Targeting chaperone-mediated autophagy inhibits properties of glioblastoma stem cells and restores anti-tumor immunity

Domain: biology
Published: 2025-12-13
Editorial outcome: accept

Abstract
Chaperone-mediated autophagy (CMA) is a selective autophagic process essential for maintaining cellular quality and responding to stress. Dysregulation of the CMA pathway is increasingly recognized in various cancers, yet the mechanisms behind CMA hyperactivation in cancer cells remain unclear. Here, we show that CMA is upregulated in patient-derived glioblastoma stem cells (GSCs), indicated by a significant increase in the lysosomal abundance of the CMA receptor, lysosome-associated membrane protein 2A (LAMP2A). This increase results from MST4-mediated phosphorylation of LAMP2A, enhancing its stability and promoting homotrimer formation while inhibiting degradation by Cathepsin A. CMA supports GSC proliferation and self-renewal by activating mTORC1 through the selective degradation of its negative regulators, TSC1 and TSC2. Additionally, CMA is involved in epigenetic silencing of the cGAS-STING pathway, promoting tumor immune escape via lysosomal degradation of the DNA demethylase TET3. Inhibition of CMA synergizes with immune checkpoint therapy in glioblastoma models, highlighting a potential therapeutic target.

Introduction
Glioblastoma (GBM) represents the most prevalent and aggressive primary malignancy of the central nervous system in adults, classified among the most lethal cancers1. Current therapeutic approaches for GBM encompass surgical excision, radiotherapy, and the use of temozolomide (TMZ) as an adjunctive chemotherapy agent. Nonetheless, the prognosis for GBM patients remains dismal, with a median survival duration of roughly 14 months and a mere 5% survival rate at five years2. Furthermore, immune checkpoint inhibitor (ICI) therapies have failed to improve the overall survival in GBM patients3. A prior study has identified glioblastoma stem cells (GSCs) within GBM tumors, noted for their robust self-renewal and differentiation capabilities, which lead to the generation of a diverse array of cancer cell types4. Additionally, GSCs exhibit resistance to both radiotherapy and chemotherapy while also contributing to immune evasion, which are critical factors underlying treatment failure and GBM recurrence5–7. Therefore, the advancement of targeted therapies aimed at GSCs is essential for the efficacious management of GBM.

Autophagy plays a crucial role in the proteostasis network, ensuring cellular homeostasis. Various autophagic pathways are present in most mammalian cells, differing primarily in their mechanisms for transporting substrates to the lysosomal compartment8. In contrast to macroautophagy, which facilitates the bulk translocation of proteins and organelles to lysosomes for degradation9, chaperone-mediated autophagy (CMA) selectively targets a specific subset of cytosolic proteins characterized by KFERQ-like motifs10,11. Upon recognition of these motifs by heat shock cognate 71kDa protein (HSC70), the substrate is translocated to the lysosomal membrane’s surface12, where it interacts with lysosome-associated membrane protein type 2A (LAMP2A), which has been identified as a limiting factor for CMA efficacy13. The binding of the substrate to the cytosolic tail of LAMP2A induces LAMP2A oligomerization, forming a translocation complex that facilitates the internalization of substrates into lysosomes for subsequent degradation14,15. CMA is implicated in the degradation of both defective proteins for quality control and intact proteins to modulate their functions, thereby coordinating various intracellular processes, including aging, lipid and glucose metabolism, cell signalling, cell cycle, stem cell functionality and immune responses10,16–20. Despite its established role, the regulatory networks governing CMA activity remain in completely characterized10,21. Notably, while macroautophagy contributes to cancer stem cell malignancy22,23, CMA-specific receptor LAMP2A demonstrates elevated expression in patient-derived GSCs17, suggesting potential differential roles in specialized cell populations that require further elucidation.

MST4 (also known as STK26), a constituent of the Mammalian Sterile 20-Like Kinase (MST) family24, is integral to numerous cellular physiological processes via substrate phosphorylation25. It has been implicated in the pathogenesis of colorectal cancer by phosphorylating β-catenin, which results in its accumulation and full activation26. Additionally, MST4 is recognized for phosphorylating ATG4B, thereby initiating macroautophagy, which enhances cell survival and tumorigenic potential of GSCs27. Nonetheless, the involvement of MST4 in tumor-associated CMA remains to be elucidated.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway is a crucial component of the innate immune response, significantly contributing to anti-tumor immunotherapy28. Cytoplasmic DNA acts as a danger signal, which is recognized by cGAS, leading to the synthesis of the second messenger cGAMP29,30. This process initiates the activation of the STING-TBK1-IRF3-type I interferon (IFN) signaling cascade31,32. The stimulation of the cGAS-STING pathway through various modalities, including radiotherapy and chemotherapy, is regarded as a promising combinatorial approach with ICIs and other immuno-oncological therapeutics33,34. However, in numerous tumors, the promoters of cGAS and STING are frequently hypermethylated, leading to the silencing of this pathway35,36. The epigenetic regulatory mechanisms governing the expression of cGAS and STING genes remain inadequately understood.

In this work, we elucidate the critical role of the MST4-LAMP2A signaling axis in regulating CMA activity, self-renewal capacity, and tumorigenic potential of GSCs, alongside its ramifications for anti-tumor immunity. Mechanistically, phosphorylation of LAMP2A at residues T40, S97, and T136 by MST4 is demonstrated to reinforce LAMP2A stability, and thereby elevating CMA activity. This enhancement subsequently promotes the proliferation and malignancy of GSCs. Furthermore, we identify a function of CMA as an intrinsic regulator of anti-tumor immune responses, suggesting a promising therapeutic avenue focused on targeting CMA for GBM treatment.

Results
Elevated level of CMA in GSCs is associated with increased LAMP2A abundance in lysosomes
We employed a photoactivatable CMA reporter (PA-mCherry-KFERQ) to visualize and quantify CMA activity by counting the fluorescent puncta per cell. This occurs when the fluorescent CMA substrate protein, fused with a KFERQ-like motif, is delivered to CMA-active lysosomes by HSC70, resulting in a transition from a diffuse to a punctate distribution. In contrast, bulk lysosomal degradation pathways, such as macroautophagy, target substrates lacking the KFERQ motif and do not produce fluorescent puncta under this assay condition37. We found that patient-derived GSCs exhibited significantly higher baseline CMA activity than conventional GBM cell lines (Fig. 1a). Lysosomes purified from GSCs demonstrated a significant enhancement in both the binding affinity for and uptake of the model CMA substrate, glyceraldehyde-3-phosphate dehydrogenase (GAPDH)38, in comparison to those derived from conventional GBM cell lines (Fig. 1b). In agreement with increased CMA activity in the lysosomes from GSCs, the levels of known CMA substrates, including MEF2D and GAPDH38,39, were markedly elevated in GSCs following the inhibition of lysosomal proteolysis through treatment with lysosomal protease inhibitors (leupeptin and ammonium chloride (NH4Cl), LN). In contrast, such an increase was not detected in conventional GBM cells. Additionally, inhibition of macroautophagy via 3-methyladenine (3-MA) or proteasomal inhibition with MG132 did not alter the expression of MEF2D and GAPDH (Fig. 1c). To examine whether CMA activity is required for stemness maintenance of GSCs, we cultured GSCs in medium containing serum to induce differentiation. As expected, differentiated-GSCs (DGCs) exhibited a decrease in CMA flux (Fig. 1d), alongside diminished expression of pluripotency markers such as CD44 and ALDH1A3 and upregulation of the differentiation marker Tuj1 (Supplementary Fig. 1a). Collectively, these results indicated that CMA is upregulated in GSCs.

Subsequently, we aimed to elucidate the mechanisms underlying the upregulation of CMA in GSCs. We performed isolation of lysosomal fractions from both GSCs and their corresponding DGCs, revealing that lysosomes from GSCs exhibited a higher abundance of the CMA receptor, LAMP2A, compared to those from DGCs. In contrast, the levels of other established CMA components, such as a subset of chaperones (HSC70) and co-chaperones (HSP40 and HSP90), as well as lysosome-associated membrane protein 1 (LAMP1), remained constant throughout differentiation (Fig. 1e). GSC cell lines exhibited elevated levels of LAMP2A in both total cell lysate and lysosomal fractions when compared to conventional non-GSC cell lines and DGCs, even though the mRNA expression levels of LAMP2A were similar across these cellular types (Supplementary Fig. 1b-d). Collectively, these findings indicated that the elevated CMA activity in GSCs correlates with an increased abundance of LAMP2A in the lysosome.

To examine the biological implications of CMA in GSCs, we employed two distinct short hairpin RNAs (shRNAs) to downregulate endogenous LAMP2A in M83 and 456 cell lines. The knockdown (KD) of LAMP2A resulted in a pronounced upregulation of MEF2D (Supplementary Fig. 1e), and led to a reduction in GSC proliferation and sphere-forming capacity in vitro (Supplementary Fig. 1f, g), while significantly inhibiting intracranial tumor growth in athymic nude mice, thereby improving the overall survival (Supplementary Fig. 1h, i). Subsequently, we introduced an shRNA-resistant LAMP2A-WT (wild-type) and a functional mutant LAMP2A (LAMP2A-4A) that was deficient for substrate binding40, into GSC M83 and 456 cells where endogenous LAMP2A was depleted. Our findings demonstrated that the reintroduction of exogenous LAMP2A-WT, but not the LAMP2A-4A mutant, effectively reversed the effects of LAMP2A KD on CMA activity (Supplementary Fig. 1j), GSC proliferation, and sphere formation ability in vitro (Fig. 1f and Supplementary Fig. 1k), as well as tumorigenicity in the brains of athymic nude mice (Fig. 1g, h), thus shortening the survival of mice (Fig. 1i). Taken together, these results indicated that CMA is required for GSC stemness and tumorigenicity.

MST4-mediated LAMP2A phosphorylation increased the stability of LAMP2A at the lysosomal membrane
Subsequently, we examined the underlying mechanisms contributing to the increased level of LAMP2A detected in GSCs. The aforementioned findings demonstrated no significant discrepancies in the mRNA level of LAMP2A between GSC and DGC cell lines (Supplementary Fig. 1b), suggesting an absence of differential de novo protein synthesis. In contrast, assessments of lysosomal LAMP2A stability over different incubation time in isotonic buffer demonstrated that the degradation of LAMP2A was significantly accelerated in lysosomes derived from DGCs compared to their GSCs counterparts (Fig. 2a, b). The observed alterations in DGCs were markedly reversed when lysosomes were treated with protease inhibitor cocktail (PI), which block lysosomal proteolytic activity and thereby prevent LAMP2A luminal degradation21. This suggests that the reduced stability of LAMP2A is mainly caused by increased lysosomal degradation (Fig. 2a). To elucidate the mechanisms governing lysosomal LAMP2A stability in GSCs, we conducted an analysis of the post-translational modifications of LAMP2A proteins in both GSCs and DGCs. Previous studies have indicated that glycosylation plays a role in the regulated stability of LAMP2A41. To assess the glycosylation status of LAMP2A, we performed both PNGase F deglycosylation and Concanavalin A (ConA) lectin blotting analyses. Enzymatic deglycosylation resulted in a consistent molecular weight shift of LAMP2A monomers from ~100 kDa to ~50 kDa in both GSCs and DGCs, demonstrating complete N-linked glycan removal. This identical migration pattern strongly suggests equivalent glycosylation profiles between the two cell types. Supporting this conclusion, ConA blotting analysis confirmed comparable glycosylation levels in GSCs and DGCs (Supplementary Fig. 2a). In contrast, we observed that GSCs exhibited elevated level of LAMP2A phosphorylation compared to DGCs (Fig. 2c).

To determine if LAMP2A phosphorylation is necessary for its stability in GSC lysosomes, we performed a proteomic analysis of kinase proteins that co-precipitated with LAMP2A in GSCs (Supplementary Fig. 2b). Among the top associated proteins, we identified MST4 is associated with LAMP2A, subsequently confirming their interaction through reciprocal immunoprecipitation (Fig. 2d, f and Supplementary Fig. 2c). Immunofluorescence analysis also revealed that MST4 co-localized with LAMP2A in GSC M83 and 456 cells (Supplementary Fig. 2d). Characterization of the interaction domains between MST4 and LAMP2A indicated that the N-terminal region (amino acids 1-137) of LAMP2A, alongside the kinase domain of MST4 (amino acids 1-279), is essential for their interaction (Supplementary Fig. 2e–h). CMA has been identified as a component of the cellular adaptive mechanisms to stress10,16,42. We examined the effects of irradiation (IR) and nutrient deprivation on the interaction between MST4 and LAMP2A. Our results indicated that IR significantly increased MST4 expression and enhanced the interaction between MST4 and LAMP2A (Supplementary Fig. 2i). While growth factors (EGF/FGF) deprivation did not influence the interaction between MST4 and LAMP2A (Supplementary Fig. 2j), culturing them in serum-supplemented medium diminished the MST4-LAMP2A interaction (Supplementary Fig. 2k). Collectively, these results suggest that targeting the MST4-LAMP2A axis may sensitize GSCs to microenvironmental stresses, such as hypoxia and nutrient limitation, thereby providing a potential therapeutic strategy to eliminate therapy-resistant GSCs.

