Compression-induced NF-κB activation sustains tumor cell survival in confinement by detoxifying aldehydes and promotes metastasis

Domain: biology
Published: 2025-12-14
Editorial outcome: major_revision

Abstract
Metastasis remains the primary cause of cancer-related mortality. During dissemination, cancer cells must navigate spatially confined microenvironments, yet the underlying metabolic adaptations that facilitate this process remain unclear. Here, through an in vivo CRISPR screen targeting metabolic enzymes, we identify aldehyde dehydrogenase 1 family member B1 (ALDH1B1) as essential for tumor cell survival in confining capillaries. Mechanistically, compressive force induces casein kinase 2 alpha 3 (CSK23) to phosphorylate kappa-B kinase subunit beta (IKKβ) at Ser177/181, which activates the nuclear factor kappa B (NF-κB) pathway and upregulates ALDH1B1. The upregulation of ALDH1B1 enhances aldehyde detoxification, which suppresses ferroptosis and promotes tumor cell survival during migration through the capillaries, thereby facilitating metastasis. Importantly, genetic or pharmacological inhibition of CSK23 or ALDH1B1 effectively impairs metastasis. In lung cancer patients, confined tumor cells exhibit higher levels of ALDH1B1 and NF-κB activation, which correlates with metastatic recurrence. Our findings reveal a mechano-metabolic pathway that promotes metastasis and suggest CSK23 and ALDH1B1 as potential therapeutic targets.

Introduction
Tumor metastasis, the movement of tumor cells from a primary site to colonize distant organs, is a significant factor in the mortality of cancer patients1. The metastatic cascade is an intricate multi-step journey that requires cancer cells to first dissociate from the primary tumor, intravasate into circulation, survive transit, extravasate at distant sites, and ultimately colonize secondary tissues2. A pivotal component of this process is cell migration, which requires navigating spatially complex microenvironments3. The capacity of cancer cells to migrate within physically restricted compartments represents a critical mode of in vivo movement, such as through pores within the tumor extracellular matrix (ECM) or pre-existing tunnel-like tracks, including paths along interstitial ECM fibers, the spaces between muscle and nerve fibers, routes adjacent to or within blood vessels, and the vascular networks of distant organs4–8. Consequently, a detailed understanding of the molecular machinery controlling this confined migration is vital for designing therapeutic approaches to block metastatic dissemination.

Cancer cells frequently alter their metabolic processes to sustain cell growth and viability9. Emerging evidence shows that metastasizing cancer cells adapt their metabolism selectively and dynamically at each step of the metastatic process10. For instance, the expression of glutamate dehydrogenase 1 (GDH1) is upregulated in circulating tumor cells (CTCs), which contributes to anti-anoikis signals and promotes metastatic behavior in liver kinase B1 (LKB1)-deficient lung cancer11. During the colonization of distant organs, breast cancer cells induce the production of α-ketoglutarate-activated collagen hydroxylation to drive collagen-based ECM remodeling in the lung metastatic niche12. We recently demonstrate that UDP-glucose 6-dehydrogenase (UGDH) enhances the stability of snail family transcriptional repressor 1 (SNAI1) mRNA to initiate the epithelial-mesenchymal transition, thus promoting the extravasation process of lung cancer cells. In contrast, it is mostly unknown how tumor cells rewire metabolism to support their migration through the spatially-confining environment, thereby accomplishing their seeding to distant organ.

In this study, we performed the in vivo CRISPR loss-of-function screening in lung cancer cells with sgRNA targeting 1685 metabolic enzymes and identified that aldehyde dehydrogenase 1 family member B1 (ALDH1B1) is required for tumor cell survival in confining capillaries. ALDH1B1 is an aldehyde dehydrogenase that detoxifies aldehydes into corresponding carboxylic acids in cells13. We demonstrate that ALDH1B1 expression enhances aldehydes detoxification and suppresses ferroptosis in confined cells, which support tumor cell survival during their migration in confining capillaries, thereby promoting lung cancer metastasis. In response to compressive force, casein kinase 2 alpha 3 (CSK23) activates nuclear factor kappa B (NF-κB) pathway to upregulate ALDH1B1 expression.

Results
ALDH1B1 is essential for tumor cell survival in capillaries and subsequent distant metastasis
To identify the metabolic enzyme that is required for tumor cell migration in confining spaces in vivo, we conducted CRISPR knockout screening using lung adenocarcinoma-derived A549 cells-an epithelial cell line with robust migratory and invasive capacities14,15. Cells were transduced with a gRNA library targeting 1685 metabolic enzymes (Supplementary Data 1) with a lung metastasis mouse model via tail vein injection. 30 min after injection, almost all tumor cells have reached the lung tissues, 24 h after implantation, over 90% of tumor cells remained within the capillaries of lung tissues and approximately 20% of these cells survived in lung capillaries and were responsible for subsequent extravasation and distant metastasis16–20. GFP-expressing living tumor cells were then isolated from dissected lung tissues and gRNA abundance was quantified by the next-generation sequencing (NGS) (Fig. 1a, Supplementary Fig. 1a, b, and Supplementary Data 2). 57 metabolic enzymes were identified to be required for tumor cell survival in confining capillaries (Fig. 1b, c). 10 candidate genes were selected for further investigation, including kynureninase (KYNU), phenylalanine hydroxylase (PAH), phosphatidylinositol-4-phosphate 3-kinase C2 domain-containing subunit alpha (PIK3C2A), myotubularin-related protein 8 (MTMR8), 6-pyruvoyltetrahydropterin synthase (PTS), diacylglycerol kinase iota (DGKI), L-histidine phosphatase (LHPP), aldehyde dehydrogenase 1 family member B1 (ALDH1B1), retinol dehydrogenase 12 (RDH12) and methylthioribose-1-phosphate isomerase 1 (MRI1) (Supplementary Fig. 1c).

We next evaluated the association of these 10 candidate genes with lung cancer metastasis by using the Gene Expression Omnibus (GEO) dataset (GSE72094), which showed that PTS, DGKI, and ALDH1B1 mRNA levels were strongly correlated with the malignancy and prognosis of lung cancer patients (Supplementary Fig. 1d, e). Furthermore, we defined the relationship between these 3 genes and metastatic recurrence by utilizing the GEO dataset (GSE37745). Among them, ALDH1B1 expression was higher in patients with metastatic recurrence compared to those without metastatic recurrence, while PTS and DGKI showed no differences in their expression between these patients (Fig. 1d and Supplementary Fig. 1f), suggesting that ALDH1B1 may play an important role in tumor metastasis. To investigate whether ALDH1B1 promotes tumor cell survival in confining spaces, we simulated confinement scenario in vitro by adopting a transwell chamber with 8μm pores (Supplementary Fig. 2a). We depleted ALDH1B1 with short hairpin RNA (shRNA) in A549 cells (Supplementary Fig. 2b), and found that ALDH1B1 depletion increased tumor cell death under confinement but did not significantly affect cell survival in unconfinement conditions (Supplementary Fig. 2c). In addition to ALDH1B1, we identified ALDH9A1 and ALDH6A1 as secondary candidates among the 18 ALDH isoforms in our CRISPR-based screen (Supplementary Fig. 2d). We established A549 cells with ALDH6A1 or ALDH9A1 depletion (Supplementary Fig. 2e, f) and found that ALDH9A1 depletion increased tumor cell death in confining pores (Supplementary Fig. 2g), though its effect was weaker than that of ALDH1B1 ablation. In contrast, ALDH6A1 depletion showed no significant effects on confined tumor cell survival (Supplementary Fig. 2h). Transcriptomic analysis of the GSE288929 dataset further demonstrated that baseline ALDH1B1 expression exceeded ALDH9A1 levels in A549 cells (Supplementary Fig. 2i), potentially explaining the dominant functional role of ALDH1B1 in confining spaces. Collectively, among ALDH isoforms, ALDH1B1 is identified as the primary regulator governing tumor cell survival under spatial confinement.

During metastatic dissemination, tumor cells must navigate two critical physical constraints, including narrow capillary lumens in distant organs and endothelial gaps during intravasation and extravasation3. To further confirm that ALDH1B1 contributes to tumor cell survival in the vasculature of distant organ, we implanted luciferase-expressing A549 cells with or without ALDH1B1 into female BALB/c athymic nude mice via tail vein injection. At 0.5 h post tail vein injection, bioluminescence imaging of the mice showed comparable tumor cell signals between the two groups. At 24 h post-injection, the ALDH1B1-depleted group showed accelerated diminishment of bioluminescence signals from tumor cells in lung tissues (Fig. 1e). Moreover, these mice were sacrificed and lung tissues were dissected at 24 h after injection. TUNEL assay on dissected lung tissues showed that ALDH1B1 depletion increased tumor cell death in lung capillaries (Fig. 1f). In addition, we also investigated whether ALDH1B1 regulates tumor cell survival during extravasation through endothelial gaps. Most surviving tumor cells begin extravasation between 24 and 36 h after intravenous injection20–22. Since visualizing the survival of cells trapped within endothelial gaps proved challenging, we instead quantified both the cells remaining in capillaries and those that had completed extravasation 48 h after injection, when most cells completed extravasation. Immunofluorescence analysis showed that ALDH1B1 depletion led to a twofold reduction in tumor cells retained within capillaries and a fivefold decrease in extravasated tumor cells (Fig. 1g), suggesting that ALDH1B1 likely also contributes to the survival of tumor cells during extravasation.

Furthermore, we implanted these cells into female BALB/c athymic nude mice via intracardiac injection. Bioluminescence imaging of the mice showed that ALDH1B1 depletion accelerated the diminishment of bioluminescence signals of tumor cells in brain 7 days after inoculation, and dampened brain metastasis of the cells 63 days after inoculation (Fig. 1h and Supplementary Fig. 2j). Hematoxylin eosin (H&E) staining of dissected brain tissues confirmed that ALDH1B1 depletion reduced the number and size of brain metastatic lesions (Fig. 1i). Consequentially, the mice bearing with ALDH1B1-depleted tumors survived longer (Fig. 1j). These results demonstrate that ALDH1B1 is required for tumor metastasis at least partially by enhancing tumor cell survival within capillaries and during extravasation.

ALDH1B1 sustains tumor cell survival in confining spaces by detoxifying aldehydes
To investigate how ALDH1B1 promotes tumor cell survival in confining spaces, we depleted ALDH1B1 in A549, H1299 cells, or H460 cells and rescued the cells with wild-type ALDH1B1 (WT) or ALDH1B1 enzymatic-dead (ED) mutant, which indeed had much lower activity than WT (Supplementary Fig. 3a–d). While rescued expression of ALDH1B1 WT almost completely restored the survival and migratory capability of ALDH1B1-depleted cells in confining pores, excluding the off-target possibility of ALDH1B1 shRNA. However, tumor cells rescued with ALDHB1 ED could not recover the survival and the migratory capability of ALDH1B1-depleted cells in confinement (Fig. 2a, b and Supplementary Fig. 3e–h). We also examined the survival of tumor cells in polydimethylsiloxane (PDMS) microchannels and found that ALDH1B1 depletion increased tumor cell death in these channels (Supplementary Movies 1–4). These results indicate that ALDH1B1 activity was essential for tumor cell survival and subsequent migration in confining spaces.

ALDH1B1 has previously been reported to metabolize aliphatic aldehydes, aromatic aldehydes, and the products of lipid peroxidation, 4-hydroxynonenal and malondialdehyde. Moreover, ALDH1B1 exhibited a higher affinity for short-chain and medium-chain aliphatic aldehydes than other type aldehydes13. We thus examined the levels of ALDH1B1 substrate aldehydes, including acetaldehyde, propionaldehyde, hexanal, and nonanal. The levels of these aldehydes were increased in confined cells and were further upregulated in the cells after ALDH1B1 depletion (Fig. 2c and Supplementary Fig. 3i). We further treated tumor cells expressing shNT or shALDH1B1 in confinement with or without aldehyde scavenger Acloproxalap23. Acloproxalap almost completely recovered the survival and the migratory capability of ALDH1B1-depleted confined cells (Fig. 2d, e and Supplementary Fig. 3j, k). Of note, wound healing assays showed that ALDH1B1 depletion did not influence the mobility of these cells under no confinement (Supplementary Fig. 4a, b). Similarly, both the proliferation and the survival of tumor cells were not influenced by ALDH1B1 depletion under no confinement (Supplementary Fig. 4c–e). These results demonstrate that ALDH1B1-catalyzed aldehydes detoxification is required for tumor cell survival in confinement.

Ferroptosis is a non-apoptotic cell death mechanism characterized by iron-dependent membrane lipid peroxidation24,25. Recently, accumulating evidence indicates that aldehydes can damage ferroptosis-associated proteins and exacerbate intracellular oxidative stress and damage to the cell membrane, accelerating lipid peroxidation and leading to ferroptosis26,27. To investigate how ALDH1B1 regulates the survival of confined cells, we thus tested whether ALDH1B1 regulates the ferroptosis. Confinement significantly increased lipid peroxidation in tumor cells, and depletion of ALDH1B1 further exacerbated these effects (Supplementary Fig. 5a, b). Additionally, ALDH1B1 depletion decreased the GSH/GSSH ratio in confined cells, while Acloproxalap greatly abrogated these changes in ALDH1B1-depleted cells (Fig. 2f, g and Supplementary Fig. 5c, d). Ferroptosis is normally inhibited by the continuous activity of coupled enzyme-metabolite systems that prevent the accumulation of membrane lipid peroxides to toxic levels. The enzyme glutathione peroxidase 4 (GPX4) reduces potentially toxic lipid hydroperoxides to less dangerous lipid alcohols. GPX4 appears to be the most critical enzyme for preventing lipid hydroperoxide accumulation in most cells28–30. Thus, we overexpressed GPX4 in ALDH1B1-depleted cells (Supplementary Fig. 5e, f). GPX4 overexpression recovered the survival and the migration of ALDH1B1-depleted cells in confining spaces (Fig. 2h, i and Supplementary Fig. 5g, h). 4-Hydroxynonenal (4-HNE) is a well-established marker of aldehyde metabolism and a known substrate of ALDH1B113,31, directly reflecting intracellular aldehyde accumulation and lipid peroxidation32. Immunofluorescence analysis of mouse lung tissues harvested 24 h after tail vein injection showed that ALDH1B1 depletion significantly increased 4-HNE levels in tumor cells confined within lung capillaries (Fig. 2j). In addition, given that aldehydes also induce apoptosis33, we examined whether ALDH1B1 regulates apoptosis under confinement by measuring caspase-3/7 activity. Caspase-3/7 activity was elevated in confined cells, and this effect was further exacerbated by ALDH1B1 depletion (Supplementary Fig. 5i). However, compared to its effect on ferroptosis, ALDH1B1 depletion had a much weaker influence on the apoptosis of confined tumor cells. These results are consistent with previous studies demonstrating that aldehydes induce both apoptosis and ferroptosis26,33, and that aldehydes derived from ALDH inhibition tend to promote ferroptosis20,27,34. Collectively, these results suggest that ALDH1B1 plays a dual protective role in confined tumor cells by suppressing both ferroptosis and apoptosis, with its anti-ferroptotic effect being mechanistically dominant.

Given the necessity of ALDH1B1 activity for the survival of the confined cells, we next evaluated the therapeutic potential of ALDH1B1 inhibitor for lung cancer metastasis. Firstly, we treated A549 or H1299 cells with ALDH1B1 inhibitor, IGUANA-135. Transwell migration assay showed that IGUANA-1 suppressed the confined migration of these cells in a dose-dependent manner without influencing their proliferation (Fig. 2k and Supplementary Fig. 5j–l). Moreover, we implanted luciferase-expressing A549 cells into randomized female BALB/c athymic nude mice via intracardiac injection, followed by the administration of IGUANA-1. Bioluminescence imaging of the mice showed that IGUANA-1 treatment dampened brain metastasis of lung cancer cells (Fig. 2l and Supplementary Fig. 5m). H&E staining of dissected brain tissues confirmed that IGUANA-1 reduced the number and size of brain metastatic lesions (Fig. 2m). Similarly, the survival duration of tumor-bearing mice was extended by IGUANA-1 treatment (Fig. 2n).

Compressive force induces ALDH1B1 upregulation in confined cells
To investigate how ALDH1B1 is regulated in tumor cells during confined migration, we examined ALDH1B1 expression in confined cells (Fig. 3a). The mRNA and protein levels of ALDH1B1 were increased in confined cells (Fig. 3b, c and Supplementary Fig. 6a, b). Cells are subjected to compressive force in confined environments36. Multiple mechanosensitive calcium channels, including Piezo-type mechanosensitive ion channel (PIEZO), Orai calcium-release-activated calcium modulator (ORAI), and transient receptor potential vanilloid (TRPV) members, play important roles in cancer biology, though their activation mechanisms differ significantly37. PIEZO1 is a well-characterized mechanosensitive ion channel that directly converts extracellular mechanical forces such as shear stress and membrane stretch into Ca²⁺ signals through plasma membrane deformation38–40. ORAI1 mediates store-operated calcium entry (SOCE) and is activated indirectly upon endoplasmic reticulum (ER) calcium depletion via stromal interaction molecule 1 (STIM1) coupling, rather than through direct mechanical stimulation41–43. TRPV4, meanwhile, functions as a polymodal sensor that responds to both mechanical and chemical stimuli44,45. Among these, PIEZO1 stands out as the primary channel responsible for rapid, force-dependent calcium signaling in response to physical perturbation. We thus depleted PIEZO1 in A549 and H1299 cells (Supplementary Fig. 6c, d). PIEZO1 depletion abrogated the upregulation of ALDH1B1 mRNA and protein levels in confined cells (Fig. 3d, e and Supplementary Fig. 6e, f). Meanwhile, we also depleted ORAI1 or TRPV4 in A549 cells (Supplementary Fig. 6g, h). In contrast, ORAI1 depletion had no significant effect on ALDH1B1 expression in confined tumor cells (Supplementary Fig. 6i, j), while TRPV4 depletion partially abrogated ALDH1B1 upregulation (Supplementary Fig. 6k, l), though unlike PIEZO1 depletion, which completely abrogated ALDH1B1 upregulation in confined tumor cells. These results suggest that under spatial confinement, PIEZO1 may serve as the primary mediator, and implicate that mechanical force drives ALDH1B1 upregulation in confined tumor cells.

