800 mg/kg lactate\nadministration(updated Figure 6Fbelow).\nWe are truly grateful for your careful attention to these details, which has\nsignificantly strengthened the clarityand quality ofour work.\nResponse Figure 23 (Updated Figure 2I, 2M, 6K and 6F). (2I) Immunofluorescence (IF)\nimages of plasma CTCs from lactate administered 4T1 bearing mice (Scale bars, 5 μm). (2M)IF\nimagesofplasma CTCs(left;scalebars,10μm),CTCnumbers(middle;n=5miceforeachgroup),\nas well as SOX2 fluorescence intensity quantification in CTCs (right; NS, n=100 cells from 5\nmice;CHC,n=50cellsfrom5mice;GNE,n=50cellsfrom5mice)fromlactateadministered4T1\nbearing mice treated with NS or CHC/GNE. (6K) SOX2 IHC staining in CTCs of No.9 breast\ncancer patient in Fig S7I. Scale bars, 10 μm. (6F) Laser scanning confocal microscopy showing\nco-localization of CD147 (red), LDHB (blue) and MCT1 (purple) in COX IV (green) labeled\nmitochondria from lung metastatic tumors in PDX1 bearing mice, with or without 800 mg/kg\nlactate. Merge1: COX IV, CD147 and LDHB. Merge2: merge1 and MCT1. Scale bars, 20 μm.\nOrange arrows show the colocalization. Data are represented as mean±SEM. Statistical analysis:\nupdated2M,one-wayANOVA.***p<0.001,****p<0.0001.\n5. Another important issue of this manuscript is the authors ’claim that lactate can\ninduce stem cell properties in non-stem cells based on the results presented in Figure\n3. It could be possible thatALDH- cells can becomeALDH+ cells in vivo, which are\nthe ones targeted by lactate, as they prove in Figure 2. This is supported by the fact\nthat the number of ALDH+ cells is basically the same in primary tumors and\nmetastasis derived from ALDH+ or ALDH- cells (compare Fig. 2C and Fig. 3F),\nindicating that the effect of lactate could be on the newly formedALDH+ cells and is\nnot driving the acquisition of stem cell properties inALDH- cells. To fully prove this\nhypothesis, they authors should dotracing experiments.\nResponse:\nWesincerely apologize forthe lack of clarity in our original description regarding\nthe phenotype of the inoculated cells. To clarify: the cells used in Figure 2C were\nunsorted 4T1 total cells rather than 4T1 ALDH⁺ cells, whereas Figure 3F utilized\nspecifically sorted4T1ALDH⁻cells.\nSince thatALDH⁺ cancer stem cells (CSCs) represent only a minor fraction ofthe\ntotal tumor cell population[36], the majority of cells within the unsorted 4T1 total\ncells in Figure 2C areALDH⁻ cells. Therefore, it is reasonable that the proportion of\nALDH⁺ cells observed in primary tumors and metastases is comparable between\nFigures 2C (unsorted cells) and 3F (sorted ALDH⁻ cells). This consistency strongly\nsupports our conclusion that lactate drives the acquisition of stem cell properties in\nALDH⁻cells.\nFollowing your valuable recommendation, we performed tracing experiments to\nrigorously validate whether lactate primarily affects the newly formed ALDH+ cells\nor it drive the acquisition of stem cell properties in ALDH⁻ cells. As shown in\nResponse Figure 24 below, we established an orthotopic breast cancer model using\nFACS-purified, mCherry-labeled 4T1 ALDH⁻ cells (A). Crucially, lactate treatment\nsignificantly increased the proportion of mCherry⁺ ALDH⁺ cells (B) and SOX2\nexpression in isolated mCherry-labeled 4T1 ALDH⁻ cells (C) within lung metastatic\ntumors. This result provides direct evidence that lactate induces the transition of\ninoculated mCherry-labeled ALDH⁻ cells into mCherry⁺ ALDH⁺ cells and the\nacquisition of stemness properties, thereby conclusively validating our original\nfindings.\nWe are deeply grateful for your insightful suggestions, which have significantly\nenhanced the robustness andmechanistic insightofourstudy.\nResponse Figure 24. Lactate promotes the transition of mCherry-labeled 4T1 ALDH- cells to\nALDH+ cells in breast cancer mouse model. (A) Experimental flowchart. ALDH- cells were\nisolated from bulk 4T1 total cells by flow cytometry and transduced with mCherry lentivirus to\nobtain mCherry-4T1 ALDH- cells. Subsequently, 1×10⁵ mCherry-4T1 ALDH- cells were\nimplanted into the mammary fat pads of BALB/c mice to establish orthotopic breast tumors.\nWhen tumor volume reached 100 mm³, mice were randomized to receive daily intraperitoneal\ninjections of 100 μL saline or lactate-Na (800 mg/kg) for 5 consecutive days. Mice were\neuthanized 15 days post-treatment initiation, and tumors were harvested for flow cytometry\n(analyzing mCherry+ ALDH+ subpopulation ratios, and sorting mCherry+ cells for western blot\nassay). (B) mCherry+ ALDH+ subpopulation analysis in primary (left) and metastatic (right)\ntumors from mCherry-4T1 ALDH- cell bearing mice. n=5 mice for each group. (C)\nSOX2/MCT1/LDHB protein levels of mCherry+ cells isolating from primary and metastatic\ntumors from mCherry-4T1 ALDH- cell bearing mice. n = 4 mice for each group. Data are\nrepresented as mean±SEM. Statistical analysis: B, t test. ****p < 0.0001. ns, not significant.\nCreated in BioRender. Song, C. (2025) https://BioRender.com/5why6l0\n6. In their in vivo experiments, they demonstrate that lactate uptake by CSCs is\nimportant for metastasis formation by injecting lactate into mice. In these conditions,\nthey show an increase in the number of metastatic nodules. However, is this\nmechanism also occurring in more “physiological” conditions? For instance, is lactate\nuptake by CSCs in non-treated mice involved in metastasis formation?Are there other\ncancer cells releasing lactate under normal conditions (for instance glycolytic cancer\ncells) and feeding this CSCs to become more metastatic? This could be easily\nassessed by staining tumorsections withspecific markers.\nResponse:\nWe sincerely appreciate your insightful comments and valuable question\nregarding the physiological relevance of lactate uptake by CSCs in metastasis\nformation. We concur that our initial in vivo experiments demonstrating the\npro-metastatic effect of lactate employed exogenous administration, rather than\nreflecting endogenous lactate dynamics in untreated mice. This approach was chosen\ndue to the typically high lactate concentrations found in the tumor microenvironment\n(TME) of many solid tumors, including breast cancer. This lactate is constitutively\nproduced by glycolytic cancer cells via the Warburg effect and actively imported by\ndetached cells via upregulatedMCT1 to maintain CSC stemness.\nTo directly assess the impact of endogenous lactate utilization on stemness and\nmetastasis under physiological conditions, we established orthotopic xenografts using\n4T1 breast cancer cells with or without MCT1 knockdown to specifically impair\nlactate uptake.As shown in Response Figure 25 below, inhibiting lactate transport via\nMCT1 knockdown in mice concurrently receiving 800 mg/kg lactate supplementation\nresulted in a significant reduction in lung metastatic nodules (Figure A-B). This was\naccompanied by attenuated stemness potential within metastatic tumors (Figure C-D)\nand a notable extension in survival (Figure E). These findings demonstrate the\nphysiological relevance ofCSC lactateuptake in metastasis.\nFollowing your valuable suggestion, we performed IF staining on primary tumors\nsections from untreated 4T1 tumor-bearing mice. Our aim was to identify glycolytic\ncancer cells (potential lactate producers) and oxidative cancer cells (potential lactate\nconsumers) under basal conditions. We employed the following markers: LDHA (a\nlactate-producing key enzyme) to mark glycolytic tumor cells, MCT1 (a critical\nlactate importer) to mark oxidative tumor cells, and SOX2 to assess stemness and\nmetastatic potential.\nAs shown in Figure F-G below, we observed distinct spatial partitioning within\nprimary tumors:glycolytic regions exhibiting high LDHA(lactate-producing)and low\nMCT1 expression, and oxidative regions exhibiting high MCT1 (lactate-importing)\nand low LDHA expression. Critically, SOX2 expression was significantly higher\nwithin oxidative, MCT1-high regions. Furthermore, these glycolytic and oxidative\nzones were spatially adjacent, suggesting local transfer of lactate produced by\nglycolytic tumor cells to neighboring oxidative tumor cells, thereby enhancing their\nstemness and metastatic potential.