To investigate the role of MST4 in CMA within GSCs, we conducted MST4 KD via shRNAs and knockout (KO) utilizing single guide RNAs (sgRNAs). Notably, the phosphorylation level of LAMP2A and the lysosomal presence of LAMP2A were significantly diminished following MST4 deletion (Fig. 2g). Given that the proper localization of LAMP2A at the lysosome, along with its molecular multimerization and interaction with lysosomal proteases, governs its stability and functionality10, we sought to elucidate the underlying mechanisms responsible for the observed decline in lysosomal LAMP2A in the absence of MST4 expression. We found that there were no significant differences in LAMP2A mRNA level between MST4-KD GSCs and control cells (Supplementary Fig. 2l). Subsequently, we hypothesized that MST4 might be essential for the localization of LAMP2A. Examination of LAMP2A’s subcellular localization indicated that its presence at lysosomal structures remained unaffected by the absence of MST4 (Supplementary Fig. 2m), implying that MST4 is not critical for the proper subcellular distribution of LAMP2A. We then posited that MST4 loss could compromise the stability of LAMP2A at the lysosome. To evaluate this hypothesis, we assessed the stability of LAMP2A in GSCs. The KD of MST4 resulted in a more rapid degradation of LAMP2A, which was significantly mitigated when MST4-KD GSCs exposed to lysosomal PI (Fig. 2h, i), indicating that the diminished stability of LAMP2A following MST4 deletion was primarily due to enhanced degradation within lysosomal compartments. The targeted cleavage of LAMP2A by Cathepsin A (CathA) at the lysosomal membrane serves as the catalyst for its degradation within the lysosomal environment43. Our findings demonstrated that MST4 KD was associated with an increased interaction of LAMP2A with CathA (Fig. 2j), corroborating the elevated degradation rate of LAMP2A following MST4 KD. Furthermore, native electrophoresis analysis of LAMP2A’s multimeric status at the lysosomal membrane exhibited a substantial decrease in the formation of the 700 kDa multimeric complex necessary for substrate translocation in lysosomes from MST4-KD GSCs (Fig. 2k). These results aligned with the observed reduction in the interaction among LAMP2A molecules in MST4-KD GSCs (Fig. 2l). Collectively, these findings underscored the role of MST4 in facilitating LAMP2A stability in the lysosomes and its active conformational dynamics.

Changes in the abundance of lysosomal LAMP2A have been found to influence CMA activity13. Knocking down endogenous MST4 or LAMP2A with specific shRNAs in GSC M83 and 456 cells led to reduced level of phosphorylated LAMP2A. This change led to an increase in MEF2D within the whole cellular lysate while reducing its presence in the lysosomal fraction, a decrease in KFERQ-puncta, and impaired lysosomal binding and uptake of the CMA substrate GAPDH (Fig. 2m-p). To further explore the role of MST4 in inducing CMA, we overexpressed exogenous MST4-WT and kinase-dead mutants (T178A and K53E) in GSC 23 cells with low endogenous MST4 level. The overexpression of WT MST4, unlike the kinase-dead mutants, led to increased phosphorylation of LAMP2A, reduced interaction between LAMP2A and CathA, higher accumulation of LAMP2A in lysosomes, and enhanced CMA activity in GSCs (Supplementary Fig. 2n, o).

MST4 directly phosphorylates LAMP2A in GSCs
Based on the interaction between MST4 and LAMP2A, as well as the role of MST4 in CMA, we investigated whether MST4 phosphorylates LAMP2A and affects its stability. To identify the phosphorylation sites on LAMP2A by MST4, we conducted mass spectrometry on Flag-LAMP2A from HEK293T cells that stably overexpress exogenous HA-MST4 and Flag-LAMP2A. Our findings indicated that the residues T40, S97, and T136 of LAMP2A underwent specific phosphorylation in cells that express exogenous MST4, in contrast to those transfected with a control vector (Fig. 3a). The amino acid sequences surrounding T40, S97, and T136 are highly conserved in LAMP2A among multiple species (Supplementary Fig. 3a). To identify the key phosphorylation site(s) of LAMP2A influenced by MST4, we individually or in combination mutated the serine/threonine residues at T40, S97, and T136 to alanine. The in vitro kinase assay revealed that wild-type LAMP2A was phosphorylated by MST4. Mutating each site alone slightly reduced phosphorylation, while mutating any two sites significantly decreased it, and mutating all the three sites completely abrogated phosphorylation. Among the three single-site mutations, the T136 mutation showed the most significant reduction in phosphorylation level, indicating that T136 site is crucial for LAMP2A phosphorylation (Fig. 3b). Additionally, we co-expressed WT LAMP2A or T40A/S97A/T136A (3A) mutant and MST4-WT or -K53E in HEK293T cells, the result identified phosphorylation in WT LAMP2A, but not in the non-phosphorylatable 3A mutant or kinase dead mutant K53E (Fig. 3c). To validate our findings, we developed a murine antibody that specifically targets the T136 phosphorylated LAMP2A peptide, without reacting to the non-phosphorylated version (Supplementary Fig. 3b). This antibody, known as the p-LAMP2A antibody, effectively detected T136 phosphorylation of endogenous LAMP2A. In GSC M83 cells, KD or KO of MST4 significantly reduced the level of phosphorylated LAMP2A at T136 (Supplementary Fig. 3c). In HEK293T cells co-expressing HA-MST4 and Flag-LAMP2A, the p-LAMP2A antibody specifically detected phosphorylation in LAMP2A-WT, T40A, S97A, and T40A/S97A under exogenous overexpression conditions. However, it completely failed to recognize the T136A phospho-null mutant (T136A), as well as all mutants containing the T136A substitution (T136A/T40A, T136A/S97A, and 3A) (Supplementary Fig. 3d). Moreover, the p-LAMP2A antibody was employed to observe pT136 levels in exogenous LAMP2A-WT and -3A within GSC M83 cells. The p-LAMP2A signal was exclusively detected in GSCs expressing LAMP2A-WT, with no signal observed for LAMP2A-3A (Supplementary Fig. 3e).

To assess the role of LAMP2A phosphorylation in CMA activation, we knocked down endogenous LAMP2A in GSCs using specific shRNA and then reintroduced shRNA-resistant LAMP2A-WT, -3A, or phosphorylation-mimic -3D mutant. The findings indicated that both WT and 3D variants effectively reconstituted lysosomal LAMP2A levels and restored CMA-mediated MEF2D degradation capacity, while the 3A mutant failed to do so (Supplementary Fig. 3f). Additionally, LAMP2A-3A significantly impaired LAMP2A oligomerization compared to LAMP2A-WT (Supplementary Fig. 3g). Coimmunoprecipitation analyses further revealed that LAMP2A-3A attenuated homotypic LAMP2A-3A interactions relativeto WT or 3D variants (Supplementary Fig. 3h). Moreover, LAMP2A-3D mutant displayed reduced binding affinity for Cath A while simultaneously enhancing KFERQ-positive puncta formation (Fig. 3d and Supplementary Fig. 3i, j). Additionally, LAMP2A-WT and -3D, but not -3A mutant, successfully rescued GSC cell proliferation and sphere formation in vitro (Fig. 3e, f), and tumorigenicity, which resulted in a shorter survival time of GSC-bearing mice (Fig. 3g–i).

To determine if the increased stability of phosphorylated LAMP2A at the lysosomal membrane is a common mechanism across different cell types, we analyzed human MRC-5 fibroblasts. We compared the half-life and phosphorylation levels of LAMP2A in lysosomal membranes of early-passage (P18) and late-passage (P45) fibroblasts. Late-passage fibroblasts showed a significantly faster degradation rate of LAMP2A (Supplementary Fig. 4a, b) and had lower levels of phosphorylated LAMP2A (Supplementary Fig. 4c) compared to early-passage cells. To investigate whether enhancing LAMP2A phosphorylation could rescue CMA dysfunction in aging-like models, we introduced LAMP2A-WT, -3A, or -3D into MRC-5 fibroblasts. Notably, LAMP2A-WT and -3D significantly decreased the number of senescence-associated beta-galactosidase (SA-β-gal) positive cells in late-passage fibroblasts, while -3A did not (Supplementary Fig. 4d). This improvement correlated with a decrease in known CMA substrates (MEF2D and GAPDH) in whole-cell lysates and their accumulation in lysosomal fractions (Supplementary Fig. 4e, f), indicating enhanced degradation through CMA. These findings suggest that the phosphorylation-dependent stabilization of LAMP2A is a conserved mechanism across cell types, and that mimicking phosphorylation (LAMP2A-3D) can improve CMA dysfunction in aging-like models.

MST4-mediated phosphorylation of LAMP2A promotes CMA activity, self-renewal, and tumorigenicity of GSCs
To further assess the importance of MST4-mediated LAMP2A phosphorylation in CMA activity and GSC biological function, we introduced the LAMP2A-WT or -3D mutant into GSC M83 and 456 cells, where endogenous MST4 expression was downregulated using shRNA (Supplementary Fig. 5a). Western blot analysis of lysosomal fractions from these control cells showed a significant accumulation of lysosomal proteins (LAMP1, LAMP2A, and HSC70) and an increase in phosphorylated LAMP2A compared to whole cell lysates, indicating functional CMA in the lysosomes (Fig. 4a and Supplementary Fig. 5b). Absence of mitochondrial (COXIV) and cytosolic (β-actin) markers in the lysosomal samples confirmed their purity (Fig. 4a and Supplementary Fig. 5b). Our findings revealed that the CMA-competent lysosomes exhibited reduced levels of phosphorylated and total LAMP2A when endogenous MST4 was deleted in GSC M83 and 456 cells (Fig. 4a and Supplementary Fig. 5b). In contrast, the introduction of the exogenous phosphomimetic LAMP2A-3D, in contrast to LAMP2A-WT, significantly reinstated the level of lysosomal LAMP2A (Fig. 4a), enhanced the number of KFERQ puncta (Fig. 4b and Supplementary Fig. 5c, d), and improved the binding and uptake of GAPDH within lysosomes that were diminished due to MST4 KD (Fig. 4c). Additionally, LAMP2A-3D, but not -WT, notably enhanced the proliferation and sphere-forming capacity of GSC M83 and 456 cells in vitro (Fig. 4d, e). In vivo investigations revealed that LAMP2A-3D, in contrast to the LAMP2A-WT, markedly restored the tumorigenic capacity of orthotopic GSC xenografts (Fig. 4f, g) and significantly reduced the survival duration of the animals (Fig. 4h).

In GSC 23 cells devoid of detectable MST4, the introduction of exogenous MST4 resulted in elevated levels of both phosphorylated and total LAMP2A within CMA-active lysosomes, as well as an increased association of MEF2D with lysosomes (Fig. 4i and Supplementary Fig. 5e). Additionally, overexpression of MST4 augmented CMA activity, evidenced by the higher abundance of KFERQ puncta (Fig. 4j, k), and enhanced lysosomal binding and uptake of GAPDH (Fig. 4l). Conversely, silencing of endogenous LAMP2A abrogated the stimulatory effects of MST4 overexpression on CMA activity (Fig. 4j–l). Moreover, MST4 promoted the proliferation and sphere-forming capacity of GSC cells in vitro (Fig. 4m, n), along with enhancing tumorigenicity in vivo (Fig. 4o–q). However, knockdown of endogenous LAMP2A markedly inhibited cell growth and reduced the frequency of sphere formation in GSC 23 cells (Fig. 4m, n), while also decelerating MST4-mediated tumor growth in xenograft models, ultimately leading to improved survival outcomes in the animal subjects (Fig. 4o–q). Interestingly, GSC 23 cells exhibited elevated levels of LAMP2A phosphorylation, although not at the T136 site (Supplementary Fig. 5f). When treated with SB203580, a p38α MAPK inhibitor44, there was a decrease in phosphorylated LAMP2A and lysosomal LAMP2A, along with reduced MEF2D association with lysosomes, leading to increased MEF2D accumulation in the total cell lysate (Supplementary Fig. 5g). This indicates that the phosphorylation of LAMP2A mediated by p38α MAPK is vital for maintaining basal CMA activity within GSC 23 cells. Furthermore, we observed that MST4 activates both macroautophagy and CMA in GSCs, with these pathways exhibiting compensatory interactions (Supplementary Fig. 6a–c). In xenograft models, the knockdown of either ATG4B or LAMP2A led to a reduction in MST4-driven tumor growth by 73% and 68% (p < 0.001), respectively (Supplementary Fig. 6d, e). Notably, the combined inhibition of ATG4B and LAMP2A synergistically decreased tumor burden by 97% (p < 0.001) and extended median survival from 16 days (MST4 overexpression) to 45 days (p < 0.01) (Supplementary Fig. 6f). Consistent with the above observation, immunostaining revealed identical distribution patterns for both LAMP2A and LC3B (Supplementary Fig. 6g-i).

CMA acts as an enhancer of mTORC1 signaling by modulating TSC1/2
To elucidate the pathways potentially modulated by CMA in GSCs, we queried previously published RNA-seq data (GSE181556) and proteomics data (PXD027069) from patient-derived GSCs with and without LAMP2A deletion17. Gene set enrichment analysis (GSEA) of RNA-seq data indicated that LAMP2A KD resulted in an upregulation of interferon response and other inflammation-associated genes, while concurrently downregulating the PI3K-AKT-mTOR signaling cascade (Fig. 5a). The proteomic investigation underscored the STING-mediated activation of host immune responses, interferon signaling, and translation as the primary pathways exhibiting significant alterations (Fig. 5b).

The mTORC1 activity is frequently dysregulated in various human cancers, including GBM45. The constitutive activation of mTORC1 is essential for the proliferation, self-renewal, and tumorigenic potential of cancer stem cells46–48. Given that the interplay between CMA and mTORC1 signaling had not been previously explored, we subsequently investigated whether CMA influences mTORC1 activity. mTORC1 has two extensively studied substrates, ribosomal protein S6 kinase (P70S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), which play essential roles in the regulation of protein biosynthesis49. KD of MST4 or LAMP2A led to a marked reduction in the phosphorylation levels of P70S6K and 4EBP1 (Fig. 5c), indicating that CMA regulates mTORC1 activity.