To test the hypothesis, we applied direct compressive force to tumor cells and examined ALDH1B1 expression, which showed that the mRNA and protein levels of ALDH1B1 were upregulated in the cells after compression in a dose and time-dependent manner (Fig. 3f–h and Supplementary Fig. 6m–t). We also examined ALDH9A1 expression under compression and revealed modest but statistical upregulation of ALDH9A1 under compression, However, this mechanosensitive upregulation was attenuated compared to ALDH1B1’s response (Supplementary Fig. 6u, v). ECM stiffness has been widely utilized as a key biomechanical parameter to mimic tissue-specific mechanical microenvironments in vitro46,47. To determine whether matrix stiffness regulates ALDH1B1 expression, we tested distinct ECM stiffness regimes based on reported lung tissue mechanics48. We found no stiffness-dependent modulation of ALDH1B1 expression, suggesting that its regulation is independent of matrix stiffness (Supplementary Fig. 6w).

Next, we investigated whether PIEZO1 contributes to compression-induced ALDH1B1 expression. PIEZO1 depletion abrogated the increase in ALDH1B1 mRNA and protein expression in the cells after compression (Fig. 3i, j and Supplementary Fig. 6x, y). Moreover, PIEZO1 depletion increased tumor cell death in confining pores (Fig. 3k). Animal experiments showed that PIEZO1 depletion accelerated the diminishment of bioluminescence signals of tumor cells in the lungs (Fig. 3l). TUNEL assay with these lung tissues showed that PIEZO1 depletion increased tumor cell death in lung capillaries (Fig. 3m). Additionally, PIEZO1 depletion inhibited ALDH1B1 expression in tumor cells confined within lung capillaries (Fig. 3n).

Collectively, these results demonstrate that the expression of ALDH1B1 is increased in tumor cells during confined migration in response to compressive force.

NF-κB upregulates ALDH1B1 expression upon compression
Given that the mRNA levels of ALDH1B1 is upregulated upon compression, we further explored how the transcription of ALDH1B1 is regulated. We constructed a luciferase reporter system containing ALDH1B1 promoter (ALDH1B1-Luc) and transfected the construct into A549 cells and H1299 cells. As shown in Fig. 4a and Supplementary Fig. 7a, the activity of ALDH1B1 promoter was enhanced in the cells upon compression. To identify the upstream signaling that regulates ALDH1B1 expression, we treated A549 cells with the inhibitors of signal pathways that have been shown to play the crucial role in cancer progression, Ravoxertinib (ERK inhibitor), Mirdametinib (MEK1/2 inhibitor), MK-2206 (AKT inhibitor), Verteporfin (YAP inhibitor), and PDTC (NF-κB inhibitor). Among them, only NF-κB inhibitor abrogated compressive force-induced increase of ALDH1B1 protein levels (Fig. 4b and Supplementary Fig. 7b). Furthermore, the depletion of RelA, the key transcription factor of NF-κB pathway, abrogated compressive force-induced increase in the promoter activity, the mRNA levels and the protein levels of ALDH1B1 (Fig. 4c–f and Supplementary Fig. 7c–f). In addition, the mutation of RelA-binding motif in ALDH1B1 promoter abrogated compression-induced increase in its promoter activity (Fig. 4g and Supplementary Fig. 7g). Chromatin immunoprecipitation (ChIP) assay showed that RelA was recruited to ALDH1B1 promoter upon compression (Fig. 4h and Supplementary Fig. 7h). These results demonstrate that RelA regulates ALDH1B1 promoter activity to increase its expression in tumor cells under compression.

To confirm whether RelA regulates ALDH1B1 expression to detoxify aldehydes in confined cells, we overexpressed ALDH1B1 in RelA-depleted cells (Fig. 4i and Supplementary Fig. 7i). The levels of aldehydes increased in RelA-depleted confined cells, while ALDH1B1 overexpression reduced the levels of the aldehydes in these cells (Fig. 4j). Similarly, lipid peroxidation levels were increased in confined cells after RelA depletion, while ALDH1B1 overexpression almost completely abrogated such increase in RelA-depleted cells (Fig. 4k and Supplementary Fig. 7j). Moreover, the survival and the migratory capability of RelA-depleted cells were also greatly recovered by ALDH1B1 overexpression (Fig. 4l, m and Supplementary Fig. 7k, l), whereas RelA depletion showed no significant impact on lipid peroxidation or survival in unconfined tumor cells (Fig. 4k, l and Supplementary Fig. 7j, k). These results elaborate that NF-κB upregulates ALDH1B1 expression to inhibit ferroptosis in confined cells, thereby promoting confined migration of tumor cells.

Compressive force activates IKKβ and NF-κB via PIEZO1
In most cell types, NF-κB complexes are retained in the cytoplasm by a family of inhibitory proteins known as inhibitors of NF-κB (IκBs). Activation of NF-κB typically involves the phosphorylation of IκB by the IκB kinase (IKK) complex, which results in IκB degradation. This releases NF-κB and allows it to translocate freely to the nucleus49. We next investigated how NF-κB is activated upon compression. Both spatial confinement and mechanical compression greatly increased IKKβ serine (S) 177/181 phosphorylation (pS177/181) that is essential for canonical IKKβ activation as well as the S32/36 phosphorylation and degradation of IκBα (Fig. 5a, b and Supplementary Fig. 8a, b). The levels of nuclear RelA were also increased upon compression (Fig. 5c and Supplementary Fig. 8c). Furthermore, we constructed luciferase reporter system containing the responsive elements of NF-κB and observed that NF-κB was activated after compression (Fig. 5d and Supplementary Fig. 8d). Importantly, we depleted IKKβ in tumor cells (Supplementary Fig. 8e, f) and observed that depletion of IKKβ suppressed the compression-induced phosphorylation and degradation of IκBα as well as ALDH1B1 expression (Fig. 5e–g and Supplementary Fig. 8g–i). In addition, we observed that PIEZO1 depletion greatly inhibited IKKβ pS177/181, IκBα pS32/36, and IκBα degradation in the cells under compression (Fig. 5h and Supplementary Fig. 8j). These results indicate that compressive force activates IKKβ and its downstream NF-κB via PIEZO1, thereby upregulating ALDH1B1 expression.

CSK23 phosphorylates IKKβ to activate NF-κB
To investigate how compressive force activates IKKβ phosphorylation, we conducted mass spectrometry analyses of IKKβ-associated proteins after compression (Fig. 6a and Supplementary Data 3). Among IKKβ-associated protein kinases, casein kinase II subunit alpha 3 (CSK23) ranked first with a twofold stronger interaction with IKKβ after compression (Fig. 6b). The interaction between CSK23 and IKKβ was validated by co-immunoprecipitation (Co-IP) experiments (Fig. 6c and Supplementary Fig. 9a). Notably, the depletion of CSK23 resulted in the inhibition of IKKβ pS177/181, IκBα pS32/36, IκBα degradation, and ALDH1B1 expression (Fig. 6d–f and Supplementary Fig. 9b–i). To assess whether CSK23 phosphorylates IKKβ, we performed the in vitro kinase assays by mixing bacterial-purified GST-IKKβ and His-CSK23, which showed that CSK23 indeed directly phosphorylated IKKβ S171/181 (Fig. 6g). Additionally, we also mixed SFB-IKKβ and FLAG-CSK23 immunoprecipitated from A549 cells to conduct in vitro kinase assays, confirming that CSK23 phosphorylated IKKβ at S171/181 (Supplementary Fig. 9j).

In addition, we investigated how CSK23 is activated upon PIEZO1 activation by compressive force. Calcium signals are normally transduced by calcium-sensing messengers such as calmodulin (CaM), calpain, and calcineurin to regulate downstream signaling networks50. Among these, CaM emerges as a central Ca²⁺ sensor and molecular integrator, dynamically regulating protein-protein interactions and scaffolding macromolecular complex assembly through its target-binding domains51,52. Thus, we tested whether CaM is the calcium messenger between PIEZO1 and CSK23. We treated A549 and H1299 cells with CaM-selective inhibitor W7 and observed that W7 treatment attenuated compression-induced interaction between CSK23 and IKKβ (Supplementary Fig. 9k, l). Meanwhile, we also assessed whether calpain or calcineurin also contributes to PIEZO1-mediated CSK23 activation. We treated A549 cells with the calpain inhibitor MDL-28170 or the calcineurin inhibitor FK506. Unlike W7, MDL-28170 treatment had no significant effect on the compression-induced CSK23-IKKβ interaction, whereas FK506 treatment partially abrogated this interaction (Supplementary Fig. 9m, n). These results suggest that CaM is likely the major calcium messenger between PIEZO1 and CSK23.

CSK23 is required for the survival of confined cells and distant metastasis
To examine whether CSK23 regulates confined cell survival by activating NF-κB, we overexpressed RelA in CSK23-depleted cells. CSK23 depletion increased lipid peroxidation levels in confined cells, while RelA overexpression completely restored lipid peroxidation levels in CSK23-depleted cells (Supplementary Fig. 10a–e). Consequently, CSK23 depletion increased cell death rate and inhibited the migratory capability of tumor cells in confining spaces, while RelA overexpression restored the survival and migration of CSK23-depleted cells in confinement (Supplementary Fig. 10f–k). Similarly, overexpression of ALDH1B1 decreased lipid peroxidation levels and restored the survival and migration of CSK23-depleted cells in confining spaces (Fig. 6h–j and Supplementary Fig. 10l–s), whereas CSK23 depletion showed no significant impact on lipid peroxidation or survival in unconfined tumor cells (Fig. 6h, i and Supplementary Fig. 10d–h and 10o–q). Moreover, we implanted CSK23-depleted tumor cells with or without ALDH1B1 overexpression into randomized female BALB/c athymic nude mice via intracardiac injection. CSK23 depletion suppressed tumor cell metastasis. H&E staining of dissected brain tissues further confirmed that CSK23 knockdown significantly reduced both the number and size of brain metastatic lesions. Notably, these effects were effectively rescued by ALDH1B1 overexpression (Fig. 6k, l and Supplementary Fig. 10t). These results indicate that CSK23 is required for the survival of confined cells and tumor metastasis by regulating RelA and ALDH1B1.

CX-4945 (Silmitasertib) is the first orally bioavailable inhibitor of casein kinase 2 (CK2) with acceptable pharmacological properties53. We next evaluated the therapeutic potential of Silmitasertib for tumor metastasis. We treated A549 or H1299 cells with Silmitasertib. Transwell migration assay showed that Silmitasertib suppressed the confined migration of these cells in a dose-dependent manner without influencing their proliferation (Fig. 6m and Supplementary Fig. 11a–c). Moreover, we implanted luciferase-expressing A549 cells into randomized female BALB/c athymic nude mice via intracardiac injection, followed by the administration of Silmitasertib. Bioluminescence imaging of the mice showed that Silmitasertib treatment dampened brain metastasis of lung cancer cells (Fig. 6n and Supplementary Fig. 11d). H&E staining of dissected brain tissues further confirmed that Silmitasertib reduced both the number and size of brain metastatic lesions (Fig. 6o). Similarly, the survival duration of tumor-bearing mice was extended by Silmitasertib treatment (Fig. 6p).

Clinical relevance of NF-κB-dependent ALDH1B1 expression in lung cancer patients
To delineate the clinical relevance of our finding, we examined the levels of ALDH1B1 or the levels of nuclear RelA in primary tumor tissues from lung cancer patients. Immunofluorescence analysis indicated that both ALDH1B1 expression levels and nuclear RelA levels were increased in tumor cells constrained in lung capillaries, compared to tumor cells outside capillaries (Fig. 7a–c). Notably, nuclear RelA levels showed a strong positive correlation with ALDH1B1 expression in confined tumor cells (Fig. 7d). Furthermore, higher 4-HNE levels were observed in intravascular tumor cells compared to extravascular areas (Fig. 7e, f). Importantly, ALDH1B1 expression demonstrated a significant negative correlation with both 4-HNE accumulation and cell death in capillary-constrained tumor cells (Fig. 7g–i). Additionally, capillary-confined tumor cells from patients with metastatic recurrence showed elevated ALDH1B1 expression and reduced 4-HNE accumulation compared to the cells from non-recurrent cases (Fig. 7j, k). These results implicate the clinical relevance of NF-κB-regulated ALDH1B1 expression and cell survival in lung cancer patients and suggest the prognostic potential of ALDH1B1 for patients with metastatic lung cancer.

Methods
Humanspecimens
The use of human lung tumor specimens and associated data was approved by the Institutional Review Boards of Shanghai Chest Hospital (KS(Y)23082) and Shanghai XinHua Hospital (XHEC-C-2025-155-1), and informed consent was obtained from all patients. No sex and gender-based analysis was performed. The study focused on characterizing the pre-existing pathological phenomenon of tumor cells within confining vasculature, an objective cellular event that is not contingent on patient sex or gender.

Mice
Six-week-old female BALB/c athymic nude mice were purchased from Lingchang Biotech (Shanghai, China). Littermates were randomly assigned to experimental groups. Sex was not considered as a biological variable in the study design. This is because the primary objective was to investigate the intrinsic behavior of A549 tumor cells under confinement in vivo. The use of a single-sex cohort was chosen to minimize experimental variability and establish a clear baseline for assessing the effects on tumor cell survival and metastasis in this specific context. Mice were housed under specific pathogen-free conditions with a standard chow diet and maintained at a room temperature of 20–26 °C with 40–70% humidity on a 12-h light/12-h dark cycle. All experimental protocols were approved by the IACUC of the Shanghai Institute of Biochemistry and Cell Biology (approval number: SIBCB-S355-2306-18) and complied with all relevant ethical regulations. Throughout the study, no tumor exceeded the IACUC-permitted maximum size of 1.5 cm in diameter.

Antibodies
Rabbit polyclonal anti-ALDH1B1 (15560-1-AP, Proteintech Group); Rabbit polyclonal anti-CSK23 (PA5-98299, Thermo Fisher Scientific); Rabbit monoclonal anti-Flag (20543-1-AP, Proteintech Group); Rabbit monoclonal anti-HA (3724, Cell Signaling Technology); Mouse monoclonal anti-Actin (60008-1-lg, Proteintech Group); Mouse monoclonal anti-Tubulin (T9026, Sigma); Mouse IgG antibody (sc-2025, Santa Cruz Biotechnology); Rabbit monoclonal anti-RelA (8242, Cell Signaling Technology); Mouse monoclonal anti-RelA (66535-1-Ig, Proteintech Group); Rabbit monoclonal anti-IKKβ (8943, Cell Signaling Technology); Mouse monoclonal anti-IκBα S32/36 phosphorylation (9246, Cell Signaling Technology); Rabbit monoclonal anti-IKKβ S177/181 phosphorylation (2697, Cell Signaling Technology); Rabbit monoclonal anti-IκBα (10268-1-AP, Proteintech Group); Rabbit monoclonal anti-Lamin B1 (12586s, Cell Signaling Technology); Goat polyclonal anti-CD31 (AF3628, Novus Biologicals); Mouse monoclonal anti-4-HNE (MAB3249, R&D Systems); Goat-anti-mouse IgG second antibody (31160, Thermo Fisher Scientific); Goat-anti-rabbit IgG second antibody (31210, Thermo Fisher Scientific); Donkey-anti-mouse Alexa Fluor 488 (A21202, Thermo); Donkey-anti-rabbit Alexa Fluor cy3 (711-165-152, Jackson ImmunoResearch); Donkey-anti-goat Alexa Fluor 647 (A21447, Thermo).

Regents
The Annexin-V-PE/7-AAD apoptosis detection kit was purchased from BD Biosciences. The DeadEnd Fluorometric TUNEL System was purchased from Promega. The fluorescent lipid peroxidation sensor BODIPY 581/591 C11 was purchased from Invitrogen. The GSH/GSSG assay kit was purchased from Beyotime. The Caspase-3/7 activity assay kit was purchased from Dalian Meilun Biotechnology. The Flag peptide was purchased from Abclonal. All antibiotics, including ampicillin, kanamycin, puromycin, and hygromycin, were purchased from EMD Biosciences. Trizol was purchased from Life Technologies. The In Vitro DNA Transfection Reagent was purchased from Signagen Laboratories. The MEK1/2 inhibitor (Mirdametinib), AKT1/2/3 inhibitor (MK-2206), and NFκB inhibitor (PDTC) were purchased from Selleckchem. The YAP inhibitor (Verteporfin), ERK inhibitor (Ravoxertinib), ALDH1B1 inhibitor (IGUANA-1), CK2α inhibitor (Silmitasertib), CaM inhibitor (W7), Calpain inhibitor (MDL-28170), Calcineurin inhibitor (FK506), and Acloproxalap were purchased from MCE. D-luciferin was purchased from Xenogen. Acetaldehyde-DNPH, Propionaldehyde-DNPH, Hexanal-DNPH, and Nonanal-DNPH reference standards were purchased from Shanghai Titan.