\nThus, as you insightfully noted, the phenomenon wherein glycolytic tumor cells\nproduce lactate to fuel oxidative tumor cells and promote their metastasis occurs\nunder endogenous, “physiological” conditions. This finding aligns with established\nliteratureonmetabolic coupling within tumors[37].\nWe are deeply grateful for your guidance, which has profoundly strengthened the\nmechanistic foundationofour study.\nResponseFigure25.MCT1knockdowninhibitsthe lactate-inducedlungmetastasisandstemness\npotentials in 4T1 bearing mice. (A-B) Representative HE staining (A, scar bars: 1 mm) and lung\nmetastaticnodulesquantitation(B,n=10mice/group)in800mg/kglactate-treated4T1MCT1WT\n(sh-NC) or MCT1 knockdown (sh-MCT1) cell bearing mice. (C-D) ALDH+ subpopulations (C,\nn=5 mice/group) and SOX2 protein levels (D, n=4 mice/group) in primary and lung metastatic\ntumors from 800mg/kg lactate-treated4T1 MCT1WT(sh-NC)or MCT1 knockdown(sh-MCT1)\ncell bearing mice. (E) Survival curve of 800 mg/kg lactate-treated 4T1 MCT1 WT (sh-NC) or\nMCT1 knockdown (sh-MCT1) cell bearing mice (n=10 mice/group). (F) Representative IF\nstaining of LDHA/MCT1/SOX2 in primary tumor from 4T1 cell bearing mice without lactate\ntreatment. Left region of the dotted line is the glycolytic region; right region of the dotted line is\ntheoxidationregion.Scalebars,50μm.Merge,mergedchannelsofDAPI,LDHAandMCT1.(G)\nQuantification of LDHA/MCT1/SOX2 fluorescence intensity in figure F. n=12 representative\nfields from 6 mice. Data are represented as mean±SEM. Statistical analysis: (B-C, G) t test; (E)\nKaplan-Meierandlog-ranktest.***p<0.001,****p<0.0001,ns:notsignificant.\n7. The authors claim that the effect of lactate is independent of histone lactoylation\nand the activation of the lactate receptor GPR81. However, Figure S4D and S4I show\nthat, in T47D cells, both the inhibitor of P300 and the silencing of GPR81 decrease\nSOX2 expression. Interestingly, the number of spheres and CSCs is not decreased,\nsuggesting an uncoupling ofthese twophenotypes.\nResponse:\nWe fully acknowledge the observation that both A485 (a p300 inhibitor) and\nsiGPR81 treatment decrease SOX2 expression in 20 mM lactate-treated T47D cells,\nbut do not in MCF7 cells (Figure S4D and S4I). This cell line-specific discrepancy may\narise from the inherent susceptibility of T47D cells to detachment, as previously\nreported [3]. Notably, despite the downregulation of SOX2 in T47D cells, no\nsignificant changes were observed in sphere formation or CSCs phenotypes (Figure\nS4Dand S4I).Wepropose twopotential explanations forthis observation:\nFirst, the reduction in SOX2 expression may not reach a threshold sufficient to\nimpair CSC maintenance. The decrease in SOX2 levels upon A485 or siGPR81\ntreatment in lactate-exposed T47D cells might be insufficient to significantly alter\nCSC stemness,thereby preserving sphere formationand CSC ratios.\nSecond, compensatory mechanisms may sustain CSC properties independent of\nSOX2. In T47D cells, other stemness-related transcription factors or signaling\npathways could compensate for reduced SOX2 levels, maintaining the CSC\nphenotype.As shown in Response Figure 26, we observed upregulation of OCT4 and\nNANOG, key stemness regulators, in AR or lactate-treated breast cancer cells, and\ntheir expression remained largely unaffected by P300 inhibition or GPR81 silencing.\nTherefore, when SOX2 is downregulated by A485 or siGPR81, compensatory factors\nsuch as OCT4 or NANOG may maintain CSC functionality, mitigating the impact of\nSOX2reduction.\nResponse Figure 26. (A) Protein levels of stem cell transcription factors OCT4 and NANOG in\nAR or 20 mM lactate cultured breast cancer cells. (B-C) Protein levels of stem cell transcription\nfactors OCT4 and NANOG in 20 mM lactate cultured breast cancer cells pretreated with P300\ninhibitorA485(B)orGPR81siRNA(C).\n8. The co-IPexperiment in Fig. S6M (upper panel) is confusing. The blot for CD147\nin the MCT1 IPshows that either CD147 is being IPed by non-specific IgGs in T47D\ncells or, if the labelling of the lanes is wrong, then CD147 is being IPed by\nnon-specific IgGsin MCF7 cells.\nResponse:\nThank you for pointing out the labeling issue in Figure S6M. We sincerely\napologize for the oversight and have corrected it by explicit labeling the T47D cells\ntoensure the figure’s accuracy and clarity.\nUpdated Figure S6M. Interaction of CD147 with MCT1 or LDHB via Co-IP assay in\nmitochondriaisolatedfromARMCF7/T47Dcells.IgG,captureantibodycontrol.\n9. Lastly, the authors did a very elegant approach to target CD147, which provided\nnew information about the role of this chaperone in controlling lactate metabolism in\nthe mitochondria. Importantly, this approach also implicates CD147 as a potential\ntarget to inhibit breast cancer metastasis. However, it is not clear what will it be the\nadvantage of targeting CD147 instead of using MCT1 or LDHb inhibition, which,\nas the authors proved in this manuscript, efficiently blocks lactate-driven metastasis\nformation. I think that the authors could discuss this further to contextualize their\nfindings.\nResponse:\nWe sincerely appreciate your insightful question and valuable suggestions. In our\nstudy, based on the established role of CD147 in regulating lactate metabolism and\npromoting lactate-induced metastasis of detached breast cancer cells, we identified a\nmitochondrial CD147/MCT1/LDHB complex that mediatesthese functions.\nAs illustrated in Response Figure 27A, molecular docking simulations revealed\nthe conformation of this complex: CD147 acts as a bridging molecule, with its\nC-terminal transmembrane region binding to MCT1 and its N-terminus interacting\nwith LDHB. Subsequent site-directed mutagenesis analyses using purified\nGST-tagged CD147 N-terminal protein and His-tagged LDHB protein (Response\nFigure 27B-E) identified two critical interaction interfaces: CD147’s Glu168 forms a\nbond with LDHB’s Arg99, and CD147’s Arg106 interacts with LDHB’s Asp56.\nFurthermore, functional studies demonstrated that CD147 overexpression or\nknockdown correspondingly promoted or inhibited MCT1 and LDHB expression (Fig\nS6F). This structural configuration and regulation mechanism establish CD147 as the\ncentral organizer of the lactate uptake and oxidation complex, critically controlling\nboth lactate transport and oxidation processes. Therefore, targeting CD147 may offer\nsuperior therapeutic advantages for suppressing lactate metabolism and inhibiting\nlactate-driven metastasis.\nNotably, our lab has previously developed some anti-CD147 therapies, including\nmetuximab to prevent tumor recurrence[38] and enhance chemosensitivity in\npancreatic CSCs[39], as well as anti-CD147 scFv-derived CAR-T cells effective\nagainst T cell acute lymphoblastic leukemia[40]. These findings underscore the\nclinical feasibility of targeting CD147 and highlight its potential for future\ntranslational studies.\nFollowing your suggestion, we have incorporated the relevant content into the\nDiscussion Section of the manuscript. Please find the revised excerpt below: \"We\nfurther revealed that the CD147/MCT1/LDHB complex adopts a conformation in\nwhich CD147 acts as a bridging molecule. Specifically,its C-terminal transmembrane\nregion interacts with MCT1, while its N-terminal domain associates with LDHB (data\nnot shown). This structural configuration highlights CD147 as the central organizer\nwithin the lactate uptake and oxidation complex, underscoring its critical role in\nregulating both lactate transport and oxidation processes.\" (Page 14, Paragraph 1,\nLine 445).