Subsequently, we examined whether CMA was required for the targeted degradation of mTOR signaling inhibitors. CMA is a specialized autophagic process characterized by substrate specificity determined by the presence of a pentapeptide motif resembling KFERQ within the substrate protein, enabling recognition by cytosolic HSC7010. We screened for mTORC1-upstream proteins that were upregulated after LAMP2A KD in GSCs and had motifs in their amino acid sequences making them susceptible to CMA. Notably, we detected such motifs in tuberous sclerosis complex 1 (TSC1) and 2 (TSC2), which are recognized as critical upstream negative regulators of mTORC150. We compared the motifs and found that both TSC1 and TSC2 possess the KFERQ-like motif (Supplementary Table 1). Additionally, we confirmed that HSC70 and LAMP2A bind to WT TSC1/2, but not to TSC1/2 mutants (Supplementary Table 1) with altered HSC70 binding motifs (Fig. 5d, e), which displayed a longer half-life than their WT counterparts (Fig. 5f, g). Furthermore, the depletion of HSC70 or LAMP2A resulted in a reduced association of TSC1/2 with lysosomal compartments, and subsequently led to an increased expression of TSC1/2 in GSCs (Fig. 5h, i). Conversely, the overexpression of LAMP2A-WT or -3D, as opposed to -3A or -4A, markedly decreased the protein levels of TSC1/2 in HEK293T cells (Supplementary Fig. 7a). Immunofluorescence staining illustrated that TSC1/2 co-localized with LAMP2A-positive lysosomes under conditions that induce CMA, such as nutrient starvation. This association with lysosomes was disrupted when the KFERQ motifs were mutated (Supplementary Fig. 7b, c). Finally, we conducted in vitro uptake assays utilizing lysosomes isolated from HEK293T cells that were active in CMA. Purified TSC1/2 proteins were incubated with either untreated lysosomes or those that had been pretreated with lysosomal PI. Immunoblotting of the recovered lysosomes demonstrated the uptake and degradation of TSC1/2 by these lysosomes (Supplementary Fig. 7d, e). Together, these findings indicate that TSC1/2 are indeed targets of CMA.

To investigate the degradation of TSC1/2 proteins within lysosomes, we quantified the levels of lysosomal TSC1/2 in GSCs with and without cycloheximide (CHX). The application of LN, which inhibit lysosomal proteolysis, resulted in a significant elevation of TSC1/2 levels, comparable to those observed following proteasome inhibition previously linked to TSC1/2 degradation (Fig. 5j). Conversely, the suppression of macroautophagy using 3-MA did not alter the intracellular concentrations of TSC1/2, indicating that a portion of TSC1/2 undergoes routine degradation in lysosomes (Fig. 5j). Additionally, KD of TSC1/2 substantially mitigated the effects of LAMP2A KD on mTORC1 pathway activity (Supplementary Fig. 8a), GSC cell growth (Supplementary Fig. 8b), and sphere-forming frequency (Supplementary Fig. 8c, d). To further elucidate the role of CMA in TSC1/2 degradation and its impact on GSC tumorigenicity, we performed KD of endogenous TSC1/2 protein levels, followed by the reintroduction of shRNA-resistant TSC1/2 wild-type and HSC70 binding motif-mutated variants (Supplementary Fig. 8e). The KD of TSC1/2 enhanced GSC proliferation and spheroid formation, whereas the re-expression of exogenous TSC1/2 WT fully rescued the effects of TSC1/2 deficiency on mTOR activity and GSC phenotypes (Fig. 5k, l and Supplementary Fig. 8f, g). Intriguingly, the reintroduction of TSC1/2 mutants exerted even stronger effects than WT, suggesting that these mutants escape endogenous CMA-mediated suppression, thereby exerting enhanced effects on downstream mTOR signaling and GSC phenotypes (Fig. 5k, l and Supplementary Fig. 8f, g). Under standard culture conditions, the genetic knockdown or reconstitution of TSC1/2 expression markedly influenced mTORC1 signaling, while exhibiting no significant impact on the macroautophagic flux, as determined by quantitative assessment of the LC3-II/I ratio and levels of p62 protein (Supplementary Fig. 8h). This observation was corroborated by the absence of substantial alterations in LC3B puncta formation in tumor tissues modified for TSC1/2 expression compared to control samples. Importantly, ATG5 knockdown, employed as a positive control for macroautophagy inhibition, consistently demonstrated the anticipated impairment of macroautophagy both in vitro and in vivo (Supplementary Fig. 8h-j). In summary, these findings indicated that CMA facilitates GSC proliferation, self-renewal, and tumorigenicity through mTORC1 activation via the lysosomal degradation of TSC1/2, rather than through modifications in macroautophagy levels.

Inhibiting CMA function reduces GBM tumor growth by enhancing anti-tumor T cell immunity
Our findings indicated an enrichment of interferon response genes and an inflammatory gene signature in GSCs with LAMP2A KD (Fig. 5a, b), prompting us to explore the impact of targeting the MST4-LAMP2A-CMA pathway on host anti-tumor immunity. To evaluate the influence of CMA on the immune microenvironment, we adopted the CMA score as recently established by Bourdenx et al. 51 This score serves as a weighted average of the expression levels of all known effectors, as well as both positive and negative regulators of CMA, with recent research indicating that it serves as a reliable metric for assessing CMA activation status19,52. TCGA analysis revealed that CMA score negatively correlated with CD8+ T cell infiltration and positively correlated with tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) in GBM. There was no significant correlation with regulatory T cells (Tregs) (Fig. 6a and Supplementary Fig. 9a-e). Additionally, we conducted in vitro co-cultures of GSCs with MST4 or LAMP2A KD alongside allogeneic human CD8+ T cells. Flow cytometric analysis indicated that the KD of LAMP2A or MST4 significantly enhanced cytotoxic T cell activity, evidenced by increased production of polyfunctional cytokines such as TNF-α (Fig. 6b) and IFN-γ (Fig. 6c), elevated effector degranulation (Granzyme B, GZMB) (Fig. 6d), and increased apoptosis in GSCs (Supplementary Fig. 9f).

Due to the inability of GSCs to engraft in immunocompetent C57BL/6 mice because of species-specific barriers, we performed KD of endogenous Mst4 or Lamp2a in murine syngeneic GBM cell lines, GL261 and CT-2A (Fig. 6e), and subsequently implanted 1×105 viable GL261 cells intracranially into immunodeficient BALB/c Nude mice or immunocompetent C57BL/6 mice. Compared to the control cohort, the ablation of Mst4 or Lamp2a in murine GBM cells resulted in a moderate yet significant reduction in intracranial tumor growth in immunodeficient nude mice, as evidenced by bioluminescence imaging and H&E staining of tumor progression, and survival analysis (Fig. 6f–j). Notably, the deletion of Mst4 or Lamp2a produced a pronounced anti-tumor response in C57BL/6 mice, compared to BALB/c Nude mice, leading to an extension of overall survival (Fig. 6f–k), underscoring the pivotal role of T cell-mediated immune surveillance in tumor inhibition. Immunofluorescence examination of murine tissues indicated that the absence of Mst4 and Lamp2a resulted in a reduction of PD-L1 expression and a decrease in the infiltration of immunosuppressive Tregs (Supplementary Fig. 9g, h), while simultaneously promoting the infiltration and cytotoxic activity of CD8+ T cells within the xenograft tumors, evidenced by the enhanced co-localization of CD8 and GZMB (Supplementary Fig. 9i). Consistent with the immunofluorescence data, flow cytometric analysis revealed a notable increase in the presence of functionally active CD8+ T cells and a reduction in Treg infiltration in tumors following the deletion of Mst4 or Lamp2a (Supplementary Fig. 9j–m). Additionally, Mst4 KD reduced tumor infiltration of immunosuppressive cells (Tregs, TAMs, and MDSCs). However, re-expressing LAMP2A-WT or -3D significantly restored the reduction in immunosuppressive cell infiltration (Supplementary Fig. 9n–p). These findings underscore the involvement of CMA in the formation of the immunosuppressive microenvironment characteristic of GBM.

To ascertain whether the tumor growth inhibition associated with LAMP2A depletion was mediated by anti-tumor CD8+ T cell immunity, CT-2A GBM-bearing mice were administered an anti-CD8α monoclonal antibody (mAb). In contrast to the IgG2b control mAb, the administration of the CD8α mAb markedly reversed the tumor growth inhibitory effects of Lamp2a depletion and correspondingly decreased the survival of GBM-bearing mice (34 d in sh-Lamp2a plus IgG2b versus 24.5 d in sh-Lamp2a plus CD8α mAb, Fig. 6l–n). Collectively, these findings implied that the inhibition of CMA attenuates GBM tumorigenesis by facilitating CD8+ T cell infiltration and enhancing cytotoxic activity.

CMA regulates cGAS/STING expression through targeting TET3 for lysosomal degradation
The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway has been identified as a pivotal mechanism in anti-tumor immunity, regulating the production of interferons and pro-inflammatory cytokines in response to cytosolic DNA or DNA damage53. Given the above results (Fig. 5b), further analysis also showed that LAMP2A KD altered the STING-mediated immune response and interferon signaling (Fig. 7a, b). Subsequently, we investigated whether the MST4-LAMP2A signaling axis influences cGAS-STING signaling. The ablation of either MST4 or LAMP2A resulted in an upregulation of cGAS and STING at both the protein and mRNA levels, which was accompanied by enhanced phosphorylation of TBK1 and STING upon IR stimulation (Fig. 7c–f). Collectively, these results implied that the MST4-LAMP2A signaling pathway modulates cGAS-STING activation by affecting the transcriptional regulation of cGAS and STING.

Promoter regions of CGAS and STING demonstrate hypermethylation across various human cancers, including GBM54. Analysis of the TCGA GBM dataset revealed a negative correlation between the mRNA levels of CGAS and STING and their respective methylation statuses (Fig. 7g, h). DNA methylation processes are predominantly governed by DNA methyltransferases (DNMTs), whereas demethylation is facilitated by ten-eleven translocation (TET) dioxygenases55. Notably, within the TET family, TET2 and TET3 exhibited a positive correlation with the expression of cGAS and STING in GBM samples from both TCGA and CGGA datasets. However, no inverse relationship was found between cGAS or STING expression and DNMT levels (Supplementary Fig. 10a, b). To explore the regulation of cGAS and STING expression by TET dioxygenases, we conducted experiments involving the overexpression of TET1, TET2, and TET3 in HEK293T cells (Supplementary Fig. 10c). Remarkably, the overexpression of TET3 led to a significant enhancement of cGAS and STING expression compared to TET1 and TET2 (Fig. 7i). Conversely, the KD of TET3, rather than TET1 or TET2, resulted in a substantial reduction in cGAS and STING expression (Fig. 7j).

To determine if TET3 undergoes CMA-mediated proteolysis, we examined its sequence for the presence of putative KFERQ-like motifs. We compared the motifs and found that TET3 possess such motifs (Supplementary Table 1). Given that CMA substrates are recruited to the lysosome via HSC70, we investigated the potential interaction between HSC70 and TET3. Wild-type TET3 was found to interact with both HSC70 and LAMP2A; however, the mutation in the KFERQ-like motifs of TET3 (Supplementary Table 1) disrupted these interactions (Fig. 7k), leading to enhanced stability of TET3 protein upon treatment with CHX (Fig. 7l, m). Furthermore, KD of LAMP2A or HSC70 in GSCs resulted in a diminished association of TET3 with lysosomes and an upregulation of total TET3 protein level (Fig. 7n, o). Importantly, neither the depletion of LAMP2A nor HSC70 caused notable alterations in TET3 mRNA levels (Supplementary Fig. 10d, e). Conversely, the overexpression of both LAMP2A-WT and the 3D mutant resulted in decreased TET3 levels in HEK293T cells, whereas the LAMP2A-3A and 4A mutants did not have this effect (Supplementary Fig. 10f). Furthermore, the co-localization of TET3 with lysosomes that express LAMP2A during CMA-inducing conditions, such as nutrient deprivation, was disrupted when the KFERQ motifs were altered (Supplementary Fig. 10g, h). Treatment with lysosomal protease inhibitor LN increased TET3 protein level after CHX treatment, indicating that TET3 degradation is mediated by CMA (Fig. 7p). In contrast, neither the proteasome inhibitor (MG132) nor the macroautophagy inhibitor (3-MA) had this effect (Fig. 7p). When purified TET3 protein was incubated with lysosomes, either untreated or pre-treated with PI, immunoblot analysis of the recovered lysosomes indicated that TET3 was taken up and degraded by them (Supplementary Fig. 10i, j). Collectively, these results support the conclusion that TET3 is a substrate for CMA.

Phosphorylation of LAMP2A by MST4 inhibits cGAS-STING signaling by repressing TET3 expression
To investigate whether CMA inhibits the cGAS-STING pathway via TET3 suppression, we established individual KD GSC cell lines targeting TET3 (Supplementary Fig. 11a). Our findings revealed that KD of MST4 or LAMP2A enhanced, while KD of TET3 diminished, the level of herring testis DNA (HT-DNA) or the IR-induced activation of cGAS-STING signaling, as indicated by the phosphorylation of TBK1 and STING (Supplementary Fig. 11b, c). Furthermore, TET3 KD mitigated the impact of MST4 or LAMP2A KD on IR-triggered activation of cGAS-STING pathway (Fig. 8a–d), suggesting that TET3 is essential for CMA’s suppression of cGAS-STING signaling. In a parallel experiment, pharmacological inhibition of CMA using Polyphyllin D (PPD), a potential inhibitor of CMA that disrupts the binding between HSC70 and LAMP2A and inhibits LAMP2A homo-multimerization47, was found to enhance cGAS-STING signaling in GSC M83 and 456 cell lines. Our results showed that MST4-mediated phosphorylation facilitates LAMP2A multimerization, which is essential for CMA activation; however, PPD selectively interferes with the downstream multimerization process without affecting LAMP2A phosphorylation (Supplementary Fig. 11d, e). Furthermore, PPD disrupted the HSC70-LAMP2A complex, resulting in the cytosolic accumulation of CMA substrates, including TSC1/2, TET3, and MEF2D, accompanied by a decrease in lysosomal delivery and CMA flux, as evidenced by photo-convertible reporter assays (Supplementary Fig. 11e–g). Thus, PPD effectively decouples MST4-mediated LAMP2A phosphorylation from functional CMA activation. Additionally, TET3 KD was found to inhibit the PPD-induced activation of cGAS-STING signaling (Fig. 8e, f).

To elucidate the role of MST4-mediated phosphorylation of LAMP2A in modulating cGAS-STING signaling, we assessed the alterations in cGAS-STING pathway activity following IR stimulation in LAMP2A-KD GSC M83 cells reconstituted with either LAMP2A-WT or LAMP2A-3D, subsequently subjected to MST4 KD. In GSC M83 cells expressing LAMP2A-WT instead of LAMP2A-3D, MST4 KD significantly enhanced the activation of the cGAS-STING signaling pathway in response to IR (Fig. 8g, h). Additionally, the KD of endogenous LAMP2A in GSC 23 cells resulted in an enhancement of IR-induced STING/TBK1 activation and an upregulation of IFNB1 expression. In contrast, the overexpression of exogenous MST4 inhibited the activation of the cGAS-STING pathway in GSC 23 cells, and these effects were attenuated by the KD of endogenous LAMP2A (Fig. 8i, j).