DNA constructs and mutagenesis
Human ALDH1B1 and GPX4 were PCR-amplified and cloned into pCDH-Flag. Human RelA and CSK23 were PCR-amplified and cloned into pCDH-Flag and pcDNA3.0-HA. Human ALDH1B1 Promoter was PCR-amplified and cloned into pGL3. The mutations of ALDH1B1 E285A and ALDH1B1 promoter were made using the QuikChange II Site-Directed Mutagenesis Kit. The pGIPZ non-targeting shRNA was generated with the control oligonucleotide 5′-CTCGCTTGGGCGAGAGTAA-3′. The pGIPZ human ALDH1B1 shRNA was generated with 5′-CAAATTAACTCTTAGAAGA-3′ oligonucleotide targeting the noncoding region of the ALDH1B1 transcript. The pGIPZ human RelA shRNA-1 was generated with 5′-AGGCGAGAGGAGCACAGAT-3′ oligonucleotide and shRNA-2 was generated with 5′-CTCAGTGAGCCCATGGAAT-3′ oligonucleotide. The pGIPZ human IKKβ shRNA-1 was generated with 5′-GCTTAGAT ACCTTCATGAA-3′ oligonucleotide, shRNA-2 was generated with 5′-TGGACATTGTT GTTAGCGA-3′ oligonucleotide and shRNA-3 was generated with 5′-AGACCGACATTGTGGACTT-3′ oligonucleotide. The pGIPZ human CSK23 shRNA-1 was generated with 5′-CAATTGTACCAGACGTTAA-3′ oligonucleotide and shRNA-2 was generated with 5′-CACAGAAAGCTACGACTAA-3′ oligonucleotide. The pGIPZ human ALDH9A1 shRNA-1 was generated with 5′-AGGTG GATCGTTTGGTTAT-3′ oligonucleotide and shRNA-2 was generated with 5′-AGGAAAAGAGACTAGGATA-3′ oligonucleotide. The pGIPZ human ALDH6A1 shRNA-1 was generated with 5′-AGGTCTT GCTCCGCTATCA-3′ oligonucleotide and shRNA-2 was generated with 5′-GCGATCATCCGGACATCAA-3′ oligonucleotide. The pGIPZ human TRPV4 shRNA-1 was generated with 5′-ACCAAGTTTGTTACCAAGA-3′ oligonucleotide and shRNA-2 was generated with 5′-TGCTCCTATG GAGTCACATAA-3′ oligonucleotide. The pGIPZ human ORAI1 shRNA-1 was generated with 5′-ACGCTGCTCTTCCTAGCTG-3′ oligonucleotide and shRNA-2 was generated with 5′-CATGTGTGTGTGACACATA-3′ oligonucleotide. The pLKO.1 human PIEZO1 shRNA-1 was generated with 5′-CTCACCAAGAAGTACAATCAT-3′ oligonucleotide and shRNA-2 was generated with 5′-GCTGCTCTGCTACTTCATCAT-3′ oligonucleotide.

Cell culture
The human lung cancer cell lines H1299, A549, H460, and the HEK293T cell line were obtained from the Type Culture Collection of the Chinese Academy of Sciences and authenticated by short tandem repeat (STR) testing. Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and cultured at 37 °C in a humidified 5% CO₂ atmosphere.

Tail vein/intracardiac injection
For the experimental metastasis models, female athymic nude mice were used. For the lung metastasis model, 6×106 cells in 100 μL of PBS were injected per mouse via the tail vein. For the brain metastasis model, a small incision was made in the chest to expose the heart, 2×105 cells in 100 μL of PBS were injected into the left ventricle. To evaluate therapeutic efficacy, mice were treated with either IGUANA-1 (2 mg/kg, administered via intraperitoneal injection) or Silmitasertib (2 mg/kg, administered via oral gavage) once every two days. Metastatic progression was monitored at the indicated time points by bioluminescence imaging using an Xenogen IVIS system (version 4.0). At the endpoint of the experiment, the mice were euthanized and dissected. The bioluminescence imaging of the extracted mouse brains was performed using a Tanon-5200 Chemiluminescent Imaging System (Tanon). Then the lungs or brains were taken out for 4% PFA fixation and embedded for H&E or IF staining.

For the mouse survival analysis, the observation period was terminated upon the death of the last control group mouse. At this endpoint, the surviving mice in the experimental group exhibited no signs of morbidity, maintaining stable body weight and showing no adverse effects. These mice were right-censored to uphold ethical standards and statistical integrity, as the Kaplan–Meier method appropriately handles censored data85–87.

CRISPR screen and data analysis
A custom sgRNA library was designed to target 1,685 metabolic enzyme genes. The sgRNAs for each gene were selected from the human genome-scale CRISPR-Cas9 knockout (GeCKO) library88. The sequences of all sgRNAs are provided in Supplementary Data 1. The pooled oligonucleotide library comprising these sgRNAs was synthesized by GENEWIZ. and then cloned into the BsmBI-digested lenti-Guide-Puro (Addgene 52963) backbone via Golden Gate assembly. The library was amplified in E. coli DH5α with a coverage of >200-fold. Plasmid DNA was prepared via maxi-preparation from the pooled bacteria to generate the final library, which demonstrated >95% sgRNA detection efficiency. The screening experiment was performed as previously described89. Briefly, A549 cells were infected with lentivirus expressing Cas9 and blasticidin S deaminase. After one week of selection with blasticidin, single-cell clones were sorted by FACS into 96-well plates. Flag-Cas9 expression in these subclones was confirmed by immunoblotting. The sgRNA library was transduced into A549-Cas9 cells at a multiplicity of infection (MOI) of 0.3. This was followed by puromycin selection for 2 days and subsequent infection with EGFP-encoding virus. On day 5, 6×107 cells were injected into the tail veins of female BALB/c athymic nude mice (n=9). Over 90% of tumor cells were retained in the lung capillaries within 24 h post-injection, consistent with prior studies19,20. At the 24-h time point, viable tumor cells were isolated from whole lungs by fluorescence-activated cell sorting (FACS), and sorted cells from every three mice were pooled to form one sample. In parallel, control samples of the input cell population (prior to injection) were also collected. Genomic DNA was extracted from all samples, and the sgRNA inserts were amplified by PCR and subjected to next-generation sequencing, which was performed by Beirui. The CRISPR screening data were analyzed using the MAGeCK algorithm (version 0.5.7) and the MAGeCKFlute R package (version 2.8.0).

Transwell assay
Transwell migration assays were performed using 6.5 mm diameter Corning BioCoat Control Inserts (8μm pore size) according to the manufacturer’s instructions. Briefly, A549 (8×104), H1299 (1×105), and H460 (1.5×105) cells were harvested post-trypsinization and seeded into the upper chambers in serum-free medium. The lower chambers were filled with medium containing 10% or 20% FBS as a chemoattractant. After incubation for 8 or 18 h, the cells were subjected to methanol fixation and staining with 0.4% crystal violet. The upper side of the membrane was subsequently destained with a cotton swab to remove non-migrated cells. Images of the migrated cells were acquired using an inverted microscope (×200 magnification), and the relative migration was determined by normalizing the total area of the migrated cells to that of the control.

Microchannel preparation and usage
Polydimethylsiloxane (PDMS, 10/1 w/w PDMS A/crosslinker B) (GE Silicones) was used to prepare 10µm high micro-channels with constrictions of varying lengths and 8µm widths. Prior to loading into the microchannels, cells were stained with DAPI at a 1:10,000 dilution. The cell suspension was then introduced into the channel inlet and allowed to load for 24 h. Subsequently, real-time cell tracking was performed for over 48 h using a Zeiss Celldiscoverer 7 platform, with images captured every 30 min. Cells that died within the microchannels exhibited positive DAPI staining, indicating loss of membrane integrity.

Wound healing assay
A549 or H1299 cells (1×106 cells per well) were seeded in 6-well plates. After 12 h, a confluent cell monolayer was scraped using a 200μL pipette tip. The initial wound width was measured 2 h after scratching, and this time point was designated as 0 h. Images were subsequently captured at 24 or 72 h. The relative cell migration was quantified by measuring the wound width and normalizing it to the width at the 0-h time point.

Quantitative real-time PCR
Total RNA was extracted using Trizol reagent (Life Technologies), and cDNA was synthesized with the HiScript IV QRT SuperMix for qPCR kit (Vazyme). Quantitative real-time PCR analysis was performed using a Roche LightCycler 384 (version 1.5.1). Data were normalized to expression of a control gene (β-actin) for each experiment. Primer sequences of human ALDH6A1 were 5′-TCAGTGCCAACTGTAAAGCTC-3′ (forward) and 5′-TGAGGGACCCGACCAATGA-3′ (reverse); human ALDH9A1 were 5′-GTCGCAGCCGCTCAATTAC-3′ (forward) and 5′-CCTTTGCATTTTGAACAGCCAA-3′ (reverse); human ALDH1B1 were 5′-CCCATTCTGAACCCAGACATC-3′ (forward) and 5′-AATGACCTCCC CGGTGGTA-3′ (reverse); human PIEZO1 were 5′-GGACTCTC GCTGGTCTACCT-3′ (forward) and 5′-GGGCACAATATGCAGGCAGA-3′ (reverse); human ORAI1 were 5′-GACTGGATCGGCCAGAGTTAC-3′ (forward) and 5′-GTCCGGCTGGAGGCTTTAAG-3′ (reverse); human TRPV4 were 5′-GATGGGCGACCAAATCTGC-3′ (forward) and 5′-GAGG ACTCATATAGGGTGGACTC-3′ (reverse); β-actin were 5′-AGAGCTACG AGCTGCCTGAC-3′ (forward) and 5′-AGCACTGTGTTGGCGTACAG-3′ (reverse).

Cell proliferation assay
A549 cells or H1299 cells were plated in 96-well plates at a density of 1000 cells per well. Following 1, 2, 3, 4, and 5 days of incubation, the plates were harvested, and the cells were fixed using trichloroacetic acid (TCA) and stained with sulforhodamine B (SRB), followed by rinsing of the plates four times with 1% acetic acid and solubilization of the protein-bound dye with 10 mM Tris base solution. The absorbance was measured at 560 nm. Relative cell proliferation was assessed by the OD560 values and normalized to day 1.

Compression experiment
Flexcell Compression System (Flexcell International Corporation) was utilized to apply the compressive load. The diameters of pulmonary capillaries range from 5 to 8 μm90. In microfluidic channels with analogous dimensions (5–8 μm), individual tumor cells experience compressive forces of approximately 80 nN36. Using the Flexcell Compression System’s standard conversion formula, P (MPa) = (5.65 × Force (lbs)) / D (mm²), an applied pressure of 5 kPa in the BioPress well system corresponds to the target cellular force load of 80 nN. A549, H1299, or H460 cells (1×106) were suspended in 5% GelMA hydrogels (EFL-GM-30), seeded onto glass slides, photo-crosslinked under ultraviolet light, and then subjected to compressive loads of 0 or 5 kPa. Hydrogels was dissolved with GelMA Lysis Buffer (EFL-GM-LS-001), and the cells were collected for immunoblotting and quantitative real-time PCR analysis.

Stiffness experiment
GelMA hydrogels were used to replicate tissue stiffness. Based on reported lung tissue mechanics (0.5–5 kPa)48, three GelMA concentrations were selected to represent distinct ECM stiffness regimes: 10% (∼3 kPa, approximating normal lung), 12.5% (∼5 kPa, mimicking fibrotic regions), and 15% (∼9 kPa, representing pathological stiffness). The hydrogels with different concentrations were added to 6-well plates and crosslinked under ultraviolet light. A549 cells (5×105 cells/well) were seeded onto the hydrogels in 6-well plates and cultured in growth media for 24 h.

H&E staining
The brains of the injected mice were removed and fixed with 4% paraformaldehyde (PFA) at 4 °C overnight. Subsequently, the brains were embedded in paraffin. Each sample was completely sectioned at 50 μm intervals with a thickness of 5 μm. For histological examination, standard hematoxylin and eosin (H&E) staining was carried out. The images of H&E staining were taken and analysed using ImageJ software.

Immunofluorescence/TUNEL staining
For the detection of tumor cell death, lungs were harvested from the injected mice at 24 h post-injection. For the analysis of tumor cell extravasation, lungs were harvested at 48 h post-injection. All harvested lungs were fixed overnight at 4 °C with 4% PFA. After cryo-sectioning, sequential 10 μm lung tissue sections were prepared at 50 μm intervals for analysis. Immunofluorescence analysis was carried out with an anti-CD31 antibody (diluted 1:100) to enable histological examination of the vasculature. Concurrently, the DeadEnd Fluorometric TUNEL System was applied according to the manufacturer’s guidelines to detect tumor cell death in the same sections.

The expression of ALDH1B1, 4-HNE, and RelA in tumor cells from both mouse and human lung tissues was evaluated using a standard immunofluorescence protocol. Briefly, tissue sections (either frozen from mice or paraffin-embedded from humans) were fixed and incubated with specific primary antibodies (1:200 dilution), followed by corresponding Alexa Fluor-conjugated secondary antibodies (1:1000) and DAPI for nuclear counterstaining. Images were acquired using an Olympus FV3000 confocal microscope. Protein expression intensity within the confining capillaries was semi-quantitatively scored by analyzing three to five randomly selected fields (at 400× magnification) per section, and the mean score was calculated for each sample. Semiquantitative scoring of intensity was calculated using Columbus (PerkinElmer) as described previously91. Briefly, immunofluorescence images were analyzed using Columbus software (version 2.8.2). Cell numbers were quantified by nuclear identification through DAPI staining (blue). Capillary regions (confining vs. nonconfining) were demarcated using CD31 staining (white). ALDH1B1 and 4-HNE intensities were measured within these regions. Nuclear RelA intensity was quantified within DAPI-defined nuclei in these regions.

Immunoprecipitation and immunoblotting analysis
For co-immunoprecipitation (co-IP) assays, a mild co-IP buffer (50 mM Tris-HCl pH 7.5, 1% Triton X-100, 0.01% SDS, 150 mM NaCl, 1 mM dithiothreitol, 0.5 mM EDTA, 0.1 mM sodium orthovanadate, 0.1 mM sodium pyrophosphate, 0.1 mM sodium fluoride, and protease inhibitor cocktail) was used to preserve native protein-protein interactions. For enzyme activity or in vitro kinase assays, a more stringent IP buffer was used, which contained 0.1% SDS was used, with all other components identical to the co-IP buffer. Flag-tagged proteins were immunoprecipitated using anti-Flag M2 affinity agarose.

For immunoblotting, cells were lysed in cold RIPA buffer supplemented with a 1× protease inhibitor cocktail for 30 min on ice, with vortexing at 10-min intervals. The lysates were centrifuged at 14,000×g for 10 min at 4 °C to collect the supernatant. These protein extracts were combined with loading buffer, heated at 95 °C for 10 min to denature the proteins, and then subjected to SDS-PAGE. These separated proteins were electrophoretically transferred onto nitrocellulose membranes. For immunodetection, the membranes were first incubated with specific primary antibodies at 4 °C overnight. The antibodies and their dilutions were as follows: ALDH1B1 (1:4000), CSK23 (1:1000), Flag (1:4000), HA (1:4000), Actin (1:4000), Tubulin (1:5000), RelA (1:2000), IKKβ (1:1000), IκBα pS32/36 (1:1000), IKKβ pS177/181 (1:1000), IκBα (1:4000), Lamin B1 (1:4000). Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies (diluted 1:5000) for 1 h at room temperature. After thorough washing with TBST, immunoreactive bands were visualized using enhanced chemiluminescence (ECL)92.

Subcellular fractionation analyses
Cytoplasmic and nuclear proteins were extracted from treated cells using a modified protocol as described previously93. Briefly, cells were incubated with a plasma membrane lysis buffer (200 mM HEPES pH 7.9, 200 mM KCl, 25 mM MgCl₂, 6 M sucrose, 50% glycerol, 0.5% NP-40, 0.5 mM DTT, and protease inhibitor cocktail) at 4 °C for 20 min to lyse the plasma membranes. The lysates were centrifuged at 1000×g for 5 min at 4 °C, and the supernatant was collected as the cytoplasmic fraction. The nuclear pellet was then resuspended in PBS and washed three times (1000×g, 5 min, 4 °C) to remove cytoplasmic contaminants. Finally, the purified nuclei were resuspended in 50 μL of cold RIPA buffer supplemented with 1× protease inhibitor cocktail and lysed overnight at 4 °C with agitation to obtain the nuclear fraction. The extracted proteins from both fractions were then subjected to immunoblotting with the corresponding antibodies.