\nResponse Figure 27. Conformational binding of the CD147/MCT1/LDHB complex. (A) The\nmolecular docking technology simulated binding conformation of the CD147/MCT1/LDHB\ncomplex,elucidatingthestructuralbasisoftheirinteractions.Yellow:CD147;Purple:MCT1;Red:\nLDHB. (B) Coomassie Brilliant Blue staining showed the successful expression and purification\nof GST-CD147 N-terminal protein (left, 46 kDa) and His-LDHB protein (right, 36 kDa). (C)\nPull-down assay confirmed the direct interaction between GST-CD147 N-terminal protein and\nHis-LDHB protein. (D) Site mutation analysis in pull-down assay identified critical residues\ninvolved in the interaction between GST-CD147 N-terminal protein (left) and His-LDHB protein\n(right). (E) Molecular docking simulations revealed that the 168th glutamic acid of CD147\ninteractswiththe99tharginineofLDHB(left),andthe106tharginine ofCD147interactswiththe\n56th aspartic acid of LDHB (right), suggesting a hydrogen-bond-mediated interaction that\nstabilizes the complex structure. Residues are colored by protein: CD147 in blue, LDHB in\nyellow.\nMinorissues\n1. Figure 5k: how is the data represented? The figure legend states that n=3\nexperiments have been done, but one representative experiment is shown. If so, how\nhas the statistics been done?\nResponse:\nWe deeply regret the inaccuracies in the original figure legend and greatly\nappreciate your careful review. The data presented in Figure 5K were obtained from\nthree biologically independent experimental replicates. Importantly, the full studywas\ndesigned that each experiment was performed with three biological replicates, with\neach biological replicate comprising more than three technical replicates. To ensure\nclarity and eliminate ambiguity, we have modified the original statement in the figure\nlegend from “Experiments repeated in triplicate, representative experiment shown.”\nto “n=3 biologically independent samples per condition.”. Furthermore, following\nyour valuable suggestion, we have carefully reviewed and updated all figure legends\nthroughoutthe manuscript to ensure consistency and accuracy.\n2. Why CD147-KD reduces the number of ALDH+ cells in primary tumors from\nMDA-MB-231cells (Fig.S6G)?\nResponse:\nWe sincerely appreciate your inquiry. Both our preliminary studies[3] and team’s\nfindings[27] revealed that CD147 is highly expressed in anoikis resistance\nMDA-MB-231 cells, which exhibit inherent high stemness and metastatic potential,\nbut is expressed at low levels in anoikis-sensitive MCF7 and T47D cells with low\nstemness and metastatic potential (Response Figure 28). Consistently, CD147\nexpression was markedly elevated in both primary tumors and metastasis sites from\nlactate-treated MDA-MB-231 tumor-bearing mice compared to untreated controls\n(Figure S6B right). These findings collectively indicate that high CD147 expression\nin MDA-MB-231 cells correlates with enhanced stemness and metastatic potential at\nboth primary and metastatic sites. Consequently, CD147 knockdown reduced the\nstemness potentials in both primarytumors and metastases.\nResponse Figure 28. CD147 is highly expressed in anoikis resistance MDA-MB-231 cell. (A)\nApoptosis analysis in breast cancer cells cultured under an attached condition in a common\nuncoated plate or a detached condition in ultra-low attachment plate for up to 12 days. (B) Flow\ncytometry analysis of membranous CD147 levels in T47D, MCF7, and MDA-MB-231 cells\ncultured under an attached or detached condition for 0, 1, and 3 days. (C) Immunoblot assay of\nCD147 protein levels in T47D, MCF7, and MDA-MB-231 cultured under an attached (Ad) or\nanoikis resistant (AR) condition for 7 days. (D) Real-time RT-PCR analysis of CD147expression\nin a series of human breast cancer cell lines and normal tissues. (E)Westernblot detection of the\nexpression of CD147, Sp1 and c-Myc in a series of human breast cancer cell lines and normal\ntissues. Data are represented as mean ± SEM. Statistical analysis: B, one-wayANOVA(1, 3 days\nvs. 0 days). *p < 0.05, **p < 0.01, ***p < 0.001. Notes: Figure A-C were adopted from our\nprevious paper meng et al.[3], while Figure D-E were adopted from our team’s paper kong et\nal.[27].\n3. In figure S6J, it would be nice to include a lane with protein form the other\nfractions (membrane, cytosol, nuclei, lysosomes) as a positive control for the\nantibodies used to claim that mitochondrialfractions are pure.\nResponse:\nWe sincerely appreciate your insightful suggestion. In response, we performed\nadditional western blot analyses for selected protein markers in both mitochondrial\nand non-mitochondrial fraction, using the non-mitochondrial fractions as a positive\ncontrol. As shown in the updated Figure S6J below, the expression levels of each\nnon-mitochondrial protein increased dose-dependently with loading amounts of 2 g,\n10 g, 20 g, and 50 g, whereas being nearly undetectable in the mitochondrial\nfraction. These data confirm that the absence of these non-mitochondrial components\nin the mitochondrial fraction reflects high purity of the isolated mitochondria rather\nthan inadequateenrichment.\nResponse Figure 29 (Updated Figure S6J). The isolated mitochondrial (mito) or\nnon-mitochondrial (non-mito) fractions from MCF7 cells at different contents (2, 10, 20, and 50\nμg) were analyzed by immunoblotting using antibodies to COX IV, α-Tubulin, Na+-K+ ATPase,\nLAMP1, GM130, GRP78, and Lamin B1 for indicating the fraction of mitochondria, cytoplasm,\nplasmamembrane,lysosome,Golgiapparatus,endoplasmicreticulumandnucleus,respectively.\n4.Awriting revision/editing ofthe manuscript is encouragedto improve its quality.\nResponse:\nWe sincerely appreciate your valuable feedback and apologize for the\ngrammatical errors in the manuscript, which may have caused difficulties in\nunderstanding. To address this issue, we have conducted a comprehensive,\nline-by-liner review of the manuscript to correct all grammatical, syntactical, and\ntypographical errors. In addition, we also sought assistance from a native English\nspeaker tofurther polish the language,ensuring natural phrasing and improved clarity.\nThe corresponding editing certificate is provided below. Please note that the article\ntitle has also been revised. The updated title, \"Lactate oxidation drive stemness\npotentials via mitochondrial CD147/MCT1/LDHB in metastatic breast cancer\", has\nnow been incorporated intothe revised manuscript.\nWe are confident that these revisions have significantly improve the linguistic\nquality and readability of the manuscript. Thank you again for your time and\nconstructive criticism—it has greatly strengthened our work.\nReviewer#3 (Remarks totheAuthor):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nWe sincerely appreciate the time and invaluable expertise you dedicated to\nco-reviewing our manuscript. We have meticulously addressed all comments from\nyou and other reviewers, which have greatly enhanced the manuscript's\nmethodological rigor and conceptual clarity. Thank you again for your unwavering\nsupport.\nReviewer#4 (Remarks totheAuthor):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nWe sincerely appreciate your dedicated time and profound expertise in\nco-reviewing our manuscript. We have comprehensively addressed all comments\nthrough additional experimental evidence, expanded methodological clarifications,\nand refined data interpretations.As a result of these efforts, the robustness and clarity\nof our study have been significantly enhanced. Once again, we extend our sincere\nappreciationforyourinvaluable supportthroughoutthis process.\nReviewer#5 (Remarks totheAuthor):\nZhang, Tian, and Song and colleagues investigate lactate catabolism role in breast\ncancer stem like cell features. Understanding tumor metabolism, cancer stem cells,\nand metastasis are highly relevant areas of clinical importance that will be of interest\nto a very broad readership. The study is fascinating and presents several novel\nfindings, withimportant biological and translational implications.\nThe ample discussion of CSC vs MIC properties, phenotypic plasticity, and context\ndependence of their properties is a strength. Numerous exciting and novel data are\npresented in this study. The finding that lactate can essentiall transform non CSC into\nCSC is provocative. Mechanistic experiments demonstrating the lactate stemness\neffect is due to its role in oxphos, and probably not p300 etc, are robust. The\nmanuscript includes copious data, most of it complementary and thorough, and a\nwealth of novel information that will be important for the field. Enthusiasm for this\nstudyis high.