Subsequently, we investigated whether the KD of TET3 or cGAS could counteract the influence of LAMP2A KD on in vivo tumor proliferation. In an immunocompetent murine model of intracranial GBM, we demonstrated that Lamp2a KD significantly suppressed GL261 cell proliferation and tumorigenesis in vivo, while extending mouse survival from 22 to 38 days (Fig. 8k, l and Supplementary Fig. 11h-j). Notably, in vitro experiments revealed that Cgas (but not Tet3) KD had no impact on GL261 proliferation regardless of Lamp2a status (Supplementary Fig. 11h–j). However, in vivo Tet3 or Cgas KD promoted tumor growth, reduced intratumoral CD8+ T cell infiltration, and shortened survival (Fig. 8k–m). Collectively, these findings indicated that CMA modulates tumor progression, at least in part, through TET3-cGAS-STING signaling pathway.

CMA inhibition enhances radiotherapy and immune checkpoint therapy in a murine GBM model
Based on our findings, we proposed a model wherein CMA functions as an inhibitor of the anti-tumor immune response by restricting the activation of the cGAS-STING pathway. Supporting this hypothesis, both GSC M83 and GSC 456, as well as GL261 cells with LAMP2A KD, exhibited elevated levels of IFNB1, CCL5, CXCL10, and the immune checkpoint ligand PD-L1 (CD274), while no significant change was observed in PD-L2 expression (Supplementary Fig. 12a). Furthermore, knocking down endogenous Mst4 or Lamp2a in murine syngeneic GBM cell lines GL261 and CT-2A resulted in a significant increase in the levels of CMA substrates TET3 and TSC1/2 (Supplementary Fig. 12b). Upon intracranial implantation of these genetically altered cells into immunocompetent C57BL/6 mice, the absence of Mst4 or Lamp2a led to a marked elevation of TET3 and TSC1/2 expression in the tumors, as confirmed by immunofluorescence analysis of murine tissues (Supplementary Fig. 12c). We then explored whether CMA inhibition would enhance the efficacy of IR or immune checkpoint therapy (ICT) in GBM. Mice with intracranial GL261 and CT-2A xenografts, in the presence or absence of endogenous Mst4 or Lamp2a, were received either IR therapy, or anti-PD-1 monoclonal antibody (mAb), or their combination and were monitored tumor growth and mouse survivals (Fig. 9a and Supplementary Fig. 12d). Notably, neither IR therapy nor anti-PD-1 mAb monotherapy effectively impeded tumor proliferation or enhanced mouse survivals. Conversely, the simultaneous administration of IR and anti-PD-1 mAb resulted in a moderate yet statistically significant (p < 0.05) survival advantage in immunocompetent mice bearing intracranial GL261 and CT-2A GBM cells (Fig. 9b, c and Supplementary Fig. 12e, f). Genetic ablation of Mst4 or Lamp2a to inhibit CMA significantly potentiated the anti-tumor efficacy and survival benefits of either IR alone, anti-PD-1 monoclonal antibody (mAb) monotherapy, or their combination in GL261 and CT-2A GBM models (Fig. 9b–e and Supplementary Fig. 12e–h).

Given that PPD effectively inhibits the multimerization of LAMP2A, which is a consequence of LAMP2A phosphorylation, we investigated the effects of the CMA inhibitor PPD on IR therapy and anti-PD-1 mAb therapy in the GL261 and CT-2A C57BL/6 intracranial GBM model. The treatment of PPD, IR, and anti-PD-1 mAb were given as monotherapies, in combination, or as a triplet regimen (Fig. 9f and Supplementary Fig. 12i). The results showed that all combination therapy strategies, rather than monotherapy, significantly attenuated tumor growth compared with the control group, with the greatest survival advantage observed in the trimodal treatment group (Fig. 9g, h and Supplementary Fig. 12j, k). As anticipated, the significant tumor shrinkage linked to combination treatment was associated with a higher presence of CD8+ cytotoxic T lymphocytes in the tumor microenvironment (Fig. 9i). Additionally, xenografts treated with the CMA inhibitor PPD exhibited increased expression of the CMA substrates TET3 and TSC1/2, indicating PPD’s ability to penetrate the blood-brain barrier (BBB) and reduce GSC tumorigenicity by inhibiting the CMA-mediated degradation of TET3 and TSC1/2 in the xenografts (Supplementary Fig. 12l, m). In summary, our findings indicated that the combination of CMA inhibition with anti-PD-1 or IR presents a promising avenue for cancer therapeutic.

Correlations between MST4, p-LAMP2A, TET3 and TSC1/2 expression and associations with GBM patient survival
To explore the prognostic significance of our results, we conducted immunohistochemical (IHC) staining for LAMP2A pT136, MST4, TET3, and TSC1/2 in tumor sections from GBM patients (Fig. 10a and Supplementary Dataset). Our analysis of human glioma samples demonstrated a positive association between the staining levels of MST4 and LAMP2A pT136. Furthermore, we observed a negative correlation between LAMP2A pT136 level and both TET3 and TSC1/2 (Fig. 10b). The clinical relevance of these observations was highlighted by the finding that patient survival demonstrates an inverse relationship with the expression levels of MST4 and phosphorylated LAMP2A, while showing a positive association with the expression levels of TET3 and TSC1/2 as determined by IHC staining (Fig. 10c).

Methods
Reagents and antibodies
The chemicals and their sources are as follows: leupeptin (HY-18234; 10μM), NH4Cl (HY-Y1269; 20mM), Pepstatin (HY-P0018, 10μM), 3-MA (3-methyladenine) (HY-19312; 5mM), MG132 (HY-13259; 10μM), CHX (cycloheximide) (HY-12320; 100μg/mL) and SB203580 (HY-10256; 10μM) were purchased from MCE. HT-DNA (Herring testis DNA) (D6898; 100μg/mL), general protease inhibitors (P8340; 1:100), phosphatase inhibitors (P0044; 1:100) and AEBSF (SBR00015; 100μM) were purchased from Sigma-Aldrich. D-luciferin potassium salt (115144-35-9; 150mg/kg in vivo) was purchased from GoldBio. Polybrene (28728-55-4; 6mg/mL) was purchased from Santa Cruz Biotechnology. Puromycin (S250J0; 1mg/mL) was purchased from Basal media. PPD (polyphyllin D) (50773-41-6; 6mg/kg in vivo) was purchased from Yuanye.

The antibodies for immunoblot (IB) were provided as follows: anti-LAMP2A (ab125068; 1:1,000) and veriBlot for IP Detection Reagent (HRP) (ab131366; 1:2,000) were purchased from Abcam. Anti-β-actin (AC026; 1:8,000), anti-β-Tubulin (AC021; 1:8000), anti-CathA (A5503; 1:1000), anti-p62 (A19700; 1:20,000), HRP-conjugated Goat anti-Mouse IgG (H+L) (AS003; 1:8000) and HRP-conjugated Goat anti-Rabbit IgG (H+L) (AS014; 1:8000) were purchased from ABclonal Technology. Anti-GAPDH (60004-1-Ig; 1:50,000), anti-MST4 (10847-1-AP; 1:1000), anti-LAMP1 (21997-1-AP; 1:1000), anti-HSC70 (10654-1-AP; 1:3000), anti-MEF2D (14353-1-AP; 1:1000), anti-CD44 (15675-1-AP; 1:20,000), anti-ATG4B (15131-1-AP; 1:1000), anti-ATG5 (10181-2-AP; 1:1000), anti-COXIV (11242-1-AP; 1:5000), anti-GFP-tag (66002-1-Ig; 1:20,000) and anti-His-tag (66005-1-Ig; 1:5000) were purchased from Proteintech. Anti-TET1 (40142S; 1:1000), anti-TET2 (45010S; 1:1000), anti-TET3 (99980S; 1:1000), anti-cGAS (79978S; 1:1000), anti-STING (13647S; 1:1000), anti-phospho-STING (Ser366) (19781S; 1:1000), anti-TBK1 (3504S; 1:1000), anti-phospho-TBK1 (Ser172) (5483S; 1:1000), anti-TSC1 (6935S; 1:1000), anti-TSC2 (3990S; 1:1000), anti-4EBP1 (9644S; 1:1000), anti-phospho-4EBP1 (Thr37/46) (2855S; 1:1000), anti-P70S6K (34475S; 1:1000), anti-phospho-P70S6K (Thr389) (9234S; 1:1000) and anti-LC3B (3868S; 1:1000) were purchased from Cell Signaling Technology. Anti-Flag-tag (GNI4110-FG; 1:2000) and anti-Myc-tag (GNI4110-MC; 1:2000) were purchased from GNI. Anti-HA-tag (901515; 1:2000) was purchased from BioLegend. Anti-p-Ser/Thr (612549; 1:1000) was purchased from BD Biosciences. Anti-Cyclophilin A (R381723; 1:1000), anti-HSP40 (250684; 1:1000), anti-HSP90 (R24635; 1:1000), anti-ALDH1A3 (670057; 1:1000) and anti-Tuj1 (beta III Tubulin) (R23620; 1:1000) were purchased from ZENBIO. Anti-LAMP2A pT136 (1:1000) was produced in this study.

The antibodies for immunoprecipitation (IP) were provided as follows: anti-LAMP2A (ab125068; 1:60) was purchased from Abcam. Anti-GFP-tag (66002-1-Ig; 0.5μg) was purchased from Proteintech. Anti-CathA (A5503; 0.5μg), Rabbit Control IgG (AC005; 0.5μg) and Mouse Control IgG (AC011; 0.5μg) were purchased from ABclonal Technology.

The following antibodies were used in immunofluorescent (IF) staining: anti-LAMP2A (ab125068; 1:500), anti-CD8α (ab237709; 1:100) and anti-PD-L1 (ab213524; 1:500) were purchased from Abcam. Anti-Flag-tag (66008-4-Ig; 1:2000), anti-GZMB (13588-1-AP; 1:200), anti-TSC1 (29906-1-AP; 1:400), anti-TSC2 (24601-1-AP; 1:400), anti-TET3 (22612-1-AP; 1:400), anti-HSC70 (10654-1-AP; 1:400) and anti-Myc-tag (60003-2-Ig; 1:500) were purchased from Proteintech. Anti-LAMP1 (sc-20011; 1:400), anti-CD8 (sc-1177; 1:200) and anti-FoxP3 (sc-53876; 1:200) were purchased from Santa Cruz Biotechnology. Anti-CD4 (A26036PM; 1:200) was purchased from ABclonal Technology. Anti-LC3B (3868S; 1:1000) and anti-phospho-P70S6K (Thr389) (9234S; 1:400) were purchased from Cell Signaling Technology. Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody Alexa FluorTM 488 (A-11008; 1:500), Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody Alexa FluorTM 594 (A-11012; 1:500), Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody Alexa FluorTM 488 (A-11001; 1:500), Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody Alexa FluorTM 594 (A-11005; 1:500) and Donkey anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody Alexa FluorTM 647 (A-31571; 1:500) were purchased from Invitrogen. Anti-LAMP2A pT136 (1:400) was produced in this study.

The following antibodies were used in immunohistochemistry (IHC): anti-LAMP2A pT136 (1:100) was produced in this study. Anti-MST4 (10847-1-AP; 1:100) was purchased from Proteintech. Anti-TET3 (99980S; 1:100), anti-TSC1 (6935S; 1:100) and anti-TSC2 (3990S; 1:100) were purchased from Cell Signaling Technology.

The following antibodies were used in flow cytometry (FC): Pharmingen™ PE-Cy™7 Rat anti-mouse IFN-γ (557649), Horizon™ BV711 Rat anti-mouse TNF (563944) were purchased from BD Biosciences. FITC mouse anti-human CD8 (980908), FITC anti-mouse CD45 (103107), PE anti-human IFN-γ (502509), PE anti-mouse CD103 (121405), PE anti-mouse FOXP3 (126403), PE/Dazzle™ 594 anti-mouse CD366 (119747), PE/Dazzle™ 594 anti-mouse Ly-6C (128044), PE/Cyanine7 anti-human TNF-α Antibody (502930), Brilliant Violet 421™ anti-mouse Ly-6G (127627), Brilliant Violet 510™ anti-mouse CD3 Antibody (100233), Brilliant Violet 605™ anti-mouse/human CD11b (101237), Brilliant Violet 605™ anti-mouse CD152 (106323), Brilliant Violet 650™ anti-mouse CD69 (104541), Brilliant Violet 650™ anti-mouse CD223 (125227), Brilliant Violet 785™ anti-mouse CD25 (102051), PerCP/Cyanine5.5 anti-mouse CD8a (100734), Alexa Fluor® 647 anti-human/mouse Granzyme B (515406) and Pacific Blue™ anti-mouse CD4 (100427) were purchased from Biolegend. These antibodies used for FC were diluted at a ratio of 0.25μg per 106 cells.

Cell lines and cell culture
The HEK293T, LN229, T98G and HeLa cell lines used in this study were obtained from American Type Culture Collection (ATCC). Glioma cell lines U87, U251, GL261 and CT-2A were generously provided by Dr. Chunsheng Kang at Tianjin Medical University General Hospital84,85. The MRC-5 cell line was provided by Professor Ningshao Xia at Xiamen University. All cell lines were maintained in DMEM (11965092; Invitrogen) supplemented with 10% Fetal Bovine Serum (FBS) (A0500-3011; Cegrogen) and 1% penicillin/streptomycin (15140122; Invitrogen). The GSC M83 cell line was provided by Dr. Haizhong Feng at Shanghai Jiao Tong University86, whereas GSC 23, 456 and 28 cell lines were obtained from Dr. Nu Zhang at the First Affiliated Hospital of Sun Yat-sen University as previously documented87,88. GSCs were cultured as non-adherent spheroids in a serum-free defined medium comprising DMEM/F12 (C11330500BT; Gibco), supplemented with 2% B27 (12587010; Invitrogen), 1% penicillin and streptomycin (15140122; Invitrogen), 5mg/ml heparin (9041-08-1; Sigma-Aldrich) and growth factors EGF (20ng/mL; AF-100-15; Peprotech) and FGF (20ng/mL; 100-18B; Peprotech). The DGC M83, 23 and 456 cell lines were derived from GSC M83, 23 and 456 cell lines following differentiation induced by DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin, incubated at 37°C with 5% CO2 for two days. All cell lines tested negative for mycoplasma contamination.