Purification of recombinant proteins
GST-IKKβ and HIS-CSK23 were expressed in bacteria and purified. Briefly, the vectors expressing GST-IKKβ and HIS-CSK23 were used to transform BL21/DE3 bacteria. Then 0.5 mM isopropyl-beta-D-thiogalactopyranoside was used to induce protein expression (20 h, 16 °C). Cell pellets were collected and sonicated in PBS with the addition of proteasome inhibitors before centrifugation at 12,000×g for 60 min (4 °C). Cleared lysates were then bound to glutathione resin (Genescript) or Ni-NTA resin (Genescript) with rolling (3 h, 4 °C). Beads were washed extensively before eluting for 1 h in GST elution buffer (50 mM Tris-HCl, 10 mg ml−1 glutathione, 300 mM NaCl, pH 8.0) or HIS elution buffer (PBS (pH 7.4) plus 500 mM imidazole). Eluted proteins were then dialysed against PBS.

In vitro kinase assay
The kinase reactions were performed as described previously94. In brief, GST-IKKβ and HIS-CSK23 were purified from bacterial. SFB-IKKβ and Flag-CSK23 were immunoprecipitated from A549 cells. The IKKβ and CSK23 proteins were mixed with 1× kinase reaction buffer (50 mM HEPES (pH 7.5), 1 mM EGTA, 0.01 mM BSA, 0.01% Tween-20, 10 mM MgCl₂, 2 mM MnCl₂, 2 mM DTT) in a 50-μL reaction containing or not containing 100 μM ATP. The samples were then incubated at 30 °C for 1 h and stopped by adding 4× SDS loading buffer. Phosphorylated IKKβ protein was detected by immunoblotting.

ALDH1B1 activity assay
The enzymatic activity of ALDH1B1 was measured as described previously13. In brief, the Flag-ALDH1B1 and Flag-ALDH1B1 E285A proteins were immunoprecipitated from A549 cells. The reaction was initiated by adding the acetaldehyde into the reaction mixture containing 0.1 M sodium pyrophosphate, 1 mM NAD+, 1 mM pyrazole, 1 mM 2-mercaptoethanol, and ALDH1B1 WT/E285A protein, then monitoring the formation of NADH at 340 nm for 25 min at 25 °C.

In situ cell death detection
After 6 h of culture in transwell inserts (6×104 cells), the cells were stained using an Annexin V-PE kit according to the manufacturer’s instructions. Images were acquired and analyzed using a BioTek Cytation 5 imaging system (version 3.15). Transwell pore-confined cells and non-pore cells were distinguished and selected based on bright-field imaging. The cell death percentage was calculated as the ratio of Annexin V-PE-positive cells (either pore-confined or non-pore) to the total number of cells counted for each population.

Lipid peroxidation measurement
After 6 h of culture in transwell inserts (6×104 cells/insert), the cells were stained with 0.5 μM BODIPY 581/591 C11 for 30 min at 37 °C to assess lipid peroxidation. This ratiometric fluorescent probe quantitatively detects ferroptosis-associated lipid peroxidation. In its reduced state, the probe emits red fluorescence (excitation/emission: 581/591 nm), which shifts to green fluorescence (ex/em: 488/510 nm) upon oxidation by lipid hydroperoxides. The relative lipid peroxidation levels in pore-confined and non-pore cells were determined by calculating the intensity ratio of oxidized (green, F510) to reduced (red, F591) fluorescence signals (F510/F591), measured using a PerkinElmer Operetta high-content imaging system. Data analysis was performed using Harmony software.

GSH/GSSG measurement
To measure intracellular GSH and GSSG levels, A549 or H1299 cells (1×106) were seeded in serum-free medium into the upper chamber of a 24 mm diameter Transwell (Corning BioCoat Control Insert, no ECM, 8 μm pore size). The lower chamber was filled with medium containing 20% FBS as a chemoattractant. After 18 h of incubation, those cells remaining on the upper surface of the membrane (non-migratory) or within its pores (confined) were collected. The intracellular concentrations of GSH and GSSG in these cells were quantified using a commercial assay kit according to the manufacturer’s instructions, with values derived from a standard curve.

Caspase-3/7 activity assay
For the caspase-3/7 activity assay, A549 cells (1×106) were seeded into a 24 mm diameter transwell chamber in serum-free medium. After 18 h, the chamber membrane was treated with trypsin, and the cells adherent to the upper surface of the membrane and confined in the pore were collected. Caspase-3/7 activity was measured using an assay kit following the manufacturer’s protocol, with quantification based on a standard curve.

GEO data analysis
Deep sequencing data from this study are publicly available in the GEO database (http://www.ncbi.nlm.nih.gov/geo) (GSE72094 and GSE37745). The RNA-seq sample expression levels were converted from fragments per kilobase of transcript per million mapped reads (FPKM) to transcripts per million (TPM), and then log2 (TPM+1) was calculated. Differences in prognostic outcomes were assessed using Kaplan–Meier analysis in R software with the “survival” and “survminer” packages95. All statistical analyses were carried out using R software (version 4.3.2).

Luciferase reporter assay
Transcriptional activation of ALDH1B1 and NF-κB in A549 or H1299 cells was measured with a luciferase reporter assay as described previously96. In brief, cells were co-transfected with a firefly luciferase reporter plasmid under the target promoter and a Renilla luciferase control plasmid. Using the Promega Dual-Luciferase Reporter Assay Kit, relative luciferase activity was normalized to that of untreated cells, empty vector-expressing cells, or the Renilla control.

Chromatin immunoprecipitation
ChIP assays were performed as previously described97. Briefly, chromatin complexes were immunoprecipitated using an anti-RelA rabbit monoclonal antibody, with rabbit monoclonal IgG serving as a negative control. Recruitment of RelA to the ALDH1B1 promoter region was quantified by real-time PCR, using 2% of the pre-immunoprecipitation, sonicated chromatin as the input control. Primer sequences used for the amplification were 5′-TTCGGTCTCCGAAAACTTTGCTG-3′ (forward) and 5′-AGGCTCCTAAGGCACTGGTTT-3′ (reverse).

Mass spectrometry analysis
Co-immunoprecipitation and Protein Separation. SFB-IKKβ protein complexes were immunoprecipitated from A549 cells subjected to 0 kPa or 5 kPa pressure stimulation. The precipitated complexes were denatured by heating at 95 °C for 10 min, followed by electrophoretic separation of IKKβ-associated proteins using SDS-PAGE.

Protein Digestion and Peptide Preparation. Proteins were precipitated from excised gel bands using cold acetone. The resulting protein pellets were resuspended in 8 M urea/100 mM Tris-HCl (pH 8.5) buffer. Reduction was performed with 5 mM TCEP (Thermo Scientific) for 30 min at room temperature, followed by alkylation with 10 mM iodoacetamide (Sigma) for 20 min. The protein solution was then diluted fourfold and digested overnight with trypsin at a 1:50 (w/w) enzyme-to-substrate ratio. The digestion was terminated by acidification with formic acid, and resulting peptides were desalted using Monospin C18 columns (SHIMADZU-GL).

LC-MS/MS Analysis. Desalted peptides were separated on a custom-packed 30 cm analytical column (75 μm ID, ReproSil-Pur C18-AQ 1.9 μm resin, Dr. Maisch GmbH) maintained at 55 °C using an Easy-nLC 1200 system (Thermo Scientific). The mobile phase consisted of 0.1% formic acid in water (buffer A) and 0.1% formic acid in 80% acetonitrile (buffer B). Peptides were eluted with a multi-step gradient: 2–10% B over 1 min, 10–35% B over 80 min, 35–60% B over 15 min, 60–100% B over 15 min, and held at 100% B for 9 min, at a constant flow rate of 300 nL/min.

Data Acquisition and Processing. MS data were acquired on a Q Exactive Orbitrap mass spectrometer (Thermo Scientific) operating in data-dependent mode. Peptides were ionized using a 2.5 kV spray voltage. Full MS scans (m/z 300–1800) were acquired at 70,000 resolution, followed by fragmentation of the top 20 most intense precursors at 17,500 resolution. Automatic gain control targets were set at 3e6 for MS and 5e5 for MS/MS scans, with maximum injection times of 50 ms and 100 ms, respectively. Dynamic exclusion was set to 15 s.

MS/MS data were processed using MaxQuant (v1.6.10.43) against the human UniProtKB database. Search parameters included: tryptic digestion with up to two missed cleavages; carbamidomethylation (C) as fixed modification; oxidation (M) and protein N-terminal acetylation as variable modifications; precursor mass tolerance of 20 ppm. Protein quantification was performed using the LFQ intensity algorithm with a minimum of six amino acids required for peptide identification.

Cell aldehyde measurement
For aldehyde measurement, A549 or H1299 (1×106) cells were collected after trypsinization and added to a transwell chamber with a 24 mm diameter, containing serum-free medium. The outside the chamber was filled with medium containing 20% FBS. After 18 h, the membrane of the chamber was immersed in trypsin to collect the cells adherent to the upper surface of the membrane and the cells confined within the pores. These cells were then sonicated for 7 min, followed by centrifugation at 3500×g for 15 min at 4 °C. The supernatant was transferred to a 1.5 mL Eppendorf tube, and 5 mM DNPH solution (1:20) was added. The mixture was incubated in the dark at 37 °C with constant agitation for 1 h. After incubation, the mixture was centrifuged at 1000×g for 5 min, and 4 mL of hexane was added. The mixture was vortexed and then centrifuged at 4000×g for 5 min. The upper layer containing hexane was collected, and the sample was subsequently evaporated at 4 °C.

Samples were reconstituted in 80% methanol solution prior to mass spectrometric analysis. Chromatographic separation was performed on a Waters ACQUITY UPLC HSST3 column (100 mm × 2.1 mm, 1.8 μm) maintained at 40 °C, using a mobile phase comprising 0.1% (v/v) formic acid in water (solvent A) and 0.1% (v/v) formic acid in methanol (solvent B). The elution protocol employed a gradient program: 10% B (0–1 min), 10–95% B (1–5 min), 95% B (5–8 min), 95–10% B (8–8.1 min), and 10% B (8.1–12 min), with a constant flow rate of 0.3 mL/min and 1 μL injection volume.

Mass spectrometric detection was carried out using an Agilent 6495 triple quadrupole instrument coupled to an Agilent 1290 Infinity II UHPLC system. Analysis was performed in negative ionization mode with the following optimized parameters: gas temperature 200 °C; drying gas flow 14 L/min; nebulizer pressure 20 psi; sheath gas temperature 250 °C; sheath gas flow 11 L/min; capillary voltage 3000 V; and nozzle voltage 1500 V. Aldehydes were quantified in multiple reaction monitoring (MRM) mode, with specific transition pairs detailed in Supplementary Data 4. All data acquisition and processing were performed using Agilent QQQ Quantitative Analysis Software (v10.2).