\nThe paper is well written, butgrammatical editing is needed.\nWe sincerely thank you for your meticulous and insightful review of our\nmanuscript.Your enthusiastic recognition of the novelty and significance of our study\nhas been a tremendous source of motivation and inspiration for us. All raised\ncomments have been addressed through comprehensive revisions, which greatly\nstrengthened the reliability and validity ofour findings.\nAdditionally, we have also engaged a native English-speaking editor to polish the\nmanuscript, enhancing grammatical accuracy and linguistic fluency. The\ncorresponding editing certificate is provided below. Please note that the article title\nhas also been revised. The updated title, \"Lactate oxidation drive stemness potentials\nvia mitochondrial CD147/MCT1/LDHB in metastatic breast cancer\", has now been\nincorporatedintothe revised manuscript.\nOnce again, we express our sincere appreciation for your invaluable support and\nguidance throughoutthis process.\nSpecific comments\n1. There are no methods in the main paper. Methods should be readily visible to the\nreadership and not relegated to the supplement as they are required to be able to\naccurately interpret the data.\nResponse:\nWesincerely appreciate yourimportant feedback and fully agree that the Methods\nSection should be clearly presented in the main paper to ensure transparency and\nreproducibility. In the revised version, we have moved the Methods Section from the\nsupplement to the main manuscript, ensuring that all critical experimental details,\nanalytical procedures, and statistical approaches are readily accessible to readers. This\nadjustment will help readersbetterinterpretourdata and validatethe findings.\n2.First results section-cell line models are inadequately described, as is the generation\nofAd vsAR cells.Adiagram would be beneficial.\nResponse:\nThank you sincerely for your time and constructive feedback on our manuscript.\nWedeeply regretany confusion caused by the initial inadequatedescriptionofourcell\nline models and would like toclarify the experimentaldesign asfollows:\nIn our study, we investigated the impact of lactate in promoting stemness\npotential in detached cancer cells using two complementary experimental models\n(see diagram below).1) Low-Attachment Model: This model replicates the detached\nstate of metastatic cells. Parental adherent cells (Ad cells) underwent anoikis when\ncultured in low-attachment plates, whereas cells surviving 7-days of continuous\nsuspension culture (AR cells) acquired robust anoikis resistance. Both Ad and AR\ncells were maintained in glucose-containing RPMI 1640 complete medium to ensure\nphenotypic comparability. 2) Glucose-Free Model with Lactate: To mimic the\nmetabolic trait of detached cancer cells that characterized by nutrient-deprived, we\nemployed a glucose-free system supplemented with lactate. This approach was based\non our findings that detached cells exhibit reduced glucose uptake (via GLUT1\ndownregulation [4]) and enhanced lactate uptake (Figure 1 and Figure S1). Notably,\nexogenous lactate (20 mM) suppressed stemness under glucose-replete conditions\n(Response Figure 30), further supporting the rationale for this model. In this setup,\ncells were cultured in glucose-free medium supplemented with 20 mM lactate (Lac\n20mM) were designated as lactate-treated cells, while in glucose-free medium alone\n(Lac 0mM)were controlcells.\nCreated in BioRender. Song, C. (2025) https://BioRender.com/pzpeq9s\nTwo cellular models were used in the study: the low-attachment model for replicating th\nedetached state of metastatic cells, and the glucose-free model for mimicking the nutrient-depriv\nedmetabolic characteristic of detachment.\nResponse Figure 30. Lactate inhibits stemness potentials of breast cancer cells under the\nglucose-containing condition. (A-B) ALDH+ (A) and CD44highCD24-/low (B) subpopulations of\nbreast cancer cells under glucose-containing condition. Data are represented as mean ± SEM.\nStatisticalanalysis: one-wayANOVA(Lac5,10,20,and40mM vs.Lac 0mM). *p<0.05,**p<\n0.01,***p<0.001,****p<0.0001.\nAdditionally, we have supplemented the diagram above and revised the relevant\nstatement in yellow highlight in the Method Section under “Cell lines and\ntreatments” as follows: “As depicted in the following diagram, there are two distinct\ncellular models in our study.One is the Low-Attachment Model. Cells surviving for 7\ndays in glucose-containing medium (3.15g/L) under detached suspension culture\nconditions inultra-low attachmentplates wereconsideredanoikis-resistant (AR)cells,\nwhile cells cultured in uncoated common plates under attached conditions with\nglucose-containing medium (3.15g/L) served as control adherent (Ad) cells[3]. The\nother is Glucose-Free Model with Lactate. Due to the reduced glucose uptake in\ndetached cells, adherent cells cultured in glucose-free medium, with or without\nexogenous lactate incubation, were designated as “Lac 20mM” and “Lac 0mM”,\nwhich weredesigned tomimic the states ofAR andAdcells,respectively.”\nThank youagain foryourrigorousreview and valuable suggestions.\n3. Fig S1,ALDH+ population data should also be provided, as they may contribute to\nmet initiation and/or may also have a similar metabolic fuel adaptation. Itis important\nto denote whether these phenotypes are dependent on ALDH status or not. I would\nexpect that ALDH+ compared to ALDH- population inherently has higher lactate\nuptake and MCT1 protein levels.This should be experimentally tested.\nResponse:\nWe sincerely appreciate your insightful and constructive suggestions. In response\nto your valuable comments, we have performed additional experiments to address\nthese key points.As shown in Response Figure 31 below, our results reveal two major\nfindings: 1) Following treatment with 20 mM lactate, extracellular lactate levels\ndecreased progressively over time (ALDH- > total >ALDH+ cells),while intracellular\nlactate accumulation concomitantly increased (ALDH- < total < ALDH+ cells). This\ndemonstrates significantly higher lactate uptake and retention specifically in ALDH⁺\ncells (Updated Figure S1H). 2) Under detachment conditions, expression of the\nlactate transporter MCT1 and the lactate oxidation-enzyme LDHB exhibited a\nstepwise increase from ALDH- to total and to ALDH+ cells (Updated Figure 1H).\nThese results collectively indicate that the cellular capacity for lactate uptake (via\nMCT1) and oxidative utilization (via LDHB) escalates hierarchically with increasing\nALDH expression and activity, underscoring the pivotal role of ALDH status in\nlactatemetabolism.\nResponse Figure31(UpdatedFigureS1H&1H).ALDHstatusregulatedlactatemetabolism\nand the expression of lactate uptake and oxidation proteins. (S1H) Extracellular and\nintracellular lactate (lac) levels in MCF7/T47D ALDH-, total and ALDH+ cells cultured in\nglucose-free media containing 20 mM lactate for 0–48 hours. Data are represented as mean ±\nSEM. (1H) MCT1 and LDHB protein levels in AR and Ad MCF7/T47D ALDH-, total, and\nALDH+cells.\n4. [lactate]in tumors as they progress would support the circulating lactate datain Fig\n14T1-luciferase.\nResponse:\nThank you sincerely foryour valuable suggestions. In response, we have repeated\nthe relevant experiments and included measurementsof lactatelevels in tumortissues.\nAs shownin Response Figure 32(updatedFigure 1D)below,we observed that plasma\nlactate levels progressively decreased during tumor progression and metastasis in\ntumor-bearing mice, while lactate concentrations in tumor tissues gradually increased.\nThis reciprocal relationship suggests that circulating lactate may serve as an energy\nsource for tumor cells during the metastatic processes, further supporting our earlier\nconclusions.Wedeeply appreciate yourconstructive feedback again.\nResponse Figure 32. Representative images of tumor burden (left) and lactate levels of plasma\n(middle) and tumor (right) in 4T1 luciferase-bearing mice (n=7 mice). Data are represented as\nmean±SEM.