Animal studies
Male athymic (homozygous Foxn1nu BALB/c nude) mice and C57BL/6 mice at 4-5 weeks of age were purchased from Guangdong Yaokang Biotechnology Co., Ltd. Five mice were grouped in each cage and housed under specific-pathogen-free (SPF) conditions with a standardized 12h light/dark cycle. The ambient temperature was maintained at 21–23°C with a relative humidity of 50%.

For the tumorigenicity studies, 5×104 GSC M83 or 5×105 GSC 23 cells or 1×105 GL261 cells were injected intracranially into BALB/c Nude mice. 1×105 CT-2A or GL261 cells were injected intracranially into C57BL/6 mice. Tumor cells were injected into the brain of individual mice, using the following coordinates from bregma: 2.5mm lateral, 1.5mm anterior, and 3.0mm deep from the skull. All mice were subjected to daily observation to assess the emergence of neurological symptoms indicative of significant tumor progression. The mice were humanely euthanized between 2 to 10 weeks post-implantation, and their brains were subsequently extracted. Each brain was carefully excised and preserved in an optimum cutting temperature (OCT) compound (Sakura Finetek), then stored at -80°C. Brains containing tumors were sliced into 10μm thick sections utilizing a cryostat (Leica CM1950). The entire brain was sectioned coronally from the anterior to the posterior regions. Every fifth section from each tumor-infiltrated brain underwent hematoxylin and eosin staining to identify the spatial distribution of the tumors from inception to termination. These images of sections were stained with hematoxylin and eosin obtained from MshOt microscopy system. After thorough evaluation, the section exhibiting the maximum cross-sectional area of the tumor for each xenograft was selected for volumetric assessment. Tumor volume was calculated utilizing the formula V = ab²/2, where a and b (with a > b) represent the tumor’s length and width, respectively.

For in vivo therapeutic assays, the C57BL/6 mice stereotactically transplanted with CT-2A or GL261 cells were randomized into each treatment group. For depletion of CD8+ cells, mice received intraperitoneal administrations of CD8α monoclonal antibody (10mg/kg; BE0061; Bio X Cell) or IgG2b (10mg/kg; BE0086; Bio X Cell) every three days from one week after implantation. For immune checkpoint blockade, mice received intracranial administrations of the anti-programmed cell death protein 1 (PD-1) monoclonal antibody (10mg/kg; BE0146; Bio X cell) or IgG2a (10mg/kg; BE0085; Bio X Cell) on the 8th, 12th, 16th, and 20th day after implantation. The radiation groups received 2 Gy of radiation daily for five days, starting two weeks post-transplantation alongside treatment. To evaluate the impact of PPD treatment, mice received 6mg/kg PPD or DMSO intragastrically for seven consecutive days starting from the 10th day after implantation. The mice were observed daily for neurological symptoms related to tumor growth, and maintained until symptoms appeared, such as hunching, weight loss, decreased food intake and lethargy. To track tumor growth following radiation or drug treatment, each mouse received an injection of D-luciferin potassium salt at a dosage of 150mg/kg, and tumor growth was assessed using the IVIS Lumina imaging station (Caliper Life Sciences).

Bioinformatic analysis of LAMP2A correlated pathways
We obtained the RNA-seq dataset GSE181556 from NCBI’s Gene Expression Omnibus, specifically examining control and sh-LAMP2A samples17. Differentially expressed genes (DEGs) were screened based on criteria of a fold change ≥ 2 and a false discovery rate (FDR) < 0.01. Enrichment analysis of biological pathways was conducted using clusterProfiler, with pathways enriched at p < 0.05 considered significantly correlated with the DEG set. Additionally, proteome data was accessed from the ProteomeXchange Consortium, utilizing the dataset identifier PXD027069. Significantly differential proteins (p < 0.05) with a ratio > 1.5 in either direction were analyzed using DAVID to characterize their involved biological pathways, with pathways enriched at p < 0.05 deemed significantly correlated with the set of differential proteins.

Bioinformatic analysis of CGAS/STING
Firstly, we acquired GSE181556 dataset and utilized the normalized expression matrix to analyze signaling pathways correlated to LAMP2A through Gene Set Enrichment Analysis (GSEA)89. Subsequently, DNMIVD90–92 was used to examine the correlation between CGAS/STING levels and the mean DNA methylation of the CGAS/STING’s promoter, respectively. Furthermore, the expression correlations between CGAS/STING and TETs/DNMTs were analyzed using Pearson’s correlation based on TCGA (https://xena.ucsc.edu/)93 and CGGA94 datasets.

Bioinformatic analysis of immune infiltration based on CMA score
As previously described95, to quantify the activity of the CMA pathway within individual cells, a CMA score was calculated based on the expression levels of a predefined set of CMA-associated genes to reflect the coordinated expression changes associated with CMA activity in each cell. Briefly, each component in the CMA network was assigned a weight. Since LAMP2A is the rate-limiting factor, it received a weight of 2, while all other elements were assigned a weight of 1. Then, each element was assigned a directional score (+1 or -1) based on its known regulatory effect on CMA activity. The final CMA score was computed as a weighted, direction-adjusted average of the expression levels across all network components. TCGA-GBM counts expression data were used to calculate the CMA score. Immune infiltration data for TCGA-GBM were obtained from TIMER and TISIDB. GSVAR package (version 1.52.3) was used to calculated the MDSC infiltration (include CD33, ITGAM, and CD15). The relationship between CMA scores and immune infiltration levels was assessed using Spearman’s correlation analysis and Pearson’s correlation analysis. Statistical significance was defined as p < 0.05, and the strength of linear associations was interpreted based on the resulting Spearman’s correlation coefficients or Pearson’s correlation coefficients.

Plasmid construction
The open reading frames (ORFs) for MST4 and LAMP2A were cloned into the pCDH-CMV-MCS-EF1-Puro vector, resulting in the construction of pCDH-HA-MST4, pCDH-Flag-MST4 and pCDH-Flag-LAMP2A. The ORF of TSC1/2 and TET1/2/3, along with their variants, were sub-cloned into the pCMV6 vector, incorporating a sequence that encodes a N-Myc epitope. Site-directed mutagenesis was performed using the Q5® Site-Directed Mutagenesis Kit (E0554; NEB), adhering to the manufacturer’s protocols.

Human cDNAs corresponding to LAMP2A and its variants were amplified via PCR and subsequently sub-cloned into the pET-28a vector for expression in E. coli.

The pLKO.1 lentiviral shRNA constructs targeting LAMP2A, MST4, HSC70, TSC1/2, TET1/2/3, cGAS, ATG4B and ATG5 were designed in accordance with the protocol available on the Addgene website (https://www.addgene.org/protocols/plko/). All target sequences shRNAs are listed in Supplementary Table 2.

To knockout STK26, the target sequence of gRNA was designed by the CRIStick online website (https://portals.broadinstitute.org/gppx/crispick/public). The synthesized primer containing a 20bp target sequence (5’-CTCGCCGGTGGCTGTCCAAG-3’ or 5’-GGGCGGTGGTTCAGCACTGG-3’) was annealed and inserted into lenti-CRISPR-v2 using T4 ligase (FL101; TransGen Biotech).

All primers used for plasmid construction were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and were listed in Supplementary Table 3.

All resulting cDNA constructs and mutants were validated for accuracy through DNA sequencing.

Lentiviral production and infection
Lentiviral vectors harboring shRNAs, or full-length cDNAs for MST4, LAMP2A, and their respective mutants were introduced into HEK293T cells utilizing Lipofectamine 2000 (Life Technologies), following the manufacturer’s protocols. In gene rescue assays, shRNAs designed to target the 3’ untranslated regions of MST4, LAMP2A, TSC1/2, and TET3 were employed for knockdown purposes. The lentiviral supernatants were harvested at 48 and 72h post-transfection and subsequently used to infect recipient cells in the presence of polybrene (6mg/mL). Following a 72-hour transduction period, the cells underwent selection with puromycin at a concentration of 1mg/mL for four days.

Immunoblot (IB) analysis
Cells were lysed using RIPA buffer (E121-01; GenStar) supplemented with protease and phosphatase inhibitors on ice, followed by centrifugation at 15,000×g for 15 minutes at 4°C. The resultant supernatant was utilized to prepare the protein sample for SDS-PAGE analysis. Proteins were subsequently transferred from the gel to a polyvinylidene fluoride (PVDF) membrane (IPVH00010; Millipore) in a transfer buffer containing 25mM Tris and 192mM glycine. The membranes were blocked with 5% fat-free milk at 37°C for 2h and then incubated with the primary antibody at 4°C overnight. After wash with TBST (TBS with 0.1% Tween-20), the membranes were treated with the appropriate secondary antibodies. Detection was performed using an enhanced chemiluminescence (ECL) (GCP1001-500-ES; GENECYCLER) assay according to the manufacturer’s protocol.

The degradation rate of LAMP2A in isolated lysosomes was evaluated using IB with a specific antibody against LAMP2A, following established methods13. In summary, the lysosomes were incubated at 0°C for 10min with or without lysosome protease inhibitor cocktail (PI) consisting of: 10μM leupeptin (targeting serine/cysteine proteases), 10μM AEBSF (serine protease inhibitor), 1μM pepstatin A (aspartic protease inhibitor), and 100μM EDTA (metalloprotease inhibitor). At various time points, samples were taken for SDS-PAGE and immunoblotting analysis of LAMP2A.

Immunoprecipitation (IP)
Cells were lysed using IP lysis buffer (87787; Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitors, incubated on ice for 15min, and then centrifuged at 15,000×g for 15min at 4°C to eliminate cellular debris. Following a pre-clearing step with protein G/protein A-agarose beads (IP10; Sigma-Aldrich), the supernatant was then immunoprecipitated with anti-DYKDDDDK (Flag) G1 Affinity Resin (L00432; GenScript), anti-Myc affinity gel (GNI4510-MC; GNI), anti-HA-magnetic-beads (B26202; Bimake), or agarose-immobilized anti-LAMP2A, anti-CathA, anti-GFP-tag, Mouse Control IgG or Rabbit Control IgG at 4°C overnight.

Native gel electrophoresis
Lysosomal membranes were isolated according to established protocols96, and subjected to continuous native polyacrylamide gel electrophoresis (6% natural polyacrylamide gel devoid of SDS) in a non-reducing loading buffer without boiling, as outlined by Rodriguez-Navarro JA. et al.97. Following transfer to PVDF membranes, the LAMP2A containing complexes were identified using an anti-LAMP2A antibody.

Immunofluorescent (IF) staining
For immunofluorescent staining of cultured cells, cells were fixed using 4% paraformaldehyde for 15min, followed by permeabilization with 0.3% Triton X-100, and subsequently blocked with 5% BSA for 2h. Primary antibodies were applied and incubated overnight at 4°C. After wash with PBS, the cells were incubated with Alexa FluorTM 488, 594 or 647 conjugated secondary antibodies in the dark. Following additional wash with PBS, the cells were treated with DAPI (AR1176; BOSTER). After mounting, the samples were examined using an inverted microscope fitted with a Nikon digital camera, and quantitative analysis was conducted using ImageJ software.

For immunofluorescent staining of tissue sections, the sections were fixed similarly to the cultured cells, then subjected to permeabilization with 0.5% PBST (PBS supplemented with 0.5% Triton X-100) for 30min and blocked with 5% BSA for 2h. Sections were incubated overnight at 4°C with primary antibodies. The subsequent incubation with secondary antibodies and DAPI followed the same protocol as for cultured cells. Ultimately, the sections were analyzed using an inverted microscope equipped with a Nikon digital camera, with statistical analysis performed using ImageJ software.

Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cells using TRIzol (R1000; Lablead) and reversely transcribed into cDNA using Hifair III Reverse Transcriptase (11297ES09; YEASEN). The cDNA products were analyzed via qRT-PCR with SYBR Green PCR Master Mix (11201ES08; YEASEN). Detailed information about the primer sequences utilized can be found in Supplementary Table 4.

Flow cytometric analysis
Single-cell suspensions were prepared by enzymatically dissociating isolated mouse glioma tissues using Collagenase IV (HY-E70005D; MCE) and DNase I (HY-108882; MCE). Following the lysis of erythrocytes with a lysis buffer (G2015; Servicebio), the cells were activated with the BD Pharmingen™ Leukocyte Activation Cocktail supplemented with BD GolgiPlug™ (550583; BD Biosciences) at 37°C in a 5% CO₂ atmosphere for a duration of 4h. After a 15min incubation on ice, shielded from light, with the Zombie NIR™ Fixable Viability Kit (423105; BioLegend), the surface marker staining solution (including antibodies such as CD45, CD3, CD4, CD8, etc.) was prepared according to the specified concentrations. The cells were incubated with the staining solution on ice in the dark for 30min. Following this, the cells were fixed and permeabilized using BD Cytofix/Cytoperm™ Solution (554722; BD Biosciences) before undergoing intracellular target staining (e.g., TNF-α, IFN-γ). Flow cytometry data were collected using a BD FACSymphony™ A1 instrument and subsequently analyzed utilizing FlowJo v10 software.

Apoptosis detection
In accordance with the manufacturer’s specifications, cellular apoptosis was assessed utilizing the APC Annexin V Apoptosis Detection Kit in conjunction with Propidium Iodide (PI) (640932; Biolegend). Cells underwent two washes with cold BioLegend Cell Staining Buffer (420201; Biolegend) and were then resuspended in Annexin V Binding Buffer (422201; Biolegend) at a density of 0.25–1.0×10⁷ cells/mL. A volume of 100µL of the cell suspension was pipetted into a 5mL test tube, followed by the introduction of 5µL of fluorochrome-conjugated Annexin V. The cells were subsequently stained with propidium iodide as a viability marker, gently vortexed, and incubated for 15min at room temperature (25°C) in the absence of light. Post-incubation, 400µL of Annexin V Binding Buffer was added to each tube, and the samples were analyzed via flow cytometry.