Statistics and reproducibility
No statistical methods were used to predetermine sample size, but our sample sizes are similar to those reported in previous publications16,22. All statistics were performed using GraphPad Prism software (version 8.0). Specific statistical tests for each experiment are described in the figure legends. A value of P<0.05 was considered statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, Liu et al. explore how cancer cells adapt metabolically to migrate through confined spaces, a key step in metastasis. Researchers conducted an in vivo CRISPR knockout screen targeting 1,684 metabolic enzymes in a lung metastasis mouse model. They identified ALDH1B1 as crucial for tumor cell survival within capillaries. Under compressive forces, CSK23 phosphorylates IKKβ, activating the NF-κB pathway, which increases ALDH1B1 expression. This upregulation enhances aldehyde detoxification, suppresses ferroptosis, and aids cell survival during capillary migration. Inhibiting CSK23 or ALDH1B1 impairs metastasis, highlighting them as potential therapeutic targets. Elevated ALDH1B1 and NF-κB activation in patient tumor tissues correlate with metastatic recurrence, underscoring their role in metastasis. While the association of ALDH1B1 upregulation with tumor metastasis and survival has been previously established, the authors have made significant strides in elucidating the mechanism by which tumors adapt to compressive forces within confined 3D spaces such as capillaries. With moderate revisions to the text and figures, this manuscript has the potential to be a strong fit for publication in a rigorous journal like Nature Communications.\nMajor concerns:\n1. Authors should explain the screen setup in more details. For example, when dissecting mouse lung tissue, how to differentiate cancer cell in the alveoli or in the lung capillary tract? Also, authors need to show some basic quality control metric of the screen, at least in the supplementary to demonstrate the coverage and selection power of the screen.\n2. PIEZO1 and its sensing of the compression force were shown to affecting the expression level of ALDH1B1. Does depletion of PIEZO1 therefore also affecting the survival of the tumor cells in tight capillaries? It seems this part of data is missing from the Figure 3.\n3. Liu et al. have elucidated the signaling pathway from compression force sensing to upregulation of ALDH1B1. However, there remain one missing piece of the puzzle: how PIEZO1 activate CSK23? PIEZO1 is a calcium channel that activated by force, could there be a calcium sensing messenger between PIEZO1 and CSK23?\n4. In Figure 6d, without any CSK23 depletion, IKKbeta pS177/181 level is lower in compression 1hr condition than the compression 0.5hr condition, which is contradicting with result in Figure 5b. What causes this discrepancy? The authors should quantify their western blot results and show statistical replicates.\nMinor concerns:\n1. The authors should introduce briefly (a sentence is sufficient) on the background of A549 cell line, which will help the reader understand rationale of using it.\n2. Figure 2a, right panel. It would be interesting to see the statistical comparison between group 1 and 3 (i.e., negative control vs shALDH1B1+ALDH1B1 rescue). It seems rescue on has even lower cell death rate.\n3. Please explain the assay briefly for Figure 2f. For example, what’s the green/red ratio. It would be easier to understand for the audience that are not familiar to ferroptosis.\n4. Although treatment is not the focus of the study, the authors should attempt to discuss about the therapeutic potential (pros and cons) of ALDH1B1 inhibitor (or any method perturbing this cancer adaption pathway.", "reviewer_2": "This manuscript presents a CRISPR knockout screen targeting 1,684 metabolic enzymes in a mouse model of lung cancer metastasis. The authors identify ALDH1B1, a member of the aldehyde dehydrogenase family, as essential for tumor cell survival within physically confining capillaries. Mechanistically, they propose that compressive forces activate casein kinase 2A3 (CSNK2A3), which in turn phosphorylates IKKβ (IκB kinase subunit beta) at serine residues 177 and 181. This phosphorylation event is suggested to activate the NF-κB pathway, leading to upregulation of ALDH1B1 expression. The increased expression of ALDH1B1 is proposed to enhance aldehyde detoxification, suppressing ferroptosis and supporting tumor cell survival in confined environments, ultimately facilitating lung cancer metastasis. They further suggest that genetic or pharmacological inhibition of CSK23 or ALDH1B1 could impair lung cancer metastasis.\nThe central novelty and impact of the study lies in linking mechanical compression within capillaries to changes in ALDH isoform expression and promoting metastasis via ferroptosis suppression. The signaling work is of high quality and rigor, and the graphical summary at the end of the manuscript is especially well-done. However, despite the novelty, there are several significant concerns that must be addressed before publication:\nConcerns\n1. These was limited scope of ALDH isoform screening. Specifically, the ALDH family includes 18 isoforms, many of which are implicated in cancer progression and metastasis. It is unclear whether all ALDH isoforms were included in the gRNA screen targeting 1,684 metabolic enzymes. Since isoform redundancy is well-documented—where suppression of one ALDH isoform results in compensatory upregulation of another—focusing solely on ALDH1B1 is insufficient. The manuscript must clarify whether other ALDH isoforms were also screened and ruled out. Controls targeting other ALDH isoforms (e.g., ALDH1A3, ALDH2, ALDH3A1) should be included to confirm the specificity of the phenotype.\n2. There are limitations of the experimental metastasis assay that are not addressed. Specifically, in experimental metastasis assays, many cancer cells accumulate in the lungs initially, but the majority are naturally cleared within 24 hours. The authors need to clarify whether the genes identified are truly mediating survival in the confined capillaries or merely affecting initial accumulation. A clear distinction between transient lodging and sustained survival in capillaries must be made.\n3. Figures as presented suggest cell line limitation and lack of generalizability. Specifically, the study relies almost entirely on the A549 lung cancer cell line. To demonstrate robustness and reproducibility, results must be validated across additional lung cancer cell line and ideally across different cancer types. Partial validation of some experimentation in only the H1299 cell line is not sufficient. Figures throughout the manuscript should be updated to include these validations.\n4. There is concern related to the scope of confinement in the metastatic cascade. Specifically, the metastatic process involves several stages of physical confinement—not just within capillaries but also during tissue migration, intravasation, circulation, and extravasation. The manuscript currently focuses only on capillary confinement. The authors must experimentally address whether the described mechanism applies more broadly to other stages of the metastatic cascade.\n5. There is major concern regarding the possible redundancy among ALDH isoforms in metastasis. Specifically, given the overlap in detoxification function across ALDH isoforms, it's likely that multiple isoforms could suppress ferroptosis under confined conditions. The manuscript must explore and experimentally demonstrate whether other ALDH isoforms are also upregulated or functionally active in response to mechanical stress and assess their potential contributions.\n6. The therapeutic implications are overstated is several parts of the manuscript. Specifically, the suggestion that ALDH1B1 could be a promising therapeutic target is problematic. Many prior efforts to target specific ALDH isoforms have failed due to functional compensation by other isoforms. A more realistic approach would be to explore broad-spectrum ALDH inhibitors, which is not currently tested or discussed in the manuscript.\n7. Calibration of the in vitro model validation for mechanical compression is a concern. Specifically, the BioPress well system used to model mechanical compression must be better validated. The authors need to demonstrate that the in vitro forces and extracellular matrix (ECM) conditions are comparable to those experienced by tumor cells in capillaries or at other sites in the metastatic cascade. The contribution of ECM stiffness to gene expression changes should also be evaluated since it is significant in the scientific literature.\n8. Western blot quality and quantification needs improvement. Specifically, several western blot images appear overexposed for the loading controls, which may not be within the linear detection range. The authors should provide lighter exposures and include quantitative analysis normalized to loading controls.\n9. There is weakness of human tumor correlation data (Figure 7). Specifically, the correlative data from human tumors presented in Figure 7 are interesting but lack depth. Stronger validation using larger datasets or orthogonal approaches (e.g., IHC, RNA-seq, or survival analysis) would improve the translational relevance of this finding.", "reviewer_3": "The paper from Liu et al (NCOMMS) proposes that compression force in capillaries activates NF-kB during metastasis to promote survival of tumor cells through upregulation of ALDH1B1. The authors have used metastasis models (tail vein injection and intra-cardiac injection) to measure metastasis and analyze tumor cells in capillaries. In vitro studies, using confined pore transwell assays indicate that CSK23 is activated to phosphorylate IKK to promote NF-kB activation which drives expression of ALDH1B1. Mutant ALDH1B1 is inactive in promoting the confinement-induced cell survival response, consistent with the effect of ALDH1B 1 on acetaldehydes.\nWhile the cell-based studies are consistent with the hypothesis, there is a concern with the tumor metastasis studies. Human lung cancer patient samples were analyzed to correlate with animal and cell-based stdies. The authors have failed to acknowledge the extensive publication history on ALDH1B1 in cancer (over a 100 publications), where it is described to promote a variety of oncogenic phenotypes such as control of cancer stem cells and beta-catenin signaling (both involved in metastasis). Lack of consideration of these oncogenic phenotypes confounds interpretation of some of the proposed results. Additional concerns are outlined below.\n1) In Fig. 1, the authors conclude that loss of ALDH1B1 in tumors promotes metastasis via effects on survival of tumor cells in capillaries. Given the significant role of ALDH1B1 in several cancer phenotypes (unreferenced, and see statement above), the authors cannot conclude that the loss on metastasis is solely through cell death in capillaries. In this regard, can the authors show that the same number of cells reach the lung for WT vs ALDH1B1 knockdown? Is that what is mean to be shown in fig. 1g? The authors need to analyze the number of mets that form and not total GFP intensity since loss of ALDH1B1 will likely reduce the growth of the metastases. The authors utilize TCGA data to indicate that elevated levels of ALDH1B1 mRNA is linked with metastasis and survival.\n2) Related to the point above, the authors use Kaplan-Meier human data to argue that ALDH1B1 promotes metastasis. In the cases with higher ALDH1B1, it could be that the primary tumor is larger, has more cancer stem cells, or is resistant to therapy. Additionally, what is measured in the Kaplan-Meier analysis is ALDH1B1 RNA expressed in the tumor and not in tissue capillaries, thus making this point disconnected from the proposed mechanism of upregulation in confined capillaries. The lack of consideration of additional oncogenic phenotypes for ALDH1B1 is a significant flaw in the manuscript.\n3. For the studies shown in Fig. 2 related to survival and other effects on confined transwell pore assays, the authors need to show side by side analysis with unconfined cells. In the cell studies related to suppression of ferroptosis by ALDH1B1, did the authors analyze apoptosis? (this relates to a point below).\n4. For the metastasis studies using the ALDH1B1 inhibitor, again the authors need to show effects on metastasis numbers and size, and not luciferase intensity (this is focused on the concept that inhibiting ALDH1b1 will likely block growth of mets – unrelated to effects on actually promoting metastasis.\n5. Fig. 3 shows that the mechanosensory protein PIEZO1 is required for activation of ALDH1B1. Effects are called “dramatic” but are 2-fold or less.\n6. PDTC blocked the upregulation of ALDH1B1, and the authors took to mean that the IKK/NF-kB pathway was involved. Note that PDTC can block a number of pathways. They authors note that levels of RelA are increased in compression/confinement but this experiment does not measure nuclear RelA. Knockdown of RelA blocked compression/confinement-induced ALDH1B1. The authors linked confinement induced cell death with ferroptosis (and see comment below). As before, confinement results with non-confinement results need to be placed together.\n7. Compression induces the phosphorylation of IKKbeta, and this correlated with p-IkBa (in the figures the authors need to refer to this correctly – IkappaB-alpha - with Greek letters). In Fig 5d, the effect on an NF-kB reporter is relatively minimal. Knockdown of IKKbeta was shown to reduce ALDH1B1 levels.\n8. The authors found that CSK3 associates with IKKbeta during tumor cell compression and promotes IKKbeta activity, Knockdown of CSK3 leads to loss of ALDH1B1 expression and promoted cell death.\n9. In Fig. 6, does knockdown of CSK3 and the CSK3 inhibitor block mets (number and size) – see points above. Like ALDH1B1, CSK3 is linked with oncogenic phenotypes – not referenced. The question is whether loss of CSK3 truly affecting metastasis or simply growth of the tumors once they have metastasized.\n10. Fig. 7 links ALDH1 levels with capillary tumor cells and metastasis in lung cancer patients. These experiments are examining migration of cells from the tumor site to relate to distal metastasis and survival. There are several questions/concerns. When the authors refer to unconstrained tumor cells, are they simply referring to tumor? The cell death measurement is TUNEL assays which is associated with apoptosis and less with ferroptosis (see point above – did they authors measure apoptosis in their cell-based studies? Lastly, the text says that the authors are measuring nuclear RelA, but there is nothing to describe how that was measured. It seems that they are staining for whole cell RelA – but only nuclear would be meaningful for the overall hypothesis.", "reviewer_4": "The manuscript by Liu et al. investigates the role of ALDH1B1 in promoting lung cancer metastasis by enhancing cell survival and motility. The authors suggest that ALDH1B1 is regulated by the CSK23-RelA/NF-κB pathway and propose Piezo1 as a mechanosensor that initiates this signaling pathway in lung cancer.\nThe use of a CRISPR knockout screening system to identify key metabolic enzymes is a sophisticated and logically sound approach. However, the method used to identify Piezo1, specifically through the application of a tumor microenvironment-mimicking platform, is not appropriate for lung cancer. Given the presence of numerous other mechanosensitive calcium channels, such as those in the Orai or TRPV families, the authors have not sufficiently explored alternative possibilities for mechanosensing in lung cancer metastasis. Consequently, the claim that Piezo1 is the primary mechanosensor driving CSK23-RelA signaling lacks a strong logical foundation.\nAdditionally, it remains unclear whether aldehyde levels are elevated in the confined space of the lung. The authors should demonstrate a clear relationship between aldehyde production and the metastatic progression of lung cancer to support their hypothesis.\nFurthermore, the study reports brain metastasis occurring within seven days after lung cancer inoculation into the lung. Is there any existing literature supporting such a rapid metastatic timeline? The authors should provide references or additional experimental validation for this observation.\nThe definition and generation of confined space in this study also require further clarification. While lung cancer cells may experience mechanical constraints within capillaries during metastasis, anoikis or pulsatile hydrostatic pressure could play a more significant role than simple confinement. The authors have not considered the possibility that different types of mechanical stress might influence lung cancer behavior. A more comprehensive discussion of these factors is necessary to strengthen the study’s conclusions.\nOverall, while the study presents an interesting perspective on ALDH1B1-mediated metastasis, the manuscript requires a more logically structured approach, further experimental validation, and consideration of alternative mechanosensors and mechanical stress factors.", "reviewer_5": "In this manuscript, Liu et al. describe metabolic adaptations that enable cancer cells to survive and metastasize within confining microenvironment. Using an in vivo CRISPR-based knockout screening system, the authors identify ALDH1B1 as a critical target for tumor cell survival within capillaries. Mechanistically, they demonstrate that compressive forces activate CSK23, which phosphorylates IKKβ at S177/181, triggering NF-κB-dependent upregulation of ALDH1B1. Elevated ALDH1B1 enhances aldehyde detoxification, suppresses ferroptosis, and promotes tumor cell survival, highlighting CSK23/ALDH1B1 as potential therapeutic targets for metastatic cancer. Overall, this work uncovers a connection between mechanical stress, NF-κB signaling, and aldehyde metabolism during metastatic progressio. Addressing the major concerns would strengthen the manuscript, the detail review is outlined below:\nMajor points and suggestions：\n1. The authors used tail vein injection of gene-edited A549 cells to observe genetic differences in cells within pulmonary capillaries. How did the authors exclude the possibility that gene knockout affected cell proliferation, leading to false-positive results of reduced vascular cell counts? Additionally, is this model original? Is there any other literature that uses this method?\n2. The authors proposed that confined cells sense mechanical stress through Piezo1 and ultimately express ALDH1B1 to mitigate aldehyde toxicity. Why is this pathway specific to confined cancer cells? Why do normally growing cells not require similar mechanisms to maintain growth? Is it because confined cells face higher ferroptotic stress? Please provide experimental data to clarify.\n3. In Patient 2’s results (not enlarged), ALDH1B1 expression showed poor co-expression with CD31, and high ALDH1B1 expression was apparently observed in non-confined regions. However, the statistical plot in Fig. 7b does not reflect this trend. The authors should check the representative results, carefully verify the statistical methods, and accurately present the statistical outcomes.\nMinor comments:\n1. Western blot images in high-quality journals should include molecular weight markers.\n2. No information on tumor patient tissues or ethical approval is provided in the manuscript.\n3. Please confirm the appropriateness of using the GEO database (GSE72094), which includes tumors with Kras and other genes mutations.\n4. In Fig. 1h, TUNEL-positive areas do not fully overlap with ALDH1B1-knockdown cells, and non-tumor dead cells may affect the result. The authors should re-select representative images or quantify the percentage of co-expression between TUNEL-positive and mCherry-positive regions.\n5. The method section should highlight the sampling of pulmonary capillaries.\n6. Please carefully verify all results for data integrity (e.g., no endpoint data for the experimental group in Fig. 2m).\n7. Nuclear and cytoplasmic fractions in Fig. 5c should be presented on the same gel to compare.\n8. Total protein loading controls are required for phosphoprotein detection in Fig. 6d.\n9.Supplementary images are excessively compressed, compromising readability.", "reviewer_6": "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_7": "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."}, "author_response": "Response: We thank the reviewer for the positive comments and constructive suggestions, which greatly strengthens the manuscript.\nMajor concerns:\nPoint 1. Authors should explain the screen setup in more details. For example, when dissecting mouse lung tissue, how to differentiate cancer cell in the alveoli or in the lung capillary tract? Also, authors need to show some basic quality control metric of the screen, at least in the supplementary to demonstrate the coverage and selection power of the screen.\nResponse: Thanks for your insightful comments.\n1. According to reviewer's suggestion, we have supplemented more detailed descriptions of the screening criteria in methods section (Page: 31, line: 869) and figure legends (Extended Data Fig. 1a), and refined the screening flowchart (Fig. 1a) with additional details to clearly highlight the key steps in the screening process.