\n5. The first few figures data (besides the 4T1 expt) are all from t47d and MCF7 cells\nand it is very important to make this clear in the text and to state that the findings are\nfrom two ER+ brca cell lines. Stating ‘breast cancer’ throughout the paper is\nmisleading and it is certainly not true that all breast cancers can be lumped together\nmetabolically speaking. Even within TNBC there are major metabolic differences\nbetweentumorsand cell lines (Gong Cell Metabolism 2021).\na. -OR- congruent results need to be provided in two TNBC cell lines and then it can\nbe statedin textthe findings are fromER+andTNBC cell lines.\nb.Also, results section needs to mention whether ER+ cells were cultured in presence\nofE2and if charcoalstripped media was used.\nResponse:\nWe sincerely appreciate your insightful comment regarding the metabolic\nheterogeneity of breast cancers.Youare absolutely correct that \"it is certainly not true\nthat all breast cancers can be lumped together metabolically speaking. Even within\nTNBCthere aremajor metabolic differences between tumors and cell lines.\"\nAs you rightly noted,our in vitroexperiments were primarily conducted using the\nER+ MCF7 and T47D cell lines. Following your valuable suggestion, we have\nexplicitly stated in the relevant Results Section that these findings were\npredominantly reflect observations from these two ER+ breast cancer cell lines,\nensuring greater precision in our conclusions. For example, in the Results Section\nsubsection \"Lactate serves as a fuel for the TCA cycle in detached breast cancer\ncells\", we reported that \"Taken together, these results suggested that lactate is\npreferentially taken up by detached ER+ breast cancer cells (may be dependent on\nALDHstatus)andincorporated intothe TCAcycle as a fuel.\".\nFor our in vivo studies, we employed 4T1, two PDXs, and the MDA-MB-231\nmodels. As these represent triple-negative breast cancer (TNBC) models, we have\nalso duly noted this in the corresponding Results sections. Forexample, in the Results\nSection subsection \"Lactate induces stemness potentials in detached breast cancer\ncells\", we reported that \"Taken together, these findings suggest that lactate increases\nthe stemness potentials in detached triple-negative breast cancer (TNBC) cells,\nincluding lung metastatic cells and CTCs, and enhances the metastatic capacity of\nMICs.\".Therefore,we propose that lactate mainly functions inER+ cells,thoughits\neffects are notexclusive to ER+ cells.\nIn our study, ER+ cells were cultured in RPMI 1640 medium supplemented with\n10% standard FBS (untreated with charcoal stripping and without exogenous E2\nsupplementation). We have highlighted this information in yellow in the Methods\nSection under \"Cell lines and treatments\" as follows: \"All cells were routinely\nsupplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and\n2% L-glutamine. The ER+ MCF7 and T47D cells were untreated with charcoal\nstripping andwithout exogenous E2 supplementation.\".\nWe appreciate your attention to these methodological details above and hope\nthese clarifications address your question. Thank you again for your thoughtful\ncritique, which has helped refine ourmanuscript’s accuracy and transparency.\n6. Fig 1- the in vitro lactate data is from MCF7 but mouse data from 4T1. Congruent\ndatasets needto be added tobe able to comparein vitro and in vivo.\nResponse:\nWe sincerely appreciate your insightful feedback. To better align our in vivo and\nin vitro models, we have added additional lactate-related assays in 4T1 cells. The\nresults demonstrate that, under detachment conditions, 4T1 cells exhibit increased\nlactate uptake and upregulated expression of key molecules involved in lactate\nmetabolism, including MCT1 and LDHB. These findings are consistent with the\ntrends previously observed in MCF7/T47D cells (Updated Figure 1C & 1H below).\nThese data further validate the robustness and reproducibility of our findings across\ndifferent cellular models.\nResponseFigure33(UpdatedFigure1C&1H).(1C) Extracellularandintracellularlactate(lac)\nlevels inAR andAd MCF7/T47D/4T1 cells. (1H)MCT1 andLDHB protein levels inAR andAd\nMCF7/T47D/4T1 ALDH-, total, and ALDH+ cells. Data are represented as mean ± SEM.\nStatisticalanalysis:1C,ttest.**p<0.01,****p<0.0001.\n7. Throughout, I would avoid stressing xyz result ‘in metastatic cells’when you have\nnot compared them to non-metastatic cells. I would only mention metastasis when\nyouare specifically assaying metastasis ordirect metastasis phenotypes.\nResponse:\nWegratefully acknowledge yourinsightful feedback regarding the use of the term\n“metastatic cells” in our manuscript. You are absolutely correct that our original\nphrasing overemphasized findings in “metastatic cells” without direct comparison to\nnon-metastatic counterparts.\nIn response, we have carefully revised the text to restrict references to metastasis\nto contexts where metastasis-specific assays or phenotypes are explicitly discussed.\nFor instance, when describing lung nodules numbers—a direct measure of metastatic\ndissemination—we retained the term “metastasis”; similarly, in sections depicting the\ncorrelation between CD147/MCT1/LDHB complex and the metastasis of patients\nwith breast cancer, we also retained it. All other instances where “metastatic cells”\nwas used lacking comparative data or direct evidence of metastasis have been\nremoved orrephrased to ensure accurateoverinterpretation.\nThese revisions ensure our claims are fully data-supported and align with the\nrigorous standards you emphasized. We are deeply appreciative of your valuable\ncritique,which has significantly enhanced the clarityand precisionof ourmanuscript.\n8. Paragraph lines 175-188 stemness potential needs to be changed to stemness\n‘markers’or ‘proteins’as not functional assay is conducted (mammosphere formation\nprimary and secondary would be ideal).\nResponse:\nWe sincerely appreciate your insightful comment regarding the terminology in\nlines 175-188. As you rightly noted, our initial data demonstrated that lactate\ndose-dependently increased the ratios of ALDH+ and CD44highCD24-/low CSC\nsubpopulations and the number of tumorspheres (Figure S2A-B). However, the\nanalysis of lactate uptake/oxidation inhibition initially focused on stemness marker\nexpression withoutfunctional validationofstemness potential.\nTo address this concern, we have now supplemented the study with functional\nevidence by performing mammosphere formation assays. The updated Figure S3E-F\nbelow shows that inhibiting lactate uptake or oxidation using MCT1/LDHB inhibitors\nsignificantly suppressed stemness potentials. This suppression is evidenced by both\ndecreased ratios of the CD44highCD24-/low CSC subpopulation and reduced\ntumorsphere formation in lactate treated cells. Together with the data in Figure\nS2A-B, these new functional results substantiate our use of the term \"stemness\npotential.\"\nWe deeply thank you for this critical suggestion, which has significantly\nstrengthened the rigorand clarityofourmanuscript.\nResponse Figure 34 (Updated Figure S3E-F). (E-F) Ratios of CD44highCD24-/lowsubpopulation\n(E)or tumorspherenumbers(F)in48hours’0mM or20mM lactateincubatedMCF7/T47Dcells\npretreated with 1mM MCT1 inhibitor CHC or 1mM LDHB inhibitor GNE for 2 hours. Data are\nrepresentedasmean±SEM.Statisticalanalysis: one-wayANOVA.*p<0.05,**p<0.01,***p<\n0.001,****p<0.0001.\n9. No information about properties of the PDXs is provided- is there a citation in\nwhich they’ve been described before? If not, this publication must do the work of\npresenting basic characteristics (patient properties of donor biopsies; hormone\nreceptor status; treatment status and met status from patient; basic histology needs to\nbe shown). How the PDX identities were tracked and validated needs to be described\n(STR?)\nResponse:\nThank you for your question. In our study, the two PDX models were established\nfrom triple-negative breast cancer patients. Detailed characteristics of these PDXs\nhave been previously reported[41].