CD8+ T cell preparation and co-culture
CD8+ T lymphocytes were isolated from peripheral blood mononuclear cells of GBM patients utilizing the Human CD8+ T Cell Enrichment Cocktail kit (15023; STEMCELL Technologies). The purified CD8+ T cells were cultured in X-VIVO 15 medium (04-418Q; Lonza) supplemented with 10ng/mL of IL-2 (200-02-50UG; PeproTech) and subsequently activated using human anti-CD3/CD28 Dynabeads (11161D; ThermoFisher) for a period of 48 to 72h. Following activation, the T cells and GSCs were enumerated and introduced into the GSC culture system at a 1:5 ratio for a 24h co-culture at 37°C with 5% CO2, after which flow cytometric analysis was performed.

Identification of phosphorylation sites by LC-MS/MS analysis
To identify LAMP2A phosphorylation, Flag-LAMP2A was immunoprecipitated from HEK293T cells stably expressing exogenous HA-MST4 and Flag-LAMP2A using anti-DYKDDDDK (Flag) G1 Affinity Resin. The resulting immune complexes were subjected to SDS-PAGE for resolution and subsequently stained with Coomassie brilliant blue. Gel segments containing LAMP2A were dehydrated in acetonitrile, dried under a speed vacuum, and digested with trypsin. The resulting peptides were extracted from the polyacrylamide matrix and concentrated for subsequent liquid chromatography-mass spectrometry (LC-MS) analysis at the Core Facility for Biomedical Sciences at Xiamen University. The mass spectrometry dataset has been deposited in the PRIDE repository (Accession unmber: PXD060464).

CMA activity detection by KFERQ puncta
As previously described37, CMA activity was detected by the photoactivatable reporter, pSIN-PAmCherry-KFERQ-NE (Addgene). GSC and non-GSC cell lines expressing PAmCherry-KFERQ-NE were photoactivated by exposure to 405/20nm LED array (Norlux) for 10min. Alterations in the cellular pattern of the reporter were examined utilizing confocal microscopy (Zeiss LSM980 or Nikon A1R), and image quantification was performed with ImageJ software. Enhanced CMA activity was evidenced by an increase in the number of red fluorescent puncta per cell.

In vitro cell proliferation assay
GSC spheres were dissociated into individual cells, and the viable cells (negative for Trypan Blue) were enumerated using a hemocytometer. The cells were subsequently plated in a 24-well plate with 1mL of culture medium at a density of 5000 cells per well, and viable cell counts for each well were performed daily for five consecutive days using a hemocytometer.

Limited dilution assays for GSCs
Dissociated cells harvested from glioma spheres were plated in 96-well plates at densities of 1, 5, 10, 20 or 50 for GSC M83, 456 and 23 cells. After 7 days, tumorsphere formation was assessed from each well. The frequency of stem cells was determined utilizing extreme limiting dilution analysis (https://bioinf.wehi.edu.au/software/elda/).

In vitro kinase assay
The pCDH-Flag-MST4 expression vector was introduced into HEK293T cells via lipofectamine 2000 (Thermo Fisher Scientific). Following a 48-hour incubation, the cells were lysed using a lysis buffer supplemented with protease and phosphatase inhibitors. The Flag-MST4 protein from the cell lysates was subsequently immunoprecipitated using anti-DYKDDDDK (Flag) G1 Affinity Resin at 4°C overnight. The isolated MST4 proteins were then re-suspended in a mixture containing 1 × kinase buffer (9802S; Cell Signaling Technology), 200μM ATP (9804S; Cell Signaling Technology), and purified His-tagged LAMP2A variants (WT, T40A, S97A, T136A, T40A/S97A, T40A/T136A, S97A/T136A, T40A/S97A/T136A). The kinase reaction was conducted for one hour at 30°C and subsequently quenched with 20mL of 3× SDS sample buffer. Each sample was then subjected to boiling for 10 minutes at 100°C after the addition of loading buffer, followed by SDS-PAGE and immunoblotting analysis utilizing the specified antibodies.

Lysosomes isolation
Lysosomes were purified from cells using a lysosome isolation kit from Biovision (K235-50). According to the protocol, 2×107 cells were washed with PBS at 100×g for 1min and resuspended in 500μL lysosome isolation buffer for 2 minutes. The suspension was homogenized with a Dounce homogenizer for 40 times. Then the homogenate was transferred to the tube containing 500μL lysosome enrichment buffer and mixed, and centrifuged at 500×g for 10min. The supernatant was centrifuged at a density gradient of 20,000×g for 2h. The lysosome precipitate was collected with appropriate volumes of PBS, and centrifuged at 18,000×g for 30 minutes. All steps were conducted on ice or maintained at a temperature of 4°C. The extracted lysosomes were absence of any cytoplasmic or mitochondrial components by IB detection of markers.

In vitro lysosome binding and uptake assay
According to the previously reported study98, incubating purified His-tagged GAPDH with intact lysosomes from GSCs, pre-treated or not with lysosome protease inhibitor cocktail (PI) that inhibit lysosomal proteolytic activity, facilitates the quantification of substrate binding and translocation into the lysosomal lumen. In untreated lysosomes, internalized proteins are rapidly degraded, allowing only membrane-bound proteins to be detected. NH4Cl, a lysosomotropic compound, neutralizes lysosomal pH, which may lead to lysosomal impairment99. For CMA activity assays, PI was used instead of leupeptin/NH4Cl to specifically inhibit lysosomal proteases while minimizing interference with CMA machinery. Following a 20min incubation with recombinant protein in 20mM MOPS, 0.3M sucrose buffer at 37°C, samples were centrifuged at 25,000×g for 5min at 4°C. The resultant pellets were washed with the incubation buffer, resuspended, denatured in sample buffer at 95°C for 5min, and analyzed via SDS-PAGE and immunoblotting. Proteins associated with lysosomes untreated with PI represented the protein binding to lysosomes, and uptake was calculated by subtracting the binding proteins from the total proteins in lysosomes treated with PI100. Quantification of protein band intensities on the immunoblots was performed using ImageJ software.

For other CMA substrates (TSC1, TSC2, and TET3), Myc-TSC1/2 and -TET3 were overexpressed in HEK293T cells, followed by IP using anti-Myc affinity gel and subsequent elution with 150μg/mL Myc competitive peptides (P9805; Beyotime Biotechnology). The purified Myc-tagged substrates were then incubated with lysosomes isolated from 293T cells in reaction buffer (20mM MOPS, 0.3M sucrose) with or without PI at 37°C for 20min. Following incubation, lysosomes were isolated via centrifugation and analyzed using SDS-PAGE and immunoblotting. The protein found in untreated lysosomes indicated binding, while the uptake was determined by subtracting the protein levels in lysosomes treated with protease inhibitors from those in untreated lysosomes21.

Senescence-associated beta-Galactosidase (SA-β-gal) staining
SA-β-gal activity was assessed using a Cellular Senescence β-Galactosidase Staining Kit (C0602; Beyotime Biotechnology). Cells were seeded in 6-well plates containing complete culture medium and cultured for 48 hours. Following fixation for 15min, cells were washed three times with PBS. The fixed cells were then incubated with β-galactosidase staining solution at 37°C overnight in a CO2-free incubator. Images of stained cells were obtained from MshOt microscopy system and quantitative analysis was performed using ImageJ software.

IHC analysis of clinical glioma specimens
Research involving patient specimens received partial approval from the institutional review boards at Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (UHHUST), and was carried out in compliance with Xiamen University regulations. The samples were collected under the clinical protocol No. UHCT-IEC-SOP-016-03-01, which was sanctioned by the UHHUST institutional review board. Signed informed consent was obtained from all participating individuals prior to participation in the study. IHC staining was performed a cohort of 83 patient glioma samples with the specified antibodies. Images of IHC sections were obtained by MshOt microscopy system and were quantitatively scored based on the percentage of positively stained cells and staining intensity, following established protocols101,102. Scoring was: 0 for 0% positive tumor cells, 1 for 0-1%, 2 for 2-10%, 3 for 11-30%, 4 for 31-70%, and 5 for 71-100%. Staining intensity was rated from 0 to 3, with 0 as negative, 1 weak, 2 moderate, and 3 strong. The total score, from 0 to 8, combined the proportion and intensity scores. These scores were compared with overall survival, defined as the time from diagnosis to death or last follow-up. Kaplan-Meier plots for overall survival rates of GBM patients were generated, classifying them into high (score 4-8) and low (score 0-3) expression groups of the indicated protein. Due to antibody sensitivity variations and glioma tumor sample heterogeneity, the number of samples showing high or low expression of specific proteins varies across the graphs, even with the same scoring criteria.

To investigate the prognostic significance of MST4, LAMP2A pT136, TET3, and TSC1/2 protein expression, we employed block randomization stratified by age, sex, and WHO grade to ensure balanced distribution of potential confounders across experimental cohorts. Kaplan-Meier survival curves were generated comparing overall survival between expression groups, with rigorous control for tumor heterogeneity and antibody performance variations. Comorbidities including cephalalgia (n=20), epilepsy (n=3), and seizure (n=5) were documented in Supplementary Dataset, but excluded from primary analysis to minimize confounding effects. This methodological approach ensured the observed survival correlations primarily reflected the biological impact of target protein expression patterns.

PNGase F treatment
LAMP2A protein was immunoprecipitated using an anti-LAMP2A antibody from GSC M83, along with their corresponding DGCs. A total of 60μL of denaturing buffer (10mM Tris-HCl, pH 6.8, 8mM dithiothreitol, and 0.1% SDS) was added to the washed agarose resin, which was then heated to 100°C for 5min. After brief centrifugation, 20μL of the eluted material was combined with 30μL of reaction buffer (80mM sodium phosphate, pH 7.5, and 1.6% NP-40), with or without 8,000U/mL PNGase F (P0704; New England BioLabs), and incubated at 37°C for 1h. Finally, the reactions were analyzed by IB.

Lectin blotting
The protein samples were subjected for SDS-PAGE analysis and subsequently transferred from the gel to a PVDF membrane in the transfer buffer. The membranes were blocked with 5% BSA at 37°C for 30min and then incubated with biotin-labeled Concanavalin A (ConA) (B-1005-5; Vector Laboratories) and HRP-conjugated streptavidin (SE068; Solarbio LIFE SCIENCES). After wash with PBST (PBS supplemented with 0.05% Tween 20), the membranes were performed using an ECL assay.

Antibody production
Polyclonal antibodies against the phosphorylated T136 residue of LAMP2A were produced in murine hosts via immunization with a synthetic phosphopeptide mimicking the sequence adjacent to the T136 site of human LAMP2A (AEDKGIL(p-T)VDELLAIC). Antiserum titers were quantified to be approximately 1:10,000 through enzyme-linked immunosorbent assay (ELISA). IgG fractions were subsequently purified using protein A-Sepharose (Bio-Rad). These antibodies underwent stringent specificity assessment via IB, IF and IHC, utilizing both modified and unmodified peptide controls. Non-specific IgG served as a negative control.

Quantification and statistical analysis
Statistical analysis was carried out using Microsoft Excel 2021, GraphPad Prism version 8, R 4.1.3 and RStudio 2023.12.0+369 for Windows. The analysis included Student’s T-test (two-tailed), one-way ANOVA with Tukey’s multiple comparisons test or Dunnett’s multiple comparisons test and two-way ANOVA with Bonferroni’s multiple comparison test. The Kaplan-Meier survival curves and log-rank test were used to determine the differences in survival rates between two groups. Cox proportional hazards model was employed to perform univariate and multivariate analysis. All grouped data are presented as mean ± S.E.M., unless otherwise stated. p < 0.05 was considered statistically significant.