\n2. Over 90% of tumor cells remained lodged in the lung capillary tract at the 24 h post-tail vein injection in our mouse model, consistent with the previous studies that the majority of tumor cells reside in the pulmonary capillaries within 24 hours after tail vein injection (PMID: 10613833, PMID: 26855844).\n3. The basic quality control data for CRISPR library: >95% of sgRNAs were effectively detected. In CRISPR screening, approximately 11.9% of sgRNAs were depleted in the control group (average reads/sgRNA = 273), while 19.5% were depleted in the experimental group (average reads/sgRNA = 231), indicating selection power of the screening system. As suggested by the reviewer, these data are presented in the Extended Data Fig. 1a.\nPoint 2. PIEZO1 and its sensing of the compression force were shown to affecting the expression level of ALDH1B1. Does depletion of PIEZO1 therefore also affecting the survival of the tumor cells in tight capillaries? It seems this part of data is missing from the Figure 3.\nResponse: As suggested by the reviewer, we depleted PIEZO1 in tumor cells and observed that PIEZO1 depletion markedly increased tumor cell death in confining pores (Fig. 3k). Animal experiments showed that PIEZO1 depletion accelerated the diminishment of bioluminescence signals of tumor cells in the lungs (Fig. 3l). Further TUNEL assay on these lung tissues showed that PIEZO1 depletion increased tumor cell death in lung capillaries (Fig. 3m). Additionally, we found that PIEZO1 depletion inhibited ALDH1B1 expression in tumor cells confined within lung capillaries (Fig. 3n). These results were included in our revised manuscript (Page: 11, line: 290).\nPoint 3. Liu et al. have elucidated the signaling pathway from compression force sensing to upregulation of ALDH1B1. However, there remain one missing piece of the puzzle: how PIEZO1 activate CSK23? PIEZO1 is a calcium channel that activated by force, could there be a calcium sensing messenger between PIEZO1 and CSK23?\nResponse: Thank you for the insightful suggestion. The calcium signals are normally transduced by calcium-sensing messengers such as calmodulin (CaM), calpain, and calcineurin to regulate downstream signaling networks (PMID: 28622523). Among these, CaM emerges as a central Ca²⁺ sensor and molecular integrator, dynamically regulating protein-protein interactions and scaffolding macromolecular complex assembly through its target-binding domains (PMID: 2479144, PMID: 29247668). Thus, we tested whether CaM is the calcium messenger between PIEZO1 and CSK23. We treated A549 and H1299 cells with CaM-selective inhibitor W7 and observed that W7 treatment markedly attenuated compression-induced interaction between CSK23 and IKKβ (Extended Data Fig. 9k,l). Meanwhile, we also assessed whether calpain or calcineurin also contributes to PIEZO1-mediated CSK23 activation. We treated A549 cells with the calpain inhibitor MDL-28170 or the calcineurin inhibitor FK506. Unlike W7, MDL-28170 treatment had no significant effect on the compression-induced CSK23-IKKβ interaction, whereas FK506 treatment partially abrogated this interaction (Extended Data Fig. 9m,n). These results along with the results of Fig. 3 and Fig. 5 suggests that CaM is very likely the calcium messenger between PIEZO1 and CSK23. These results were included in our revised manuscript (Page: 13, line: 367).\nPoint 4. In Figure 6d, without any CSK23 depletion, IKKbeta pS177/181 level is lower in compression 1hr condition than the compression 0.5hr condition, which is contradicting with result in Figure 5b. What causes this discrepancy? The authors should quantify their western blot results and show statistical replicates.\nResponse: We apologized for the confusion. We reviewed the experimental records and concluded that this discrepancy likely resulted from using higher-passage A549 cells in original Fig. 6d. Thus, we revived low-passage A549 cells and repeated the experiments, which showed that IKKβ pS177/181 level was higher at 1 hr than 0.5 hr upon compression (Fig. 6d), consistent with the result of Fig. 5b.\nMinor concerns:\nPoint 1. The authors should introduce briefly (a sentence is sufficient) on the background of A549 cell line, which will help the reader understand rationale of using it.\nResponse: This point was well taken. We included a brief description of A549 cells (Page: 5, line: 90). “we conducted CRISPR knockout screening using lung adenocarcinoma-derived A549 cells-an epithelial cell line with robust migratory and invasive capacities.” (PMID: 9743595, PMID: 33659549).\nPoint 2. Figure 2a, right panel. It would be interesting to see the statistical comparison between group 1 and 3 (i.e., negative control vs shALDH1B1+ALDH1B1 rescue). It seems rescue on has even lower cell death rate.\nResponse: We reanalyzed the data and observed no significant difference between negative control group and shALDH1B1+ALDH1B1 rescue group.\nPoint 3. Please explain the assay briefly for Figure 2f. For example, what’s the green/red ratio. It would be easier to understand for the audience that are not familiar to ferroptosis.\nResponse: This point was well taken. We have expanded details in the Methods section (Page: 36, line: 1031). BODIPY 581/591 C11 is a ratiometric fluorescent probe that quantitatively measures lipid peroxidation during ferroptosis. In its reduced state, the probe emits red fluorescence (ex/em: 581/591 nm), which shifts to green fluorescence (ex/em: 488/510 nm) upon oxidation by lipid hydroperoxides. The relative lipid peroxidation (originally termed “green/red ratio”) was calculated as the intensity ratio of oxidized (green) to reduced (red) fluorescence signals (F510/F591). We have uniformly designated “green/red ratio” as “Rel. lipid peroxidation” in the current manuscript.\nPoint 4. Although treatment is not the focus of the study, the authors should attempt to discuss about the therapeutic potential (pros and cons) of ALDH1B1 inhibitor (or any method perturbing this cancer adaption pathway.\nResponse: As suggested by the reviewer, we added a discussion on the therapeutic promise and clinical challenges of ALDH1B1 inhibitor as well as CSK23 inhibitor (Page: 17, line: 475):\nOur multimodal validation establishes ALDH1B1 as a therapeutically viable target for metastatic intervention. Both genetic depletion and pharmacological inhibition of ALDH1B1 significantly reduced metastatic burden and extended median survival in nude mouse models, highlighting its potential to suppress metastatic recurrence in post-operative cancer patients. However, clinical translation requires careful mitigation of on-target toxicities, primarily due to ALDH1B1's physiological roles in intestinal stem cell maintenance (PMID: 24789370) and pancreatic regeneration (PMID: 31548432). We propose targeting circulating tumor cells during early dissemination-a phase characterized by limited population size and unique metabolic dependencies, which may enable effective metastasis blockade via transient, submaximal ALDH1B1 inhibition. Additionally, ALDH1B1 inhibitors must address the challenge of compensatory activation by other ALDH isoforms, a critical drawback of isoform-selective agents, to enhance therapeutic efficacy and minimize potential resistance mechanisms.\nCurrent broad-spectrum aldehyde dehydrogenase (ALDH) inhibitors, such as disulfiram (DSF) and diethylaminobenzaldehyde (DEAB), exhibit promising antitumor activity by simultaneously targeting multiple ALDH isoforms. Although their pan-ALDH inhibitory mechanism circumvents functional compensation, clinical advancement is still hampered by on-target toxicities stemming from broad-spectrum activity, including dose-limiting hepatotoxicity, cardiotoxicity, and off-tissue damage to vulnerable cell populations (PMID: 31515079).\nAlthough CSK23, a member of the CK2 family, has been associated with lung cancer susceptibility (PMID: 20625391) and exhibits debated tumor-suppressive activity in hepatocellular carcinoma (PMID: 33582094), its broader role in cancer-particularly in metastasis-remains poorly characterized. In our study, we demonstrate the critical role of CSK23 in brain metastasis of lung cancer cells. Pharmacological inhibition by silmitasertib (CX-4945), a clinically available inhibitor targeting CSK23, significantly reduced metastatic burden and improved survival, suggesting that targeting CSK23 represents a promising strategy to prevent post-surgical metastatic recurrence in lung cancer.\nReviewer #2 (Remarks to the Author):\nReviewer Summary:\nThis manuscript presents a CRISPR knockout screen targeting 1,684 metabolic enzymes in a mouse model of lung cancer metastasis. The authors identify ALDH1B1, a member of the aldehyde dehydrogenase family, as essential for tumor cell survival within physically confining capillaries. Mechanistically, they propose that compressive forces activate casein kinase 2A3 (CSNK2A3), which in turn phosphorylates IKKβ (IκB kinase subunit beta) at serine residues 177 and 181. This phosphorylation event is suggested to activate the NF-κB pathway, leading to upregulation of ALDH1B1 expression. The increased expression of ALDH1B1 is proposed to enhance aldehyde detoxification, suppressing ferroptosis and supporting tumor cell survival in confined environments, ultimately facilitating lung cancer metastasis. They further suggest that genetic or pharmacological inhibition of CSK23 or ALDH1B1 could impair lung cancer metastasis.\nThe central novelty and impact of the study lies in linking mechanical compression within capillaries to changes in ALDH isoform expression and promoting metastasis via ferroptosis suppression. The signaling work is of high quality and rigor, and the graphical summary at the end of the manuscript is especially well-done. However, despite the novelty, there are several significant concerns that must be addressed before publication:\nResponse: We thank the reviewer for the positive comments and insightful suggestions, which greatly strengthens the manuscript.\nPoint 1. These was limited scope of ALDH isoform screening. Specifically, the ALDH family includes 18 isoforms, many of which are implicated in cancer progression and metastasis. It is unclear whether all ALDH isoforms were included in the gRNA screen targeting 1,684 metabolic enzymes. Since isoform redundancy is well-documented—where suppression of one ALDH isoform results in compensatory upregulation of another—focusing solely on ALDH1B1 is insufficient. The manuscript must clarify whether other ALDH isoforms were also screened and ruled out. Controls targeting other ALDH isoforms (e.g., ALDH1A3, ALDH2, ALDH3A1) should be included to confirm the specificity of the phenotype.\nResponse: Thank you for the insightful suggestions.\n1. Our CRISPR screening targeting 1684 metabolic enzymes comprehensively covered all 18 ALDH isoforms, including ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH1L1, ALDH1L2, ALDH2, ALDH3A1, ALDH3A2, ALDH3B1, ALDH3B2, ALDH4A1, ALDH5A1, ALDH6A1, ALDH7A1, ALDH8A1, ALDH9A1, ALDH18A1.\n2. To identify the metabolic enzyme required for tumor cell migration in confining spaces in vivo, we performed CRISPR screening, which identified ALDH1B1 as the most significantly enriched ALDH isoform, with ALDH6A1 and ALDH9A1 emerging as secondary candidates (Extended Data Fig. 2d). To determine the role of ALDH6A1 and ALDH9A1 on tumor cell survival in confining spaces, we established ALDH6A1 and ALDH9A1 depleted A549 cells (Extended Data Fig. 2e,f). ALDH9A1 depletion increased tumor cell death in confining pores (Extended Data Fig. 2g), though its effect was much weaker than ALDH1B1 ablation (Extended Data Fig. 2c). In contrast, ALDH6A1 depletion showed no significant effects on confined tumor cell survival (Extended Data Fig. 2h). We also examined ALDH9A1 expression under mechanical compression. Quantitative PCR (qPCR) analysis revealed modest but statistically upregulation of ALDH9A1 under compression (Extended Data Fig. 6u,v). However, this mechanosensitive upregulation was markedly attenuated compared to ALDH1B1's response (Fig. 3g and Extended Data Fig. 6m). Transcriptomic analysis of the GSE288929 dataset (A549 cells) further demonstrated that baseline ALDH1B1 expression exceeded ALDH9A1 levels (Extended Data Fig. 2i), potentially explaining the dominant functional role of ALDH1B1 in confining spaces. Collectively, among ALDH isoforms, ALDH1B1 emerges as the master regulator governing tumor cell survival under spatial confinement. These results were included in the revised manuscript (Page: 6, line: 124 and page: 10, line: 277).\nPoint 2. There are limitations of the experimental metastasis assay that are not addressed. Specifically, in experimental metastasis assays, many cancer cells accumulate in the lungs initially, but the majority are naturally cleared within 24 hours. The authors need to clarify whether the genes identified are truly mediating survival in the confined capillaries or merely affecting initial accumulation. A clear distinction between transient lodging and sustained survival in capillaries must be made.\nResponse: Thank you for the constructive comment.\n1. Initial lodging (30 min post-tail vein injection): No significant difference in fluorescence intensity was observed between ALDH1B1 depletion and control groups, indicating that ALDH1B1 depletion does not affect transient cell lodging (Fig. 1e, right, top lane).\n2. Sustained survival (24 h post-tail vein injection): However, in sustained survival assays, ALDH1B1 depletion significantly accelerated the decline in the bioluminescence signals of tumor cells in lungs (Fig. 1e, right, bottom panel). Moreover, TUNEL assay on dissected lung tissues showed that ALDH1B1 depletion dramatically increased tumor cell death in lung capillaries (Fig. 1f). These results establish ALDH1B1 as a regulator of cell survival in confinement, rather than a mediator of transient lodging.\nPoint 3. Figures as presented suggest cell line limitation and lack of generalizability. Specifically, the study relies almost entirely on the A549 lung cancer cell line. To demonstrate robustness and reproducibility, results must be validated across additional lung cancer cell line and ideally across different cancer types. Partial validation of some experimentation in only the H1299 cell line is not sufficient. Figures throughout the manuscript should be updated to include these validations.\nResponse: This point was well taken.\n1. The H1299 cells have been comprehensively validated for all critical experimental findings in original manuscript, including: (1) the role of ALDH1B1 in tumor cell survival in confining space (Extended Data Fig. 3 and Extended Data Fig. 5); (2) NF-κB-mediated transcriptional regulation of ALDH1B1 in confining space (Extended Data Fig. 6, Extended Data Fig. 7 and Extended Data Fig. 8) and (3) the mechanotransduction cascade wherein confinement activates ALDH1B1 through the CSK23-IKKβ-NF-κB axis (Extended Data Fig. 9 and Extended Data Fig. 10).\n2. As suggested by the reviewer, we expanded our validation to the H460 lung cancer cells.\n1) We depleted ALDH1B1 in H460 cells and rescued the cells with wild-type ALDH1B1 (WT) or ALDH1B1 enzymatic-dead (ED) mutant (Extended Data Fig. 3d). We found that ALDH1B1 depletion markedly increased tumor cell death in confining pores (Extended Data Fig. 3f). Transwell migration assay showed that ALDH1B1 depletion greatly suppressed the confined migration of tumor cells (Extended Data Fig. 3h), while rescued expression of ALDH1B1 WT almost completely restored the survival and migratory capability of ALDH1B1-depleted cells in confining pores, excluding the off-target possibility of ALDH1B1 shRNA (Extended Data Fig. 3f,h). However, tumor cells rescued with ALDHB1 ED could not recover the survival and the migratory capability of ALDH1B1-depleted cells in confinement.\n2) We also explored whether compressive force induces ALDH1B1 upregulation in confined H460 cells. Our results showed that the mRNA and protein levels of ALDH1B1 were dramatically upregulated in the cells after compression in a time-dependent manner (Extended Data Fig. 6o,p).\n3) We also established CSK23-depleted H460 cells (Extended Data Fig. 9d), and found that CSK23 depletion inhibited IKKβ S177/181 and IκBα S32/36 activation under compressive force (Extended Data Fig. 9g).\n4) We also examined whether CSK23 regulates cell survival in confined cells by activating NF-κB, we overexpressed RelA in CSK23-depleted H460 cells (Extended Data Fig. 10c). We found that CSK23 depletion increased cell death rate and inhibited the migratory capability of tumor cells in confining spaces, while RelA overexpression restored the survival and migration of CSK23-depleted cells in confinement (Extended Data Fig. 10h,k). Meanwhile, overexpression of ALDH1B1 can also restored the survival and migration of CSK23-depleted H460 cells in confining spaces (Extended Data Fig. 10n,q,s).\nPoint 4. There is concern related to the scope of confinement in the metastatic cascade. Specifically, the metastatic process involves several stages of physical confinement—not just within capillaries but also during tissue migration, intravasation, circulation, and extravasation. The manuscript currently focuses only on capillary confinement. The authors must experimentally address whether the described mechanism applies more broadly to other stages of the metastatic cascade.\nResponse: Thank you for the constructive suggestions.\nAs suggested by the reviewer, we also investigated whether ALDH1B1 regulates tumor cell survival during extravasation through endothelial gaps. Most surviving tumor cells begin extravasation between 24 and 36 hours after intravenous injection (PMID: 31243371, PMID: 20530574, PMID: 26855844). Since visualizing the survival of cells trapped within endothelial gaps proved challenging, we instead quantified both the cells remaining in capillaries and those that had completed extravasation 48 hours after injection, when most cells completed extravasation. Immunofluorescence analysis showed that ALDH1B1 depletion led to a 2-fold reduction in tumor cells retained within capillaries and a 5-fold decrease in extravasated tumor cells (Fig. 1g), suggesting that ALDH1B1 likely also contributes to the survival of tumor cells during extravasation. These data are now included in the revised manuscript (Page: 6, line: 137).\nPoint 5. There is major concern regarding the possible redundancy among ALDH isoforms in metastasis. Specifically, given the overlap in detoxification function across ALDH isoforms, it's likely that multiple isoforms could suppress ferroptosis under confined conditions. The manuscript must explore and experimentally demonstrate whether other ALDH isoforms are also upregulated or functionally active in response to mechanical stress and assess their potential contributions.\nResponse: We thank the reviewer for their insightful suggestions.\n1. Our CRISPR screening targeting 1684 metabolic enzymes comprehensively covered all 18 ALDH isoforms, including ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH1L1, ALDH1L2, ALDH2, ALDH3A1, ALDH3A2, ALDH3B1, ALDH3B2, ALDH4A1, ALDH5A1, ALDH6A1, ALDH7A1, ALDH8A1, ALDH9A1, ALDH18A1.\n2. To identify the metabolic enzyme required for tumor cell migration in confining spaces in vivo, we performed CRISPR screening, which identified ALDH1B1 as the most significantly enriched ALDH isoform, with ALDH6A1 and ALDH9A1 emerging as secondary candidates (Extended Data Fig. 2d). To determine the role of ALDH6A1 and ALDH9A1 on tumor cell survival in confining spaces, we established ALDH6A1 and ALDH9A1 depleted A549 cells (Extended Data Fig. 2e,f). ALDH9A1 depletion increased tumor cell death in confining pores (Extended Data Fig. 2g), though its effect was much weaker than ALDH1B1 ablation (Extended Data Fig. 2c). In contrast, ALDH6A1 depletion showed no significant effects on confined tumor cell survival (Extended Data Fig. 2h). We also examined ALDH9A1 expression under mechanical compression. Quantitative PCR (qPCR) analysis revealed modest but statistically upregulation of ALDH9A1 under compression(Extended Data Fig. 6u,v). However, this mechanosensitive upregulation was markedly attenuated compared to ALDH1B1's response (Fig. 3g and Extended Data Fig. 6m). Transcriptomic analysis of the GSE288929 dataset (A549 cells) further demonstrated that baseline ALDH1B1 expression exceeded ALDH9A1 levels (Extended Data Fig. 2i), potentially explaining the dominant functional role of ALDH1B1 in confining spaces. Collectively, among ALDH isoforms, ALDH1B1 emerges as the master regulator governing tumor cell survival under spatial confinement. These results were included in the revised manuscript (Page: 6, line: 124 and page: 10, line: 277).\nPoint 6. The therapeutic implications are overstated is several parts of the manuscript. Specifically, the suggestion that ALDH1B1 could be a promising therapeutic target is problematic. Many prior efforts to target specific ALDH isoforms have failed due to functional compensation by other isoforms. A more realistic approach would be to explore broad-spectrum ALDH inhibitors, which is not currently tested or discussed in the manuscript.