\nTo maintain genetic fidelity, PDX1 and PDX2 were expanded in NOD-SCID\nmice and cryopreserved with 10 backup copies. For experimental purposes, PDX\nmodels were recovered from these backups and inoculated into mice, without being\nrefrozenpost-experiment. Specifically, the 6thand 7th generations of PDX1 and PDX2,\nrespectively, wereutilized in this study.Additionally, the identities ofthe PDX models\nwere tracked and validated using short tandem repeat (STR) profiling to ensure\nauthenticityand exclude contamination.\nIn response to your inquiry, we have now explicitly referenced the literature\ndescribing the properties of the PDXs and supplemented the text with detailed\nprotocols for tracking and validating their identities in the Methods Section under\n\"Patient-derived xenograft (PDX) models and tumor cell suspension separation\".\nThe revised content reads: \"Breast cancer PDX1/2 were obtained from Suling Liu’s\nlab[41]. Both PDX models were derived from triple-negative breast cancer patients.\nToensuregenetic fidelity andexclude contamination,the identities of the PDX models\nwere rigorously monitored through the establishment a backup library, control of\npassaging number,and performingshort tandemrepeat (STR)profilingspecificity.\".\n10.Primary tumorgrowthforFig 2needs tobe reported. Itis notclear if the affects of\nlactatewould equally impact primarytumor.\nResponse:\nThank you for raising this question. We have carried out a comprehensive\ninvestigation into how lactate impacts both primary tumor growth and metastasis.\nRegrettably, due to the space constraint, these data were not included in the submitted\nmanuscript. As shown in Response Figure 35 below, our data showed that\ntreating PDX1 or 4T1 tumor-bearing mice with lactate did not lead to any significant\nalterations in the growthrateofprimary tumors.\nFor the reason behind this phenomenon, we hypothesize that tumor cells within\nprimary tumors have sufficient glucose supply and thus predominantly utilize glucose\nas their energysource.In contrast,metastatic breast cancer cells have been reportedto\nhave the reduced capability of glucose uptake[4] and rely mainly on lactate, a\nmetabolic byproduct, to sustain their survival and maintenance their stemness.\nTherefore, it can be reasonably concluded that lactate primarily exerts its influence on\nthe formation of metastatic tumor, while having minimal effect on the growth of\nprimary tumor.\nResponse Figure35.Representative tumor images (up)andtumor growthcurve (down)of PDX1\n(A)and4T1 (B)tumor-bearingmice(n=5mice foreachgroup)with orwithout800mg/kglactate\nadministration. Data are represented as mean ± SEM. Statistical analysis: repeated measures\ntwo-wayANOVA.ns:notsignificant.\n11.Fig 4-I would expect that adding any carbonfuel tocell culture media would have\npotential to elevate oxphos, despite whatever preference the cells/mitochondria may\nhave. Fig 1 results indicate selectivity for lactate in these MICs, so authors should test\nglucose / glutamine / lipid as negative controls to see if OCR is elevated in the\nrelevant cell populations under investigation. Same goes for checking SOX2 protein\nlevel.\nResponse:\nThank you for your thoughtful question. In response, we have conducted\nadditional experiments using glucose, glutamine, and lipids as negative controls in\nMCF7/T47D ALDH- cells cultured under adherent (Ad) or anoikis-resistant (AR)\nconditions. As shown in Response Figure 36 below, under Ad conditions, exogenous\nsupplementation with glucose, glutamine, or lipids enhanced both OCR and SOX2\nexpression in breast cancer cells to varying degrees, whereas lactate had no significant\neffect on either parameter.\nIn contrast, under AR conditions, exogenous glucose, glutamine, or lipids\nsupplementation did not significantly alter OCR or SOX2 expression. Intriguingly, in\nT47D cells, exogenous lipid supplementation even suppressed both OCR and SOX2\nexpression. Strikingly, however, lactate supplementation under AR conditions\nmarkedly increased both OCR and SOX2 expression. These results indicate that\nbreast cancer cells preferentially utilize lactate as an energy source under AR\nconditions,further supporting ourearlier conclusions.\nResponse Figure 36. (A-B) OCR (A) and SOX2 protein levels (B) of Ad or AR MCF7/T47D\nALDH-cells culturedwith2.5mM glucose,2mMglutamine, 2mMlipidor20mM lactatefor48\nhours.Dataarerepresentedasmean±SEM.\n12. Results suggest that both oxphos and aKG-mediated histone methylation are\nimportant for the lactate driven stemness phenotype. Authors need to clarify- when\nthey inhibit ETC proteins, is aKG also reduced? Experiments to determine if both\nbranches are necessary to supportstemness are needed.Alternatively perhaps only the\nmethylation aspect is important and the elevated oxphos is a ‘side effect’of increased\nTCA cycling but the redox balance/ATP generation from oxphos is not actually\nimportant forthe CSC phenotype.\nResponse:\nWe appreciate your insightful questions regarding the respective roles of\nOXPHOS and α-ketoglutarate (α-KG) in mediating the lactate-induced stemness\nphenotype. To address these points, we first investigated whether inhibition of the\nETC affects intracellular a-KG levels. As shown in Response Figure 37A,\npharmacological inhibition of mitochondrial Complex I (rotenone) or Complex V\n(oligomycin) did not significantly alter α-KG levels in lactate-treated cells, suggesting\nthatOXPHOS disruptiondoes not impair α-KG availability.\nWe further compared the functional contributions of OXPHOS and a-KG in\nstemness maintenance. Intriguingly,Response Figure 37B-D demonstrate that D-2HG,\na competitive inhibitor of α-KG-dependent dioxygenases, more potently suppressed\nstemness phenotypes, including the ALDH+ CSC subpopulation, tumorsphere\nformation, and SOX2 expression, in lactate-treated breast cancer cells compared to\nrotenone. These findings indicate that α-KG-mediated epigenetic regulation is more\ncritical forsustaining stemness than OXPHOS-derived bioenergetics.\nConsistent with these observations, proteomic profiling revealed that carbon\nmetabolism and TCA cycle, rather than downstream OXPHOS, were commonly\nenriched in AR and lactate-treated MCF7/T47D cells (Figure 4A, Figure S4J).\nMechanistically, we found that α-KG directly attenuates DNMT3B binding to the\nSOX2 promoter(Figure 5 and Figure S5), inducing a DNAhypomethylation statethat\nfacilitates stemness.\nTaken together, our data support a model wherein lactate-induced stemness is\nprimarily governed by α-KG-dependent inhibition of DNA methylation, with\nOXPHOSplaying a secondary orsupportive role.\nResponse Figure 37. (A) α-KG levels in lactate treated MCF7/T47D cells cultured with 1 mM\nrotenone or 1 mM oligomycin for 48 hours. (B-D) ALDH+ subpopulations (B), tumorsphere\nnumbers (C), and SOX2 protein levels (D) in lactate treated MCF7/T47D cells cultured with 1\nmM D-2HG or 1 mM rotenone for 48 hours. Data are represented as mean ± SEM. Statistical\nanalysis:A-C,one-wayANOVA.**p<0.01,***p<0.001,****p<0.0001.ns:notsignificant.\n13. Fig6 TEM and IF indicate drastic change in mitochondrial structure and perhaps\nsubcellular localization upon lactate treatment. This should be quantified and\nreported.\nResponse:\nThank you very much for your meticulous review. Regarding the TEM image in\nFigure 6E, we admit that the image of AR group displayed a reduced number of\nmitochondrial cristae. However, after comprehensive examination and quantification,\nwe found no statistically significant difference in the quantity of mitochondrial cristae\n(Response Figure 38A). It is likely that the image presented in Figure 6E represents\nan isolated instance, probably due to inter-sample variation. To further substantiate\nthis, we present a TEM image of the AR group with normal mitochondrial cristae\n(Response Figure 38B).\nFor the fluorescence image in Figure 6F, the signals of CD147, MCT1, and\nLDHB in the lactate-treated group seem to indicate increased localization to\nmitochondria. Following your suggestion, we performed co-localization analysis of\nCD147, MCT1, LDHB with the mitochondrial marker COX IV respectively. The\nresults demonstrated enhanced co-localization of all three proteins with COX IV after\nlactate treatment (Response Figure 38C), suggesting an increase in their\nmitochondrial expression. This finding is consistent with the results we observed in\nFigure S7C.