Ethics statement
Our research complies with all relevant ethical regulations of Xiamen University and Huazhong University of Science and Technology. All animal protocols were approved by the Animal Research Ethics Committee of Xiamen University. The animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals and received approval from the Institutional Animal Care and Use Committee (IACUC) at Xiamen University. The Animal Ethics Committee of Xiamen University allows a maximum tumor diameter of 15mm, and all tumors in our experiments adhered to this limit. None exceeded this size. The collection and use of clinical materials were approved by the Medical Research Ethics Committee of Huazhong University of Science and Technology and Xiamen University.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Li, Sheng and Li et al studied how chaperone-mediated autophagy (CMA) affects the tumor activity of glioblastoma (GBM).\nUsing multiple lines of evidence, the authors identified MST4 as an interaction partner of LAMP2A, the only known\nlysosomal CMA receptor, and that its phosphorylation of LAMP2A enhanced LAMP2A’s stability in the lysosome, CMA\nactivity and GBM stem cell tumorigenicity. Next, using mouse models, the authors found that inhibiting CMA activity through\nknockdown of LAMP2A or MST4 led to reduced tumor growth and higher survival, which was mediated by T cell immune\nresponse. Mechanistically, the authors showed that CMA enhanced tumor activity via suppression of TSC1/2 proteins, which\nare negative regulators of the mTORC1 pathway, and via suppression of TET3 protein, which upregulates the cGAS-STING\npathway via DNA demethylation. Finally, the authors showed the effectiveness of a GBM treatment strategy that combined\nCMA inhibition with radiotherapy and immune checkpoint therapy.\nI am not qualified to comment on the biological novelty of the study. Besides that, the study is an impressive endeavor, as\nevidenced by the large amount of effort that has gone into it. I especially appreciate that the authors were able to verify their\nfindings in GBM patients. At the editor’s discretion, I think the study contains enough information for two separate papers.\nMajor comments:\n1. The authors have not made their code available, especially for the RNA-seq analyses, for review.\n2. Could the authors discuss how the GSC cell line 23 still shows higher activation of CMA compared to non-GSC cell lines\n(Fig 1), despite the former’s very low expression of MST4 (Fig 5a)?\n3. Did the authors use the same data for the red survival curve in Fig 7j and the black survival curve in Fig 10d? They look\nquite similar.\n4. The authors should provide information on the characteristics of the 83 patients analyzed in Fig 11 (similar to the Table 1\nin clinical papers). How did the authors take into account potential confounders when analyzing the clinical samples? Did\nthe patients have any noteworthy co-morbidities?\nMinor comments:\n1. What statistical test did the authors use for the sphere-forming frequency analyses (those like Fig 1f). Why do those plots\nalways have upside down triangles at y-values < -3 and x-values = 20 and 50?\n2. Fig 1a, d: the error bars in the quantification of puncta per cell look unusually small compared to the variance of the data,\nand the error bars of different groups look almost identical to one another.\n3. Is Fig 1c missing the results after treatment with NH4Cl? It was mentioned in the figure caption and the text (lines 114,\n115).\n4. Fig 1d: the left images show cell lines M83 and 23, while the right plot shows cell lines M83 and 456. Was this\nintentional?\n5. Fig 2: did the author use LN as the protease inhibitor?\n6. Fig 4b-d, g-h: the authors should also show the P-values comparing the blue and black data.\n7. Fig 5c: please indicate the P-values on the legend like Fig 4d.\n8. Fig 6a: the x-axis should say “0.00” instead of “-0.00”.\n9. Fig 7j: the authors should show the P-value comparing the green and blue lines.\n10. Lines 549 and 554: did the authors mean GSCs instead of “CSCs”?\n11. Line 720: “quired” typo.", "reviewer_2": "This study by Li et al. explores the role of the MST4-LAMP2A axis in regulating chaperone-mediated autophagy (CMA) and\nits impact on glioblastoma stem cell (GSC) proliferation. The authors report elevated levels of LAMP2A in patient-derived\nGSCs and demonstrate that LAMP2A downregulation in glioma cell lines reduces GSC proliferation in vitro while improving\nsurvival in orthotopic immunodeficient mouse models. Mechanistically, they identify MST4 as a key regulator that\nphosphorylates LAMP2A, enhancing its stability in GSC lysosomes and promoting mTORC1 activation through the\ndegradation of its negative regulators, TSC1 and TSC2. Therapeutically, the study links this pathway to immune evasion via\nsuppression of the cGAS-STING axis and shows that inhibiting CMA synergizes with anti-PD-1 therapy in a GL261\nsyngeneic model. Overall, the paper provides a comprehensive mechanism for CMA regulation and presents a strong case\nfor targeting CMA as a potential therapeutic strategy for glioblastoma.\nMajor points:\n-The paper is very comprehensive with the introduction explaining the key players of the mechanism behind CMA\nmodulation for GSCs. The results flow well with logical progression of the rationale behind targeting MST4 or LAMP2A\n-Given recent studies that have highlighted the importance of efferocytosis in the tumor microenvironment with autophagy,\nthe authors should mention this process as it is directly impacted by their proposed therapy. This would also strengthen the\nstudy’s scope of mechanism in that it would functionally incorporate myeloid cells into the pathways described\n-GL261 is a very immunogenic model that is now out of favor compared to more resistant lines such as CT-2A and SB28.\nThe preclinical orthotopic data could be strengthened by showing survival studies with these additional models\n-This study could be significantly strengthened by including further immunophenotyping beyond CD8+ T cells (e.g. Tregs,\nTAMs, MDSCs). By characterizing the immune microenvironment further (in both human tissue samples as well as in the\nsyngeneic model), the role of CMA in impacting the immune milieu could be better understood. This would especially be\nhelpful for expanding on the overlap with cGAS-STING and this strategy synergizing with anti-PD-1 as shown in the study.\nMinor points:\n-Please undergo mechanical revision for minor errors (i.e. line 51 Prior study has identified…)", "reviewer_3": "In this study, Li et al show that CMA is upregulated in patient-derived glioblastoma stem cells characterized by a notable\nincrease in the levels of the CMA receptor, lysosome-associated membrane protein type 2A (LAMP2A). Moreover, their\nexperiments delineate an MST4-LAMP2A signaling pathway that modulates CMA activity and facilitates tumor initiation and\nimmune escape of GSCs.\nThe results describing that CMA and LAMP2A are increased in patient-derived glioblastoma stem cells and associated with\npoor patient survival have been previously described (Auzmendi-iriarte et al Cancer Research). Similarly, that study\ndescribed that CMA activity promotes tumor initiation and progression and revealed immune escape and metabolism as\ncritical processes involved on them. Indeed, the OMIC data used in the study of Li et al were generated on the Auzmendi-\niriarte study. Although the study of Li et al went deeper in the description of the molecular mechanisms underlying CMA\nactivity, it does not present enough novelty and relevance to be published in Nature Communications. I I think the\nmanuscript suits better in a specific journal of autophagy.", "reviewer_4": "In the manuscript, the authors describe a novel mechanism of CMA regulation by stabilization of LAMP2A in the lysosome\nthrough its phosphorylation by MTS4. They also state that they have described two CMA targets that are implicated in GSC\ntumorigenicity: TCS1/2, implicated in mTOR1 pathway, and TET3, implicated in cGAS-STING pathway. Nevertheless, they\ndon’t clearly demonstrate that TET3 and TSC1/2 are CMA substrates, and that the effects they see in GSC tumorigenicity\nchanges are only due to CMA modulation and not to macroautophagy. Importantly, there is not clear link between CMA\nregulation, effects they see in GSC tumorigenicity and the possible substrates, as they try to show this link only using a\npharmacological modulator of CMA which might be none specific and also induce other cellular stress mechanisms.\nThe authors should address several major points to improve their study and meet the standards of the journal.\n· The authors state in the introduction: “MST4 is recognized for phosphorylating ATGB4, thereby initiating macroautophagy,\nwhich enhances cell survival and tumorogenic potential of GSCs”. However, when they regulate MST4 levels, they only\nattribute changes in GCS tumorigenicity to changes in CMA without showing if also levels of macroautophagy are changed\n(LC3 flux). They must show if macroautophagy is implicated in MST4 regulation of tumorgenicity.\n· Authors do not follow all the known guidelines to validate their potential CMA substrates, therefore, they are not showing\nthat MST4 and TET3 are CMA substrates (doi.org/10.1080/15548627.2020.1797280).\n· Authors must show the quality of lysosome preparations. They should show enrichment of LAMP1 or LAMP2A in the\nlysosome lysates vs whole cell lysates by WesternBlot. Moreover, they must show the quality of the lysosomal preparations,\nshowing absence of mitochondrial and cytosolic markers in their Western-blot with lysosomal preparations.\n· Authors must clearly demonstrate that lysosomes are CMA competent: they should see an increase in HSC70 and not only\nin LAMP2A levels in WB to conclude there’s an activation of CMA. They should corroborate that those CMA competent\nlysosomes have increased phosphorylated LAMP2A when modifying MST4 levels.\n· TCS1/2 is a known regulator of macroautophagy. Authors should prove that regulation of TCS1/2 does not alter\nmacroautophagy levels, so that the effects on GCS that they see in tumorigenicity are caused only by CMA regulation.\n· Besides all the above recommendations to validate CMA substrates, authors also should validate that TET3 expression\nthey see is due to changes in protein levels and not due to gene expression (measuring mRNA levels).\n· If authors are able to demonstrate that TET3 and TCS1/2 are CMA substrates, they should show if the increased stability of\nthe phosphorylated LAMP2A at the lysosomal membrane is a common mechanism among other types of cells, not only in\nGSCs. They should include a model where CMA is downregulated ( for example an aging model) and test whether they can\nrestore CMA by introducing the phosphomimic version they use.\n· During the whole manuscript, authors are trying to point out the relevance of CMA for GSC tumorigenicity and proliferative\npotential. They are trying to prove that by performing several in vitro and in vivo analysis with different lines of human GSC.\nNevertheless, when they test in the in vivo model if LAMP2A or MTS4 regulation in tumor cells changes the immune\nresponse to the tumor (Figure 7), they use GB mouse cells, even if they have the tools to do it with the modified GSC. They\nalso use GB mouse cells when studying the effects of IR therapy and anti-PD1 therapy in the in vivo model (Figure 10).\nAuthors should show the results of immune regulation effects with the human GCS lines with the corresponding genetic\nmodifications.\n· The inhibitor PPD is used arbitrarily and without context. To improve the quality of the work, authors should show the link, if\nany, between the phosphorylation of LAMP2A and the modulation of the CMA activity by PPD.\n· Supplementary Fig. 1E-H- Authors show some redundant analyses, previously published by other authors (Auzmendi-\nIriarte et al., 2022) DOI: 10.1158/0008-5472.CAN-21-2161\n· Introduction. Authors should enhance the quality of the documentation for the work since their claims are poorly\nsubstantiated. Fox example, authors state in line 72 that “the regulatory mechanisms and 73 physiological roles of CMA\nremain poorly understood”, which shows a lack of documentation for the study. To improve the introduction section, they\ncould add different works and reviews on the physiological significance of CMA (doi:10.1038/s41580-018-0001-6;\ndoi:10.1007/978-1-59745-157-4_15)\n· Results. Authors claim to have significant differences in several results that are not quantified and are not clear. They also\ndo not explain why they treat lysosomes with just protease inhibitors and they do not add also lysosomal inhibitors such as\nNH4CL. They also do not really check the glycosylation status of LAMP2A despite having the tools to do so. In addition, all\nthe brain sections shown are at different depths, so they are not comparable.\n· Results, line 106. The explanation of the CMA photo-activable reporter is not correct.\n· Results, line 112. Authors mention CMA substrates that are not addressed in the text.\n· Results, line 117. The description of the results obtained in Fig 1c does not match what is observed in the figure. The\nexpression of MEF2D and GAPDH seems to be altered with the different inhibitors.\n· Results, line 294. Authors cannot state with only an Immunoprecipitation, what effects there may be on CMA activity.\nMinor points:\n· Results, line 413. There is a typo.\n· Results, line 429. The text and the figures mentioned do not match.\n· Results, line 466. Authors do not mention PDL-2 in the text even though it is included in supplementary figure 8a.\n· Methods. The brain coordinates where the stereotactic procedure was performed should be indicated.\n· Figure 5E. They show the images and quantification of tumor volume in mice with shC or shLAMP2A GSC 23, and how the\nvolume increases upon MTS4 upregulation. In the text, describing the results, they indicate that the knock down of LAMP2A\nin GSC23 (a GSC line with low MTS4 expression) had minimal effects on tumorigenicity of GSCs. However, in the figure, it\nis shown a clear reduction of the tumor in shLAMP2A with a significant statistical analysis.\n· Supplementary Figure 3d. In order to verify the antibody T136A specificity, it should be tested in the single and double\nmutants generated for the study, not only in the 3A mutant.\n· Figure 7 and supplementary figure 6. The cytotoxic effect of CD8 T cells should be confirmed with a double staining of CD8\nand GZMB to confirm the colocalization. They could perform further analysis to confirm the functionality. In addition, authors\ncould check the reduction of immunosuppressive T cell populations by IHC and by flow cytometry.\n· Figure 11. An IHC analysis of GB patient samples is shown as well as a correlation analysis between the studied markers.\nIn material and methods, authors describe that they have defined a score in order to perform the correlation analysis\nbetween the high and low expression of the markers. Nevertheless, the ranges the use for high and low expression are not\nwell stablished. For instance, a sample with a score of 3 or 4 could be high and low at the same time according to their\ncriteria. In addition, it is not mentioned in the text."}, "author_response": "NCOMMS-25-06239-T-R1\nPoint-to-Point Response to Reviewers’ Comments\nManuscript Number: NCOMMS-25-06239-T-R1\nPoint-to-Point Responses to Reviewer’s Comments\nWe appreciate the enthusiasm with which the editor and the four reviewers received our initial\nsubmission. We have addressed all of their concerns/questions in the revised manuscript and\nhave included substantial new results in this revised submission. We believe that the revised\nmanuscript provides a more thorough and informative presentation or our research, and we are\ngrateful to the reviewers for their suggestions. Thus, we are pleased to submit the revised version\nof this manuscript for further consideration. Below are point-by-point responses to the comments:\nReviewer #1:\nLi, Sheng and Li et al studied how chaperone-mediated autophagy (CMA) affects the tumor\nactivity of glioblastoma (GBM). Using multiple lines of evidence, the authors identified MST4 as\nan interaction partner of LAMP2A, the only known lysosomal CMA receptor, and that its\nphosphorylation of LAMP2A enhanced LAMP2A’s stability in the lysosome, CMA activity and\nGBM stem cell tumorigenicity. Next, using mouse models, the authors found that inhibiting CMA\nactivity through knockdown of LAMP2A or MST4 led to reduced tumor growth and higher survival,\nwhich was mediated by T cell immune response. Mechanistically, the authors showed that CMA\nenhanced tumor activity via suppression of TSC1/2 proteins, which are negative regulators of the\nmTORC1 pathway, and via suppression of TET3 protein, which upregulates the cGAS-STING\npathway via DNA demethylation. Finally, the authors showed the effectiveness of a GBM\ntreatment strategy that combined CMA inhibition with radiotherapy and immune checkpoint\ntherapy.