\nResponse: We appreciate the reviewer’s concerns on the challenges of ALDH isoform-specific targeting. A discussion on the therapeutic promise and clinical challenges of broad-spectrum ALDH inhibitors were included in the revised manuscript (Page: 17, line: 475):\nWhile broad-spectrum ALDH inhibitors such as disulfiram (DSF) and diethylaminobenzaldehyde (DEAB) can overcome functional compensation among isoforms, their clinical translation has been limited by on-target toxicities-including dose-limiting hepatotoxicity, cardiotoxicity, and off-tissue damage to vulnerable cell populations-due to indiscriminate inhibition of multiple ALDH family members (PMID: 31515079).\nIn contrast, our study provides multimodal evidence that ALDH1B1 may represent a more tractable target. For example, CRISPR/Cas9 screens specifically identified it as one of the most enriched essential genes for tumor cell survival under spatial confinement compared to other ALDH isoforms. Both genetic depletion and pharmacological inhibition of ALDH1B1 significantly reduced metastatic burden and extended median survival in preclinical models without observed compensatory mechanisms by other ALDH isoforms. These findings demonstrate ALDH1B1's critical role in sustaining tumor cell survival within capillary networks, while simultaneously revealing its therapeutic promise as a target for inhibiting metastatic dissemination.\nFurthermore, a dual-inhibition strategy targeting both ALDH1B1 and ALDH9A1 may enhance therapeutic efficacy while demonstrating a more favorable toxicity profile compared to pan-ALDH inhibitors.\nPoint 7. Calibration of the in vitro model validation for mechanical compression is a concern. Specifically, the BioPress well system used to model mechanical compression must be better validated. The authors need to demonstrate that the in vitro forces and extracellular matrix (ECM) conditions are comparable to those experienced by tumor cells in capillaries or at other sites in the metastatic cascade. The contribution of ECM stiffness to gene expression changes should also be evaluated since it is significant in the scientific literature.\nResponse: Thank you for the constructive comments.\n1. The BioPress well system has been extensively used in multiple peer-reviewed studies (e.g., PMID: 36444299), establishing it as a reliable platform for precise modeling of mechanical compression in cellular microenvironments.\n2. The diameters of pulmonary capillaries range from 5 to 8 μm (PMID: 23606929). In microfluidic channels with analogous dimensions (5-8 μm), individual tumor cells experience compressive forces of approximately 80 nN (PMID: 33060332). Using the FlexCell system’s standard conversion formula, P (MPa) = (5.65 × Force (lbs)) / D (mm²), we determined that an applied pressure of 5 kPa in BioPress well system is equivalent to achieve the target cellular force load of 80 nN. We have included more detailed descriptions of the selection of in vitro applied forces in the Methods section (Page: 33, line 924).\n3. To determine whether matrix stiffness regulates ALDH1B1 expression, we used a photo-crosslinked GelMA 3D hydrogel system replicating physiological stiffness gradients. Based on reported lung tissue mechanics (0.5-5 kPa; PMID: 29274784), we tested three GelMA concentrations representing distinct ECM stiffness regimes: 10% (∼3 kPa, approximating normal lung), 12.5% (∼5 kPa, mimicking fibrotic regions), and 15% (∼9 kPa, representing pathological stiffness). Western blot analyses revealed no stiffness-dependent modulation of ALDH1B1 expression (Extended Data Fig. 6w), suggesting that ALDH1B1 regulation is independent of matrix stiffness. These results were included in the revised manuscript (Page: 10, line: 281).\nPoint 8. Western blot quality and quantification needs improvement. Specifically, several western blot images appear overexposed for the loading controls, which may not be within the linear detection range. The authors should provide lighter exposures and include quantitative analysis normalized to loading controls.\nResponse: This point was well taken. As suggested by the reviewer, we have now presented images with optimized exposure time (Fig. 3h, 3J, 4b, 5a, 5b, 5e, 5h, Extended Data Fig. 7b and Extended Data Fig. 8j). Additionally, we included quantitative analysis normalized to the loading control. (Fig. 3e, 3h, 4b, 4f, 6f, Extended Data Fig. 6p and Extended Data Fig. 9i).\nPoint 9. There is weakness of human tumor correlation data (Figure 7). Specifically, the correlative data from human tumors presented in Figure 7 are interesting but lack depth. Stronger validation using larger datasets or orthogonal approaches (e.g., IHC, RNA-seq, or survival analysis) would improve the translational relevance of this finding.\nResponse: Thank you for the constructive suggestion. Since conventional IHC and RNA-seq cannot reliably distinguish between intravascular tumor cells and extravascular tumor cells, we strengthened the clinical relevance of NF-κB-dependent regulation of ALDH1B1 expression by expanding the cohort to include 61 primary lung cancer samples. Immunofluorescence staining and quantitative image analysis were performed on all tumor samples. The updated results are presented in Fig. 7. These results were included in the revised manuscript (Page: 15, line: 420).\nReviewer #3 (Remarks to the Author):\nReviewer Summary:\nThe paper from Liu et al (NCOMMS) proposes that compression force in capillaries activates NF-kB during metastasis to promote survival of tumor cells through upregulation of ALDH1B1. The authors have used metastasis models (tail vein injection and intra-cardiac injection) to measure metastasis and analyze tumor cells in capillaries. In vitro studies, using confined pore transwell assays indicate that CSK23 is activated to phosphorylate IKK to promote NF-kB activation which drives expression of ALDH1B1. Mutant ALDH1B1 is inactive in promoting the confinement-induced cell survival response, consistent with the effect of ALDH1B 1 on acetaldehydes.\nWhile the cell-based studies are consistent with the hypothesis, there is a concern with the tumor metastasis studies. Human lung cancer patient samples were analyzed to correlate with animal and cell-based studies. The authors have failed to acknowledge the extensive publication history on ALDH1B1 in cancer (over a 100 publications), where it is described to promote a variety of oncogenic phenotypes such as control of cancer stem cells and beta-catenin signaling (both involved in metastasis). Lack of consideration of these oncogenic phenotypes confounds interpretation of some of the proposed results. Additional concerns are outlined below.\nResponse: We thank the reviewer for the constructive comments and insightful suggestions, which greatly strengthens the manuscript. In our discussion, we have incorporated existing reports on ALDH1B1's functional roles in cancer and contextualized these findings alongside our own discoveries.\nPoint 1. In Fig. 1, the authors conclude that loss of ALDH1B1 in tumors promotes metastasis via effects on survival of tumor cells in capillaries. Given the significant role of ALDH1B1 in several cancer phenotypes (unreferenced, and see statement above), the authors cannot conclude that the loss on metastasis is solely through cell death in capillaries. In this regard, can the authors show that the same number of cells reach the lung for WT vs ALDH1B1 knockdown? Is that what is mean to be shown in fig. 1g? The authors need to analyze the number of mets that form and not total GFP intensity since loss of ALDH1B1 will likely reduce the growth of the metastases.\nResponse: Thank you for the constructive comments.\n1. Accumulating evidence highlights that tumor cell survival in confining space represents a critical bottleneck step in metastatic progression (PMID: 27909339, PMID: 35256052, PMID: 37296072). Our study thus focused on the role of ALDH1B1 in tumor cell survival under spatially confining spaces. We agree with the reviewer that ALDH1B1 may also contribute to metastasis through other mechanisms, such as the regulation of cancer stem cell and β-catenin signaling. We thus modified the original statement to \"These results demonstrate that ALDH1B1 is required for tumor metastasis at least partially by enhancing tumor cell survival within capillaries and during extravasation\" (Page: 7, line: 165). Additionally, we have incorporated existing reports on the role of ALDH1B1 in metastasis and contextualized these findings alongside our own discoveries in the revised discussion (Page: 16, line: 458).\n2. At 0.5 h post tail vein injection, bioluminescence imaging of the mice showed comparable luciferase activity was observed between control cells and ALDH1B1-depleted cells (Fig. 1e), indicating a similar number of tumor cells reach the lung tissue. We have incorporated this result in the revised manuscript (Page: 6, line: 142).\n3. As suggested by the reviewer, we re-analyzed the effects of ALDH1B1 depletion on the number of brain metastases, which showed that ALDH1B1 depletion significantly reduced the number and size of brain metastatic lesions (Fig. 1i). These results are described in the revised manuscript (Page: 7, line: 162).\nPoint 2. The authors utilize TCGA data to indicate that elevated levels of ALDH1B1 mRNA is linked with metastasis and survival. Related to the point above, the authors use Kaplan-Meier human data to argue that ALDH1B1 promotes metastasis. In the cases with higher ALDH1B1, it could be that the primary tumor is larger, has more cancer stem cells, or is resistant to therapy. Additionally, what is measured in the Kaplan-Meier analysis is ALDH1B1 RNA expressed in the tumor and not in tissue capillaries, thus making this point disconnected from the proposed mechanism of upregulation in confined capillaries. The lack of consideration of additional oncogenic phenotypes for ALDH1B1 is a significant flaw in the manuscript.\nResponse: Thank you for the constructive comments.\n1. We have fully acknowledged that ALDH1B1 can contribute to tumor metastasis through multiple mechanisms. Our study uncovers a new mechanism of ALDH1B1-promoted tumor metastasis by supporting tumor cell survival under spatial confinement. In Discussion section (Page: 16, line: 458), we have systematically discussed multifaceted mechanisms orchestrated by ALDH1B1 in tumor metastasis (e.g., cancer stemness maintenance, therapy resistance).\n2. We agree with the reviewer that the correlation of ALDH1B1 expression with metastasis and survival in TCGA data may be due to its possible associations with primary tumor growth, cancer stem cell expansion, and therapy resistance. However, this TCGA data analysis, conducted immediately after the screening (Fig. 1a-1c), mainly serves as a crucial initial filter to exclude metabolic enzymes less relevant to metastasis, as identified in the CRISPR screen.\n3. We acknowledge the reviewer's point that, due to the limitations of bulk sequencing data from TCGA, the Kaplan-Meier analysis reflects ALDH1B1 RNA expression in tumor tissue rather than specifically in capillaries. To address this limitation, we performed immunofluorescence staining of ALDH1B1 in lung cancer patient specimens, which demonstrated that elevated ALDH1B1 expression specifically in intravascular tumor cells strongly correlates with metastatic recurrence (Fig. 7).\nPoint 3. For the studies shown in Fig. 2 related to survival and other effects on confined transwell pore assays, the authors need to show side by side analysis with unconfined cells. In the cell studies related to suppression of ferroptosis by ALDH1B1, did the authors analyze apoptosis? (this relates to a point below).\nResponse: Thank you for the constructive comments.\n1. As suggested by the reviewer, we reinstated the data from unconfined cells, which showed that ALDH1B1 depletion did not significantly influence lipid peroxidation (Extended Data Fig. 5a,b) or survival (Fig.2a and Extended Data Fig. 3e,f) in unconfined tumor cells. These results are described in the revised manuscript (Page: 6, line: 122 and page: 8, line: 205).\n2. We really appreciate this constructive comment from the reviewer. As suggested by the reviewer, we determined whether ALDH1B1 regulates cell apoptosis under confinement by examining caspase-3/7 activity in ALDH1B1-depleted tumor cells, which showed that caspase-3/7 activity was elevated in confined cells, which was exacerbated by ALDH1B1 depletion (Extended Data Fig. 5i). However, compared to its effect on apoptosis, ALDH1B1 depletion exerts a significantly stronger impact on ferroptosis in confined tumor cells (Extended Data Fig. 5a,b). These results are consistent with previous studies demonstrating that aldehydes induce both apoptosis (PMID: 16040627) and ferroptosis (PMID: 36274088), and that aldehydes derived from ALDH inhibition tend to promote ferroptosis (PMID: 37552043, PMID: 40233740, PMID: 32458004). Collectively, these results suggest that ALDH1B1 plays a dual protective role in confined tumor cells by suppressing both ferroptosis and apoptosis, with its anti-ferroptotic effect being mechanistically dominant. These results are described in the revised manuscript (Page: 9, line: 222).\nPoint 4. For the metastasis studies using the ALDH1B1 inhibitor, again the authors need to show effects on metastasis numbers and size, and not luciferase intensity (this is focused on the concept that inhibiting ALDH1b1 will likely block growth of mets – unrelated to effects on actually promoting metastasis.\nResponse: Thank you for the insightful suggestion. We re-analyzed the effects of ALDH1B1 inhibitor treatment on the number of brain metastases and found that pharmacological inhibition of ALDH1B1 significantly reduced the number and size of brain metastatic lesions. These results are described in the revised manuscript (Page: 9, line: 242).\nPoint 5. Fig. 3 shows that the mechanosensory protein PIEZO1 is required for activation of ALDH1B1. Effects are called “dramatic” but are 2-fold or less.\nResponse: We have modified the description to remove “dramatically” in the manuscript to more precisely reflect the quantitative changes (Page: 10, line: 264).\nPoint 6. PDTC blocked the upregulation of ALDH1B1, and the authors took to mean that the IKK/NF-kB pathway was involved. Note that PDTC can block a number of pathways. They authors note that levels of RelA are increased in compression/confinement but this experiment does not measure nuclear RelA. Depletion of RelA blocked compression/confinement-induced ALDH1B1. The authors linked confinement induced cell death with ferroptosis (and see comment below). As before, confinement results with non-confinement results need to be placed together.\nResponse: Thank you for the insightful comments.\n1. We appreciate the reviewer’s note regarding PDTC, a known inhibitor of NF-κB signaling (PMID: 28712932, PMID: 10491379, PMID: 2103049). Although PDTC may have off-target effects, our subsequent experiments using RelA-depleted cells confirmed the NF-κB pathway’s involvement. Specifically, RelA depletion significantly downregulated ALDH1B1 expression in confined tumor cells (Fig. 4f and Extended Data Fig. 7f).\n2. In addition, as shown in Fig. 5c, compression induced a clear increase in nuclear RelA levels.\n3. As suggested, we reinstated the data from unconfined RelA-depleted cells, which showed that RelA depletion did not significantly affect lipid peroxidation (Fig. 4k and Extended Data Fig. 7j) or survival (Fig. 4l and Extended Data Fig. 7k) in unconfined tumor cells. These results are described in the revised manuscript (Page: 12, line: 328).\nPoint 7. Compression induces the phosphorylation of IKKbeta, and this correlated with p-IkBa (in the figures the authors need to refer to this correctly – IkappaB-alpha - with Greek letters). In Fig 5d, the effect on an NF-kB reporter is relatively minimal.\nResponse:\n1. Thanks for pointing it out. The corrections have been made in the revised manuscript.\n2. Given that RelA nuclear translocation was enhanced as early as 0.5 hours post-compression (Fig. 5c), we subsequently evaluated NF-κB activity at the earlier 2-hour time point (compared to our previous 6-hour assessment). Our results demonstrated significantly elevated NF-κB activity at this earlier 2-hour time point. These results are included in the revised manuscript (Page: 12, line: 342).\nPoint 8. The authors found that CSK3 associates with IKKbeta during tumor cell compression and promotes IKKbeta activity, Knockdown of CSK3 leads to loss of ALDH1B1 expression and promoted cell death.\nResponse: We are a bit confused about the question. We speculate that the reviewer maybe want us to include the data of unconfined CSK23-depleted cells. Accordingly, we have reinstated the data from unconfined CSK23-depleted cells, which showed that CSK23 depletion did not significantly affect lipid peroxidation levels (Fig. 6h and Extended Data Fig. 10d, e and o) or survival (Fig. 6i and Extended Data Fig. 10f-h, p and q) in unconfined tumor cells. These results are described in the revised manuscript (Page: 14, line: 393).\nPoint 9. In Fig. 6, does knockdown of CSK3 and the CSK3 inhibitor block mets (number and size) – see points above. Like ALDH1B1, CSK3 is linked with oncogenic phenotypes – not referenced. The question is whether loss of CSK3 truly affecting metastasis or simply growth of the tumors once they have metastasized.\nResponse: Thank you for the insightful suggestion.\n1. We re-analyzed the effects of CSK23 depletion and CSK23 inhibitor treatment on the number of brain metastases. The results demonstrate that both CSK23 depletion and pharmacological inhibition of CSK23 significantly reduced the number and size of brain metastatic lesions. These results are described in the in the revised manuscript (Page: 14, line: 398; Page: 14, line: 414).\n2. We also included some discussion about other oncogenic functions of CSK23 (Page: 17, line: 496):\nAlthough CSK23, a member of the CK2 family, has been associated with lung cancer susceptibility (PMID: 20625391) and exhibits debated tumor-suppressive activity in hepatocellular carcinoma (PMID: 33582094), its broader role in cancer-particularly in metastasis-remains poorly characterized. In our study, we demonstrate the critical role of CSK23 in brain metastasis of lung cancer cells. Pharmacological inhibition by silmitasertib (CX-4945), a clinically available inhibitor targeting CSK23, significantly reduced metastatic burden and improved survival, suggesting that targeting CSK23 represents a promising strategy to prevent post-surgical metastatic recurrence in lung cancer.\nPoint 10. Fig. 7 links ALDH1 levels with capillary tumor cells and metastasis in lung cancer patients. These experiments are examining migration of cells from the tumor site to relate to distal metastasis and survival. There are several questions/concerns. When the authors refer to unconstrained tumor cells, are they simply referring to tumor? The cell death measurement is TUNEL assays which is associated with apoptosis and less with ferroptosis (see point above – did they authors measure apoptosis in their cell-based studies? Lastly, the text says that the authors are measuring nuclear RelA, but there is nothing to describe how that was measured. It seems that they are staining for whole cell RelA – but only nuclear would be meaningful for the overall hypothesis.\nResponse: Thanks for the constructive comments.\n1. In this study, “unconstrained tumor cells” specifically refer to tumor cells located outside CD31-positive regions, indicating tumor cells that are not confined within capillaries. A more detailed description of this analytical process has been provided in the Methods section (Page: 34, line: 950).\n2. We agree with the reviewer that TUNEL staining is more closely associated with apoptosis. In Extended Data Fig. 5i, we determined whether ALDH1B1 regulates cell apoptosis under confinement by examining caspase-3/7 activity in ALDH1B1-depleted tumor cells, which showed that caspase-3/7 activity was elevated in confined cells, which was exacerbated by ALDH1B1 depletion. However, compared to its effect on apoptosis, ALDH1B1 depletion has a much stronger influence on the ferroptosis of confined tumor cells (Extended Data Fig. 5a,b). These results are consistent with previous studies demonstrating that aldehydes induce both apoptosis (PMID: 16040627) and ferroptosis (PMID: 36274088), and that aldehydes derived from ALDH inhibition tend to promote ferroptosis (PMID: 37552043, PMID: 40233740, PMID: 32458004). Collectively, these results suggest that ALDH1B1 plays a dual protective role in confined tumor cells by suppressing both ferroptosis and apoptosis, with its anti-ferroptotic effect being mechanistically dominant. To strengthen the clinical relevance of ALDH1B1 expression to ferroptosis, we analyzed specific markers of ferroptosis. Since 4-hydroxynonenal (4-HNE) levels directly reflect intracellular aldehyde accumulation and lipid peroxidation (PMID: 28456642). Multiplex immunofluorescence analysis of primary lung cancer lesions revealed significantly higher 4-HNE expression in intravascular tumor cells (CD31+ regions) compared to extravascular areas (Fig. 7f). Moreover, ALDH1B1 expression exhibited a significant negative correlation with 4-HNE levels (Fig. 7g). Notably, tumor cells confined within capillaries from patients with metastatic recurrence displayed much lower 4-HNE levels than those from patients without metastatic recurrence (Fig. 7k). These results were included in the revised manuscript (Page: 15, line: 426).