\nResponse Figure 38. (A) Quantification of mitochondrial cristae numbers in Figure 6E (n=30\nmitochondria per group). (B) Co-localization of CD147 (10 nm) with MCT1 (20 nm) or LDHB\n(20 nm) in mitochondrial inner membrane of AR MCF7 cells observed under TEM. Scale bars:\nleft, 1μm; right, 500 nm. Red asterisks show the mitochondria, and yellow arrows indicate the\nco-localization between CD147 and MCT1/LDHB. (C) Fluorescence colocalization analysis\nbetween CD147/MCT1/LDHB and COX IV in Figure 6F (n=30 fields from 8 mice per group).\nDataarerepresentedasmean±SEM.Statisticalanalysis:ttest.***p<0.001,****p<0.0001.\nMinorcomments\n1. Line 70- the statement ‘90% of tumor cells detached from the primary tumor’ is\nconfusing and is certainly not a generalizable fact. The cited review does not prove\nthis.\nResponse:\nWe sincerely appreciate your perceptive observation and deeply apologize for the\nconfusion brought about our initial phrasing. After a thorough re-examination of the\ncited review (Chambers et al., 2002[42], Page 7, Paragraph 2),we acknowledge that\nit fails supportouroriginal statement as clearly as we had anticipated.\nPreviously, Luzzi et al. reported that 83% of the initially injected B16F1 murine\nmelanoma cells left the primary site, entered the circulation and extravasated into the\nliver parenchyma.However, only less than 0.02%ofdisseminated tumor cells (DTCs)\nwere capable ofsuccessfully seeding metastases[43-45].\nIn response, we have carefully revised the controversial sentence. The original\nstatement \"During the metastatic cascade, 90% of tumor cells detached from the\nprimary tumor, but only 0.02% of them can survive and form growing metastatic foci\neffectively\" has been replaced with \"During the metastatic cascade, millions of tumor\ncells detached from the primary tumor, but very few of them (less than 0.02%) can\nsurvive and form growing metastatic foci effectively.\" This revision ensures strict\ncompliance with the referenced literature. The revised text now precisely reflects the\nconclusions ofthe sourcewhile upholding the scientific rigorofourintroduction.\nWe are truly grateful for your meticulous review, which has significantly\nimproved the clarity ofthe manuscript.\n2. Especially since this is a metabolism study, cells should be cultured in physiologic\nglucose concentration.The [gluc] is not reported in the methods. 2.5-5 nM is standard\ntoachieve near physiologic glucose level.\nResponse:\nWe sincerely appreciate your valuable feedback on our manuscript.We apologize\nfor overlooking the reporting of the glucose concentration ([gluc]) in the Methods\nSectionand foranyinconvenience this oversight may have caused.\nIn our study, we utilized RPMI 1640 medium for the routine culture of AR and\nAd cells. The glucose concentration in this medium is 17.5 mM (3.15 g/L), notably\nhigher than the 2.5-5 mM range you suggested for approaching near-physiologic\nglucose levels. We opted for RPMI 1640 due to its widespread use and\nwell-established status in the majority of cell culture research. It provides a stable and\nconsistent environment for investigations into cell growth and metabolism[46, 47].\nMoreover, our study primarily focuses on the functional role of lactate in relation to\nstemness (Because detached cells exhibit reduced glucose uptake[4], glucose is not\nthe primary energy substrate); thus, we did not deliberately adjust the glucose\nconcentrationin the RPMI 1640 media to be close tothe physiologic levels.\nTo mimic the nutrient-deprived metabolic state commonly associated with cell\ndetachment, we cultured the cells in glucose-free medium. Specifically, one group\nwas supplemented with 20 mM lactate (Lac 20 mM), while the other group had no\nlactatesupplementation (Lac 0mM).\nTo facilitate your and other readers' understanding of our cell line model, we\nhave revised the \"Cell lines and treatments\" subsection in the Methods Section. The\nrevised content are reads: \"MCF7, T47D, MDA-MB-231, and 4T1 cells were cultured\nin RPMI 1640 medium with 3.15g/L glucose.\" and \" One is the Low-Attachment\nModel. Cells surviving for 7 days in glucose-containing medium (3.15g/L) under\ndetached suspension culture conditions in ultra-low attachment plates were\nconsidered anoikis-resistant (AR) cells, while cells cultured in uncoated common\nplates under attached conditions with glucose-containing medium (3.15g/L) served as\ncontrol adherent (Ad) cells[3].\" We are confident that this additional information will\nenhance the comprehensiveness and reliabilityof ourresearch.\nThank you once again for your constructive comments, which have played a\ncrucialrole in improving the quality ofourwork.\n3. Incorporation of lactate into TCA cycle has been reported in TNBC cells and\nshould be mentioned, in the context of therapeutic stress. Potential similarities\nbetween stress of therapy and of metastasis should be discussed (Baek et al Oncogene\n2023).\nResponse:\nThank you very much for your valuable suggestion. As recommended, we have\nexpanded the Discussion Section tocover two keypoints:(1)\"Incorporationof lactate\ninto the TCAcycle in TNBC cells under therapeutic stress has been reported\", and (2)\n\"the similarities between the stress oftherapyand ofmetastasis\".\nThe specific additions are highlighted in yellow in the third paragraph of the\nDiscussion Sectionas follows:\n\"Of note, Baek et al. revealed that under chemotherapy-induced stress, lactate\nincreasingly enters the TCA cycle of TNBC cells, accompanied by enhanced\nmitochondrial OXPHOS[48]. In fact, there are substantial similarities between\ntherapeutic stress and metastasis stress. First, tumor cells encounter comparable\nsurvival challenges during both therapeutic stress and metastasis, which may trigger\nmetabolic plasticity that drives a shift from glycolytic metabolism to OXPHOS to\nsustain cell survival[49, 50]. Thus, our observation that \"lactate fuels OXPHOS\nmetabolism in anoikis-resistant breast cancer cells via TCA cycle entry\" further\nstrongly support the findings by Baek et al. Second, CSCs play a role in both therapy\nresistance and metastatic dissemination[51, 52]. The research by Baek et al. on\ntherapy stress-induced metabolic reprogramming offers a mechanistic basis for\nunderstanding how does lactate regulate stemness potential of breast cancer cells\nduring metastasis.\"\nReferences\n1. Bu,J.,etal.,KK-LC-1asatherapeutic targettoeliminateALDH(+)stemcellsintriple\nnegativebreastcancer.NatCommun,2023.14(1):p.2602.\n2. 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Gerstberger,S.,Q.Jiang,andK.Ganesh,Metastasis.Cell,2023.186(8):p.1564-1579.\n52. Shibue,T.andR.A.Weinberg,EMT,CSCs,anddrugresistance:themechanisticlinkand\nclinicalimplications.NatRevClinOncol,2017.14(10):p.611-629.\nResponses to the reviewers’comments\nManuscriptID:NCOMMS-24-84609B\nQuestions and answers:\nReviewer#1 (Remarks totheAuthor):\nGeneral: The authors have done a comprehensive job of addressing the comments\nfromthe first rebuttal.\nResponse:\nThank you once again for reviewing our manuscript and for your valuable\nfeedback, which has significantly improved the quality of our work. In this revision,\nwe have carefully addressed all your comments and incorporated the suggested\nchanges into the manuscript. Below,we provide a point-by-point responses to each of\nyourrecommendations.\nCommentswiththe same numbering system ofthe original review and rebuttal:\n2. We appreciate the additional clarification. The authors state the results indicate\nlactate’s effects on stemness in vitro are not determined by glucose availability, but\nrather by cellular attachment status.This is contradicted by the data. In the absence of\nglucose, lactate increases the ALDH+ population in breast cancer tumorspheres\n(Figure S2A), but in the presence ofa physiological level ofglucose, lactate decreases\nthe percentage ofALDH+ cells (original Response Figure 1). This suggests lactate has\nopposing effects on stemness in vitro depending on the availability of glucose. Forthe\nfinal manuscript the authors should include the ALDH+ population percentage with\nand without lactate in the presence and absence of glucose for the breast cancer\ntumorspheres forclarity.