\n3. Although we stained for total cellular RelA (including both cytoplasmic and nuclear localization), our quantitative analysis focused specifically on nuclear RelA intensity. Nuclear regions were defined based on DAPI staining, and RelA intensity was measured exclusively within these demarcated areas. As suggested, we have now provided a more detailed description of this methodology in the Methods section (Page: 34, line: 971).\nReviewer #4 (Remarks to the Author)\nReviewer Summary:\nThe manuscript by Liu et al. investigates the role of ALDH1B1 in promoting lung cancer metastasis by enhancing cell survival and motility. The authors suggest that ALDH1B1 is regulated by the CSK23-RelA/NF-κB pathway and propose Piezo1 as a mechanosensor that initiates this signaling pathway in lung cancer. The use of a CRISPR knockout screening system to identify key metabolic enzymes is a sophisticated and logically sound approach. However, the method used to identify Piezo1, specifically through the application of a tumor microenvironment-mimicking platform, is not appropriate for lung cancer. Given the presence of numerous other mechanosensitive calcium channels, such as those in the Orai or TRPV families, the authors have not sufficiently explored alternative possibilities for mechanosensing in lung cancer metastasis. Consequently, the claim that Piezo1 is the primary mechanosensor driving CSK23-RelA signaling lacks a strong logical foundation. Additionally, it remains unclear whether aldehyde levels are elevated in the confined space of the lung. The authors should demonstrate a clear relationship between aldehyde production and the metastatic progression of lung cancer to support their hypothesis. Furthermore, the study reports brain metastasis occurring within seven days after lung cancer inoculation into the lung. Is there any existing literature supporting such a rapid metastatic timeline? The authors should provide references or additional experimental validation for this observation. The definition and generation of confined space in this study also require further clarification. While lung cancer cells may experience mechanical constraints within capillaries during metastasis, anoikis or pulsatile hydrostatic pressure could play a more significant role than simple confinement. The authors have not considered the possibility that different types of mechanical stress might influence lung cancer behavior. A more comprehensive discussion of these factors is necessary to strengthen the study’s conclusions.\nOverall, while the study presents an interesting perspective on ALDH1B1-mediated metastasis, the manuscript requires a more logically structured approach, further experimental validation, and consideration of alternative mechanosensors and mechanical stress factors.\nResponse: We thank the reviewer for the constructive comments and insightful suggestions, which greatly strengthens the manuscript.\nPoint 1. The use of a CRISPR knockout screening system to identify key metabolic enzymes is a sophisticated and logically sound approach. However, the method used to identify Piezo1, specifically through the application of a tumor microenvironment-mimicking platform, is not appropriate for lung cancer. Given the presence of numerous other mechanosensitive calcium channels, such as those in the Orai or TRPV families, the authors have not sufficiently explored alternative possibilities for mechanosensing in lung cancer metastasis. Consequently, the claim that Piezo1 is the primary mechanosensor driving CSK23-RelA signaling lacks a strong logical foundation.\nResponse: Thank you for the insightful suggestions.\nMultiple mechanosensitive calcium channels, including ORAI (PMID: 38672434) and TRPV family members (PMID: 15922584), play important roles in cancer progression (PMID: 37762011, PMID: 30086406). In response to the reviewer’s comment, we have evaluated their contributions to confinement-induced ALDH1B1 upregulation. We generated two A549 cell lines with depletion of key ORAI1 (PMID: 22108917, PMID: 30414508) or TRPV4 (PMID: 21204499) subtypes (Extended Data Fig. 6g,h), we found that ORAI1 depletion had no significant effect on ALDH1B1 expression in confined tumor cells (Extended Data Fig. 6i,j). TRPV4 depletion partially abrogated ALDH1B1 upregulation (Extended Data Fig. 6k,l), though unlike PIEZO1 depletion which completely abrogated ALDH1B1 upregulation in confined tumor cells (Fig. 3d,e and Extended Data Fig. 6e,f). These results suggest that under spatial confinement, PIEZO1 may serve as the primary mediator of ALDH1B1 expression regulation. These results have been incorporated into the revised manuscript (Page: 9, line: 250).\nBesides the tumor microenvironment (TME)-mimicking platform, we further confirmed the role of PIEZO1 in lung cancer metastasis with animal experiments, which showed that PIEZO1 depletion accelerated the diminishment of bioluminescence signals of tumor cells in the lungs (Fig. 3l). TUNEL assay with these lung tissues showed that PIEZO1 depletion increased tumor cell death in lung capillaries (Fig. 3m). Additionally, PIEZO1 depletion inhibited ALDH1B1 expression in tumor cells confined within lung capillaries (Fig. 3n). These results have been incorporated into the revised manuscript (Page: 11, line: 291).\nPoint 2. Additionally, it remains unclear whether aldehyde levels are elevated in the confined space of the lung. The authors should demonstrate a clear relationship between aldehyde production and the metastatic progression of lung cancer to support their hypothesis.\nResponse: Thank you for the insightful suggestion. As suggested, we investigated whether aldehyde levels are elevated in the confined lung microenvironment. 4-hydroxynonenal (4-HNE) is a well-established marker of aldehyde metabolism (PMID: 12893006) and a known substrate of ALDH1B1 (PMID: 20616185). Given that 4-HNE levels directly reflect intracellular aldehyde accumulation and lipid peroxidation (PMID: 28456642), we used 4-HNE as a surrogate measure for aldehyde production in spatially confined tumor cells. Immunofluorescence analysis of primary lung cancer lesions revealed significantly higher 4-HNE levels in intravascular tumor cells (CD31+ regions) compared to extravascular areas (Fig. 7f). Moreover, ALDH1B1 expression exhibited a significant negative correlation with 4-HNE levels (Fig. 7g). Notably, tumor cells confined within capillaries from patients with metastatic recurrence displayed much lower 4-HNE levels than those from patients without metastatic recurrence (Fig. 7k). Furthermore, we found that ALDH1B1 depletion increased 4-HNE accumulation in tumor cells confined in lung capillaries (Fig. 2j). These results demonstrate that aldehyde levels are elevated under confinement, while ALDH1B1 protects tumor cells by detoxifying aldehydes, thereby promoting metastatic progression. This result was included in our revised manuscript (Page: 8, line: 217 and page: 15, line: 426).\nPoint 3. Furthermore, the study reports brain metastasis occurring within seven days after lung cancer inoculation into the lung. Is there any existing literature supporting such a rapid metastatic timeline? The authors should provide references or additional experimental validation for this observation.\nResponse: We apologized for the confusion. In the study, we employed an intracardiac injection metastasis model, where A549 cells were directly introduced into the left ventricle to facilitate systemic circulation and subsequent brain metastasis formation. This established methodology is specifically designed to study late-stage hematogenous metastasis, particularly brain metastasis (PMID: 39037653). In this model, tumor cells reach the brain within 30 minutes and undergo gradual cell death. By day 63 post-injection, we could observe the formation of significant brain metastatic lesions. Based on our observation and published literature using this model (PMID: 39708324), the signals detected on day 7 were more likely from residual tumor cells rather than established metastatic lesions. The data from day 7 was used to support that ALDH1B1 depletion accelerates tumor cell death in brain. Detailed description of this mouse model of brain metastasis was provided in the Methods section (Page: 35, line: 1008).\nPoint 4. The definition and generation of confined space in this study also require further clarification. While lung cancer cells may experience mechanical constraints within capillaries during metastasis, anoikis or pulsatile hydrostatic pressure could play a more significant role than simple confinement. The authors have not considered the possibility that different types of mechanical stress might influence lung cancer behavior. A more comprehensive discussion of these factors is necessary to strengthen the study’s conclusions.\nResponse: Thank you for the insightful suggestions. A more comprehensive discussion of various mechanical stresses that tumor cells may experience within capillaries was included in the revised discussion section (Page: 18, line: 518):\nDuring metastatic dissemination, tumor cells encounter diverse mechanical stresses in distinct vascular environments. For example, anoikis primarily occurs in large vessels; pulsatile hydrodynamic pressure dominates in arteries; and in narrow capillaries, tumor cells mainly experience both fluid shear stress and mechanical compression (PMID: 28898694, PMID: 39917412, PMID: 11136289). Our study mainly focuses on mechanical compression, demonstrating that the CSK23-IKKβ-ALDH1B1 pathway is critical for tumor cell adaptation to compressive forces, enabling their survival in constricted capillaries. Targeting this pathway impairs mechanical stress adaptation and suppresses distant metastasis.\nIn contrast, fluid shear stress has been extensively studied for its role in tumor metastasis, as it not only disrupts circulating tumor cells (CTCs) from settling but may also induce cell-cycle arrest or even cell death. To overcome this mechanical barrier, CTCs enhance their phenotypic plasticity (e.g., through EMT or cytoskeletal remodeling) to improve survival (PMID: 18310319, PMID: 28054593, PMID: 33238151). Additionally, they form aggregates with platelets, neutrophils, and other cells via adhesive interactions, which protect their surface from shear forces and NK-cell-mediated lysis (PMID: 24862136). Meanwhile, CTCs upregulate bulky glycoproteins on their surface to increase physical adhesion to endothelial walls, thereby resisting shear forces. These adaptive mechanisms enable tumor cells to overcome shear stress and successfully establish distant metastases (PMID: 25030168).\nHowever, whether the CSK23-IKKβ-ALDH1B1 pathway is also critical for tumor cell adaptation to fluid shear stress within capillaries requires further investigation.\nReviewer #5 (Remarks to the Author):\nReviewer Summary:\nIn this manuscript, Liu et al. describe metabolic adaptations that enable cancer cells to survive and metastasize within confining microenvironment. Using an in vivo CRISPR-based knockout screening system, the authors identify ALDH1B1 as a critical target for tumor cell survival within capillaries. Mechanistically, they demonstrate that compressive forces activate CSK23, which phosphorylates IKKβ at S177/181, triggering NF-κB-dependent upregulation of ALDH1B1. Elevated ALDH1B1 enhances aldehyde detoxification, suppresses ferroptosis, and promotes tumor cell survival, highlighting CSK23/ALDH1B1 as potential therapeutic targets for metastatic cancer. Overall, this work uncovers a connection between mechanical stress, NF-κB signaling, and aldehyde metabolism during metastatic progression. Addressing the major concerns would strengthen the manuscript, the detail review is outlined below:\nResponse: We thank the reviewer for the constructive comments and insightful suggestions, which greatly strengthens the manuscript.\nMajor points:\nPoint 1. The authors used tail vein injection of gene-edited A549 cells to observe genetic differences in cells within pulmonary capillaries. How did the authors exclude the possibility that gene knockout affected cell proliferation, leading to false-positive results of reduced vascular cell counts? Additionally, is this model original? Is there any other literature that uses this method?\nResponse: Thanks for the comments.\n1. In the animal experiment (Fig. 1a), observations and sample collections were performed at 24 h. In circulation, most of the cells do not proliferate at 24 h. Moreover, as shown in Extended Data Fig. 4c and d, ALDH1B1 depletion did not significantly influence the proliferation of A549 cells.\n2. The method of the visualization of tumor cell viability in lung tissues in tumor metastasis mouse model via tail vein injection was extensively used in many previous studies (PMID: 33993094, PMID: 30926774, PMID: 37296072).\nPoint 2. The authors proposed that confined cells sense mechanical stress through Piezo1 and ultimately express ALDH1B1 to mitigate aldehyde toxicity. Why is this pathway specific to confined cancer cells? Why do normally growing cells not require similar mechanisms to maintain growth? Is it because confined cells face higher ferroptotic stress? Please provide experimental data to clarify.\nResponse: Thank you for the question. Indeed confinement significantly increased lipid peroxidation in tumor cells (Extended Data Fig. 5a,b) and elevated cell mortality (Fig. 2a, and Extended Data Fig. 3e,f), consistent with the upregulated aldehyde levels in confined tumor cells (Fig. 2c and Extended Data Fig. 3i). These results have been incorporated into the revised manuscript (Page: 6, line: 122 and page: 8, line: 205).\nPoint 3. In Patient 2’s results (not enlarged), ALDH1B1 expression showed poor co-expression with CD31, and high ALDH1B1 expression was apparently observed in non-confined regions. However, the statistical plot in Fig. 7b does not reflect this trend. The authors should check the representative results, carefully verify the statistical methods, and accurately present the statistical outcomes.\nResponse: Thank you for the comment. Upon re-examining the representative results from Patient 2, we confirmed that ALDH1B1-high regions predominantly co-localized with CD31-positive areas. The poor co-localization visibility in the merged image was due to the much stronger ALDH1B1 and RelA signals than CD31 signals in some regions. To avoid the confusion, we have now replace it with more clearly distinguishable representative results (Fig. 7a).\nMinor comments:\n1. Western blot images in high-quality journals should include molecular weight markers.\nResponse: As suggested by the reviewers, we have now added molecular weight markers to all western blot results.\n2. No information on tumor patient tissues or ethical approval is provided in the manuscript.\nResponse: This point was well taken. Detailed clinical information of the cancer patients along with the ethical approval number (KS(Y)23082) and (XHEC-C-2025-155-1) have been documented in the Methods section (Page: 34, line: 972).\n3. Please confirm the appropriateness of using the GEO database (GSE72094), which includes tumors with Kras and other genes mutations.\nResponse: As suggested by the reviewers, we have confirmed that the GSE72094 dataset contains lung adenocarcinoma samples with KRAS and other gene mutations.\n4. In Fig. 1h, TUNEL-positive areas do not fully overlap with ALDH1B1-knockdown cells, and non-tumor dead cells may affect the result. The authors should re-select representative images or quantify the percentage of co-expression between TUNEL-positive and mCherry-positive regions.\nResponse: Thank you for the comments. Our quantification specifically measured the proportion of mCherry+/TUNEL+ tumor cells relative to total mCherry+ tumor cells. To avoid confusion, we have added new representative images and modified the axis label in Fig. 1f to “mCherry+ tumor cell death (%)”.\n5. The method section should highlight the sampling of pulmonary capillaries.\nResponse: As suggested by the reviewers, we have added more detailed descriptions of the tumor cell sampling process in the Methods section (Page: 31, line: 869). Our sampling encompassed tumor cells from the entire lung, with over 90% of tumor cells remained lodged in the lung capillary tract at the 24 h post-tail vein injection in our mouse model, consistent with previous studies (PMID: 10613833, PMID: 26855844). We also updated the screening workflow diagram (Figure 1a) to improve understanding of our approach.\n6. Please carefully verify all results for data integrity (e.g., no endpoint data for the experimental group in Fig. 2m).\nResponse: In original Fig. 2m, the observation period was terminated upon the death of the last control group mouse. At this endpoint, the surviving mice in the experimental group remained in good condition, with no significant weight loss or other adverse effects observed. These surviving mice were right-censored to ensure both ethical compliance and statistical validity, as the Kaplan-Meier method inherently accounts for censored data (PMID: 30015653, PMID: 27713848, PMID: 21367692). We have now added a detailed description of this approach in the Methods section (Page: 36, line: 1018).\n7. Nuclear and cytoplasmic fractions in Fig. 5c should be presented on the same gel to compare.\nResponse: Thank you for the suggestion. The nuclear and cytoplasmic fractions were loaded and imaged on the same gel. However, because the cytoplasmic fraction had a significantly higher protein concentration (leading to overexposed p65 signals compared to the nuclear fraction), we used different exposure times for the cytoplasmic and nuclear fractions.\n8. Total protein loading controls are required for phosphoprotein detection in Fig. 6d.\nResponse: Thank you for the suggestions. As suggested by the reviewer, we have added actin as the reference.\n9. Supplementary images are excessively compressed, compromising readability.\nResponse: As suggested by the reviewer, we have revised the supplementary images to improve readability.\nReviewer #6 (Remarks to the Author):\nI 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.\nResponse: Thank you for your help.\nReviewer #7 (Remarks to the Author):\nI 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.\nResponse: Thank you for your help."}, {"round": 2, "reviewer_comments": {"reviewer_3": "The authors have made a thorough effort at responding to the critique. Their responses address have satisfactorily addressed my concerns.", "reviewer_4": "In this manuscript, the authors identify a role of ALDH1B1 regulated by CSK23-RelA/NF-kB and Piezo1 during lung cancer metastasis. Overall, the authors have performed a thorough and comprehensive study supporting their hypothesis that CSK23 and ALDH1B1 play an important role in metastatic environment of lung cancer. Over the past several years, studies have highlighted the critical role of biomechanical forces in cancer metastasis. In this context, this work is an important study that elucidates how the compression forces experienced within extremely narrow blood vessels in lung cancer specific metastasis niches influence lung cancer dissemination and proliferation, as well as the underlying mechanisms. In the revised manuscript, the authors have included additional control data that address my concerns and have strengthened the manuscript.", "reviewer_5": "This is an excellent piece of work. The authors have provided a thorough and in-depth mechanistic study, and their well-designed experiments have fully addressed the concerns I raised previously.", "reviewer_6": "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_7": "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_8": "The comments by reviewer #2 were satisfactorily addressed by the authors, and the manuscript as revised is appropriate for publication."}, "author_response": "Response: We thank the reviewer for the positive comments and constructive suggestions, which greatly strengthens the manuscript.\nMinor concerns:\nPoint 1. In their response to Major Point 4 raised by Reviewer 1, the authors attribute the observed discrepancies to the use of higher-passage cells. While this explanation is plausible, it also demonstrates the significant experimental variation across batches. Without additional evidence, it is difficult to determine whether the observed effects are due to cell passage or are confounded by batch-specific factors. To strengthen the validity of their conclusion, I strongly recommend that the authors include biological replicates of the relevant experiments and provide appropriate statistical analyses. This will allow for a more rigorous assessment of reproducibility and help clarify the source of the observed variation.\nResponse: As suggested by the reviewer, we quantified the ratio of phospho-IKKβ S177/181 to total IKKβ expression of Fig. 6d from three biologically independent experiments. Quantitative analysis and statistical comparison (Fig. 6d, right) showed that higher phospho-IKKβ S177/181 levels at 1 hr compared to 0.5 hr in the cells upon compression are reproducible and statistically significant.\nPoint 2. The identification of Calmodulin as a potential mediator in the signal transduction pathway between PIEZO1 and CSK23 is a noteworthy and potentially impactful finding. If the authors are confident in the robustness of this result, I suggest that they incorporate this finding into the graphical abstract presented in Figure 8.\nResponse: As suggested by the reviewer, we have updated Figure 8 to include Calmodulin (CaM) as a mediator in the signaling pathway between PIEZO1 and CSK23."}]

Decision letter