\nResponse:\nThank you for your question, and we sincerely apologize for the lack of clarity in\nourinitial explanationregarding the context-dependenteffect oflactateonstemness in\nvitro. We fully agree with your observation that lactate exerts the opposing effects on\nstemness in vitro depending on the availability of glucose. The dual role can be\nexplained as follows:\nUnder glucose-depleted conditions, lactate serves as a critical alternative\nOXPHOSsubstrate,thereby enhancing stemness potential in detached cells.\nConversely, in our glucose-replete conditions (RPMI1640 medium with 17.5 mM\nglucose—far exceeding the upper limit of physiological blood glucose levels of ~7.8\nmM), exogenous lactate does not function as an efficient alternative fuel source.\nInstead, it likely inhibits glucose utilization via negative feedback, exacerbating\nenergy stress and metabolic imbalance caused by glycolysis, ultimately reducing the\nproportion of ALDH⁺ cells (updated Figure S2A). Notably, endogenous lactate levels\nin this condition are already high, suggesting that cells may be near saturation for\nlactate uptake and utilization. Consequently, additional exogenous lactate leads to\nextracellular lactate accumulation, significantly acidifying the tumor\nmicroenvironment (low pH). This acidosis can suppress overall cellular metabolism,\nimpair cell survival, and indirectly compromise stemness maintenance[1,2].\nThis opposite effect highlight the metabolic plasticity of TICs or CSCs,\nparticularly during the metastatic cascade under detachment-induced glucose uptake\nimpairment[3],as demonstratedin ourstudy.\nWe sincerely appreciate your valuable suggestions. In response, we have now\nadded the data of ALDH+ population percentage (with and without lactate in\nglucose-containing medium) into Figure S2A. The revised text now reads:\n\"Incubation of breast cancer cells in glucose-free medium supplemented with lactate\ninduced a dose-dependent increase in ALDH+ and CD44highCD24-/low CSC\nsubpopulations, as well as tumorsphere formation, with the most pronounced effects\nobserved at 20 mM lactate (Fig S2A-B). In contrast, these effects were attenuated in\nglucose-containing conditions.\" (Page 7,Paragraph 1,Line 179-182)\nIn addition, we have now included in our manuscript a discussion on the\ndifferential effects of lactate on stemness depending on the availability of glucose,\nas follows:\n\"Notably, lactate exerts context-dependent regulatory effects on stemness,\neither\nsuppressing or promoting it according to glucose availability (Figure S2A). These\nfindings highlightthe metabolicplasticity of tumor-initiatingcells, particularly during\nmetastasis, a process characterized by detachment-induced glucose limitation[3].\nIntriguingly, under such glucose-deprived conditions, lactate may shift from an\ninhibitory to a supportive role in maintaining stemness during metastasis.\" (Page 14,\nParagraph 2,Line 470)\n10. Response figure 5 demonstrating increased lactylation of NDUFS1 is interesting.\nThe text of the manuscript lines 251-260 should reflect these findings. While the\nrebuttal response is adequate, the manuscript states lactate predominantly induces\nstemness potential by providing fuel for the TCA cycle and thus promoting oxphos.\nThis is contradicted by the isolated complex 1 assay which is not dependent on\nNADH generation by the TCA cycle. The text should be changed to reflect the\nNDUFS1 lactylation results as a potential explanation for the enhanced Complex I\nactivity observed in lactate treated cells. This should be sufficient to address this\nissue.\nResponse:\nWe sincerely appreciate your insightful suggestion and deeply regret that the data\non increased NDUFS1 lactylation were not reflected in the text of manuscript. In\nresponse to this valuable advice, we have incorporated these findings into Figure S4N\nand revised the manuscript text (Lines 251-260) as follows: \" Notably,while complex\nI assembly and mitochondrial mass remained unchanged, we observed significant\nupregulation of pan-Kla (lysine lactylation) on the complex I subunit NDUFS1 (Fig\nS4L-N). In contrast, the suppression of lactate utilization by MCT1/LDHB inhibitors\nor siRNAs significantly weakened ATP production, OCR, and complex I activity in\nlactate-treated cells (Fig 4F-H, Fig S4O-P). Moreover, the ATP synthase inhibitor\noligomycin and the complex I inhibitor rotenone both significantly decreased the\nratios of ALDH+ or CD44highCD24-/low CSC subpopulations (Fig 4I-J), tumorsphere\nnumbers (Fig 4K), and SOX2 expression in AR and lactate-treated ALDH- or total\ncells (Fig 4L). Taken together, these data suggest that lactate induces stemness\npotentials in detached ER+ breast cancer cells by enhancing the Complex I activity\nandlactylation ofits critical subunitNDUFS1.\"\nComment onnew text\n1. Lines 209-214 in the revised manuscript and corresponding figures S3 O-P. No\nevidence is provided for a paracrine lactate flux as described in the text and this\nshould be omitted. The oxidative and glycolytic regions of the tumors may simply\nexist dueto localdifferences in perfusion and oxygen availability.\nResponse:\nWe sincerely thank you for your insightful and constructive feedback. And, we\nfully agree with you that \"No evidence is provided for a paracrine lactate flux as\ndescribed in the text\", and acknowledge that our initial interpretation of the data was\nsomewhat overstated. In fact, the data presented in Figures S3O-P were incorporated\nat the specific request of another reviewer.Topresent our findings in a more objective\nand accurate manner, we have revised the text in Lines 209-214 as follows:\n\"Interestingly, within tumor tissues of 4T1-bearing mice, we observed spatially\ncoexistence of lactate-producing glycolytic tumor cells and lactate-importing\noxidative tumor cells. Notably, oxidative tumor cells displayed significantly higher\nMCT1 and SOX2 expression (Fig S3O-P), implying that these oxidative cells might\nenhance their stemness potentialthroughlactate consumption.\"\nReviewer#2 (Remarks totheAuthor):\nIntheir second revision, the authors have addressed all my concerns.\nPlease, note that there is a mistake in Figure 6L.The zoomed image (right panel) does\nnotmatch the originalimage (middle panel).\nResponse:\nWe would like to express our most sincere and profound gratitude once again for\nyour meticulous review of our manuscript. We truly value your astute observation in\npointing out this error and deeply apologize for this oversight. In response, we have\nrectified the image (middle panel)in Figure 6L.\nReviewer#3 (Remarks totheAuthor):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nResponse:\nWe sincerely extend our heartfelt gratitude to you once again for reviewing our\nmanuscript.Yourvaluable comments have significantly elevated the overall quality of\nourwork.\nReviewer#4 (Remarks totheAuthor):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nResponse:\nWe sincerely thank you once again for your guidance and support throughout the\nreview process. Your thoughtful and constructive comments have been of\nimmeasurable value, leading to a substantial enhancement in the overall quality of our\nmanuscript.\nReferences\n1. Michl, J., et al., CRISPR-Cas9 screen identifies oxidative phosphorylation as\nessential for cancer cell survival at low extracellular pH. Cell Rep, 2022.\n38(10):p.110493.\n2. Ge, H., et al., Targeting ASIC1a Promotes Neural Progenitor Cell Migration\nand Neurogenesis in Ischemic Stroke.Research (Wash DC),2023.6: p.0105.\n3. Uen, W., et al., Detachment stress mediated bioenergetic switch of malignant\nmelanoma cells into anti-Warburg phenotype. Aging (Albany NY), 2022.\n14(13):p.5511-5522."}]

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