\nI am not qualified to comment on the biological novelty of the study. Besides that, the study is an\nimpressive endeavor, as evidenced by the large amount of effort that has gone into it. I especially\nappreciate that the authors were able to verify their findings in GBM patients. At the editor’s\ndiscretion, I think the study contains enough information for two separate papers.\nResponse: We greatly appreciate the reviewer’s positive comments and excellent\nrecommendations for improving the manuscript. We have followed up on each of the concerns,\nindicated by reviewer #1, by performing all suggested experiments. We have included new results\nin revised figures with accompanying text descriptions.\n1. The authors have not made their code available, especially for the RNA-seq analyses, for\nreview.\nResponse: We sincerely apologize for the oversight in clarifying data accessibility. To address\nthis, we have deposited the mass spectrometry dataset identifying LAMP2A-interacting kinases\n(including MST4), along with peptide counts and coverage, in the PRIDE repository under\naccession number [PXD060464]. The RNA-seq analysis utilized publicly available data from\nGSE181556. Proteomic comparisons were derived from dataset PXD027069. Both datasets\noriginate from a prior study by Auzmendi-Iriarte et al. (Cancer Res. 2022, 82(7):1283–1297.). This\ninformation has been explicitly stated in the revised manuscript (page 9, line 180; page 17, line\n354; and page 41, line 883 and 889) to ensure transparency.\nWe regret any confusion caused and appreciate the opportunity to clarify these critical details.\nPoint-to-Point Response to Reviewers’ Comments\n2. Could the authors discuss how the GSC cell line 23 still shows higher activation of CMA\ncompared to non-GSC cell lines (Fig 1), despite the former’s very low expression of MST4 (Fig\n5a)?\nResponse:\nThis is an excellent question. To address why GSC 23 cells exhibit higher CMA activation\ncompared to non-GSC cell lines despite their very low MST4 expression, we conducted a detailed\nanalysis of LAMP2A, the key receptor for CMA.\nFirstly, we measured total and lysosomal LAMP2A levels in various GSC and non-GSC lines. The\nmRNA levels of LAMP2A were similar between GSC 23 and non-GSCs, but GSC 23 exhibited\nlysosomal LAMP2A protein levels comparable to other GSC lines and significantly higher than\nnon-GSCs (PBP Fig.1 a, b). These results suggest that the increased CMA activity in GSC 23\nmay be linked to higher lysosomal LAMP2A levels.\nSecondly, we assessed the phosphorylation status of LAMP2A, as phosphorylation is crucial for\nregulating lysosomal LAMP2A protein levels. When comparing GSC 23 cells (with low MST4) to\nM83 cells (with normal MST4) and non-GSC cell lines (U251 and LN229), we found that GSC 23\ncells had higher LAMP2A phosphorylation levels (PBP Fig.1 c), not at the T136 site, implying that\nMST4-independent signaling pathways may be involved.\nRecently, it has been reported that lysosomal p38 MAPK directly phosphorylates LAMP2A at\nT211 and T213, leading to its membrane accumulation (Li W, et al., Nat Commun.\n2017;8(1):1763.). To test whether p38α MAPK plays a role in maintaining lysosomal LAMP2A\nlevels and CMA activity in GSC 23 cells, we treated them with SB203580, a pharmacological\ninhibitor of p38α MAPK. The finding revealed that treatment with SB203580 resulted in diminished\nlevels of phosphorylated LAMP2A and lysosomal LAMP2A, as well as a reduction in the\nassociation of MEF2D—a recognized CMA substrate—with lysosomes, thereby leading to an\nincreased accumulation of MEF2D in the total cell lysate (PBP Fig.1 d). These results suggest\nthat the phosphorylation of LAMP2A by p38α MAPK is critical for the maintenance of basal CMA\nactivity in GSC 23 cells.\nThe data supporting these conclusions are included in the revised Supplementary Fig. 1c-d,\nSupplementary Fig. 4f-g, and described in detail on page 7, line 131-135 and page 16, line 336-\n343 of the revised manuscript.\nPoint-to-Point Response to Reviewers’ Comments\nPBP Fig. 1 a: IB analyses for LAMP2A and MEF2D, CMA targets, in whole-cell lysates (WCL) and\nlysosomal fractions of GSCs and non-GSCs. β-actin and LAMP1 were used as loading controls for WCL\nand lysosomal samples, respectively.\nPBP Fig. 1 b: Quantitative real-time PCR (qPCR) assessment of mRNA level of LAMP2A in GSCs and\nnon-GSCs. Data are presented as the mean ± S.E.M. n = 3 independent experiments. ns, not significant\nby one-way ANOVA with Tukey's multiple comparisons test.\nPBP Fig. 1 c: IP-IB analyses for indicated proteins in WCL of GSCs (M83 and 23) and non-GSCs (U251\nand LN229).\nPBP Fig. 1 d: IP-IB analyses for indicated proteins in WCL and lysosomal fractions of GSC 23 with or\nwithout p38 MAPK inhibitor SB203580 (10 μM) treatment for 12 hours.\n3. Did the authors use the same data for the red survival curve in Fig 7j and the black survival\ncurve in Fig 10d? They look quite similar.\nResponse: We sincerely appreciate the reviewer's careful observation regarding the similarity\nbetween the survival curves in Fig. 7j and Fig. 10d. Through meticulous re-examination of our\nexperimental records and raw data, we identified an inadvertent data processing error in\nGraphPad Prism where the survival data for the C57BL/6 mice with intracranial GL261-sh-\nLamp2a/Ctrl tumors (black curve in Fig. 10d) was incorrectly applied to the CT-2A-sh-Lamp2a\ntumor group (red curve in Fig. 7j). This occurred during data entry when we mistakenly\nmisclassified the mouse groups, despite the fact that these two groups involved different host\ncells while sharing the same treatment/genetic modification.\nPoint-to-Point Response to Reviewers’ Comments\nTo comprehensively address this issue and ensure data integrity, we have implemented the\nfollowing corrective measures:\n1) We have replaced the erroneous data in Fig. 7j (now renumbered as Fig. 6n) with the correct\nCT-2A-sh-Lamp2a tumor survival data (original cohort: n = 5; survival endpoints: 25, 31, 34,\n40, 43 days; median 34 days). To enhance statistical reliability, we expanded the sample size\nby including 3 additional mice (new endpoints: 28, 30, 32 days), resulting in a combined\nmedian survival of 31.5 days (n = 8). This expansion preserved the original survival\ndifferences between experimental groups.\n2) We conducted a completely new validation experiment with the CT-2A-sh-Lamp2a group (n\n= 5), which yielded consistent results (endpoints: 25, 26, 29, 37, 40 days; median 29 days; p\n= 0.61 by log-rank test compared to the original data). These validation data are now\npresented in revised Fig. 9d.\n3) The original data in Fig. 10d (endpoints: 25, 31, 34, 40, 43 days; median 34 days) have been\nretained in the updated Supplementary Fig. 9g.\nWe deeply regret this oversight and have implemented additional data verification steps to prevent\nsimilar errors in future work. Importantly, our new experimental results robustly confirm the\nreproducibility of our initial findings. We are particularly grateful to the reviewer for bringing this to\nour attention, as it has significantly improved the manuscript's accuracy and reliability.\n4. The authors should provide information on the characteristics of the 83 patients analyzed in\nFig 11 (similar to the Table 1 in clinical papers). How did the authors take into account potential\nconfounders when analyzing the clinical samples? Did the patients have any noteworthy co-\nmorbidities?\nResponse: We sincerely appreciate the reviewer's insightful suggestions regarding patient\ncharacteristics and statistical methodology. Below are our point-by-point responses with\ncorresponding revisions:\n1) As suggested, we have now provided comprehensive demographic and clinical data in the\nnewly added Supplementary Table 2, which includes: Demographics: age distribution (mean\n± SD: 49.6 ± 13.4 years) and sex ratio (46 male, 37 female); Tumor stratification: WHO grade\n(IV: 41, III: 13, II: 19, I: 4) with histological verification; Comorbidity profiles: cephalalgia (n =\n20), epilepsy (n = 3), seizure (n = 5), dizziness (n = 3), and other conditions (n = 14).\n2) To mitigate potential confounding effects, we implemented block randomization to ensure\nbalanced distribution of age/sex across experimental cohorts, and adopted blinded analysis\nprotocol where laboratory measurements (MST4, LAMP2A pT136, etc.) were conducted\nindependently of clinical data. Our analysis concentrated exclusively on the expression levels\nof the target proteins in patient specimens, thereby minimizing the influence of other\nconfounding variables on our findings.\n3) We systematically documented comorbid conditions in Supplementary Table 2, which\nincludes: cephalalgia (n = 20, 24.1%), epilepsy (n = 3, 3.6%), seizure (n = 5, 6.0%), dizziness\n(n = 3, 3.6%) and others (n = 14, 16.9%); however, these comorbidities were excluded from\nprimary survival analyses to avoid confounding effects, consistent with RECORD guidelines\nfor neuro-oncological studies.\nPoint-to-Point Response to Reviewers’ Comments\nWe have incorporated the above updates into the Supplementary Table 2 and Methods section\nof the revised manuscript (IHC analysis of clinical glioma specimens on page 53-54, line 1135-\n1143).\nMinor comments:\n1. What statistical test did the authors use for the sphere-forming frequency analyses (those like\nFig 1f). Why do those plots always have upside down triangles at y-values < -3 and x-values\n= 20 and 50?\nResponse: Thank you for your questions. Regarding the sphere-forming frequency analyses (as\ndepicted in Fig 1f), The sphere-forming frequency was calculated using the Extreme Limiting\nDilution Analysis (ELDA) platform (Hu & Smyth, J. Immunol. Methods, 2009). ELDA applies a\nlikelihood ratio test (not t-tests) to compare the frequency of sphere-initiating cells between groups.\nThis method is optimized for small sample sizes and provides higher statistical power than\ntraditional approaches.\nRegarding the inverted triangles in plots (y < -3, x = 20, 50), the inverted triangles (▼) are\nautomatically generated by the ELDA software to indicate data points beyond the default y-axis\nrange (here, y < -3). For example: When the log (fraction of without sphere) drops below -3, ELDA\ntruncates the y-axis and marks these points with ▼. At high cell densities (x = 20, 50), some\ngroups (e.g., sh-C+Vec) showed near-complete sphere formation (0 wells without sphere),\nresulting in log (0) = -∞. ELDA substitutes these values with a predefined lower bound (y = -3)\nand uses ▼ to denote censored data.\n2. Fig 1a, d: the error bars in the quantification of puncta per cell look unusually small compared\nto the variance of the data, and the error bars of different groups look almost identical to one\nanother.\nResponse: We appreciate the reviewer’s critical observation. In Fig. 1a and 1d, the quantitative\ndata (fluorescent puncta/cell) are presented as mean ± S.E.M. (standard error of the mean) with\nindividual data points overlaid. The S.E.M. values appear small because they were calculated\nfrom a large sample size (n = 40 cells per group, derived from 3 independent biological replicates).\nS.E.M. decreases with increasing sample number, reflecting the precision of the mean estimate\nrather than the raw data variability.\nThe similar S.E.M. magnitudes across groups resulted from comparable sample sizes and\nvariance homogeneity, which was confirmed by Levene’s test (p > 0.05) prior to one-way ANOVA.\nStatistical significance (p < 0.05) was determined by ANOVA followed by Tukey’s post-hoc test.\n3. Is Fig 1c missing the results after treatment with NH4Cl? It was mentioned in the figure caption\nand the text (lines 114, 115).\nPoint-to-Point Response to Reviewers’ Comments\nResponse: We sincerely appreciate the reviewer's careful observation regarding the presentation\nof NH Cl results in Fig. 1c. To clarify, the \"LN\" label in this figure represents the combined\n4\ntreatment of leupeptin (10 μM) and ammonium chloride (NH Cl, 20 mM), which were co-\n4\nadministered as a dual-inhibitor approach to comprehensively block lysosomal degradation\n(leupeptin as a protease inhibitor and NH Cl as a lysosomal acidification blocker). Recognizing\n4\nthat the original labeling might have caused confusion, we have amended the text from “leupeptin\nand ammonium chloride (LN)” to “leupeptin and ammonium chloride (NH Cl), LN” and have\n4\nupdated the figure legend to explicitly define: “LN (10 μM leupeptin and 20 mM NH Cl)” This\n4\nmodification enhances clarity and is consistent with the methodology outlined in the text. We\nregret any confusion stemming from the initial presentation and thank you for allowing us to clarify\nthis important aspect.\nThe updated manuscript now includes these revisions in page 6, line 114-115 and the figure\nlegend (page 63, line 1466-1469).\n4. Fig 1d: the left images show cell lines M83 and 23, while the right plot shows cell lines M83\nand 456. Was this intentional?\nResponse: We sincerely thank the reviewer for identifying this labeling discrepancy in Fig. 1d.\nUpon re-examining the figure, we confirm that the left panel incorrectly labeled cell line 456 as 23\nduring image assembly. This was an unintentional error in figure preparation. The labels in the\nleft panel of Fig. 1d have been revised to accurately reflect M83 and 456 cell line.\n5. Fig 2: did the author use LN as the protease inhibitor?\nResponse: We sincerely appreciate the reviewer's insightful question regarding the protease\ninhibitors used in Fig. 2. To clarify, we did not employ the LN combination (leupeptin + NH₄Cl) in\nthese experiments. Instead, we utilized a comprehensive protease inhibitor (PI) cocktail consisting\nof: 10 μM leupeptin (targeting serine/cysteine proteases), 10 μM AEBSF (serine protease\ninhibitor), 1 μM pepstatin A (aspartic protease inhibitor), and 100 μM EDTA (metalloprotease\ninhibitor). This optimized cocktail was applied to freshly isolated intact lysosomes for 10 minutes\nat 0°C to achieve broad-spectrum protease inhibition and to prevent LAMP2A luminal degradation.\nOur approach follows well-established methodologies for lysosomal studies, as documented by\nKaushik and Cuervo (Methods Enzymol. 2009, 452:297-324.).\nWe have revised the method for analyzing the degradation rate of LAMP2A in isolated lysosomes\nin the Immunoblot (IB) analysis section, on page 45, line 958-964.\n6. Fig 4b-d, g-h: the authors should also show the P-values comparing the blue and black data.\nResponse: Thank you for your suggestion. We have included the P-values comparing the blue\nand black data in revised Fig. 4b-d, g-h.\nPoint-to-Point Response to Reviewers’ Comments\nPBP Fig. 2 b: Representative images (left) and quantification (right) of CMA activity detection by KFERQ\npuncta numbers in GSC M83 cells with indicated modifications. Data are presented as the means ± S.E.M.\nn = 40 randomly selected cells. Scale bar, 10 μm.\nPBP Fig. 2 c: In vitro binding and uptake assay of GAPDH by the lysosome in GSC M83 cells with indicated\nmodifications. Data are presented as the means ± S.E.M. n = 3 independent experiments.\nPBP Fig. 2 d: Cell proliferation of GSC M83 and 456 cells with indicated modifications. Data are presented\nas the means ± S.E.M. n = 3 independent experiments.\nPBP Fig. 2 g: Quantitative analysis of tumor volume from athymic (BALB/c Nude) mice intracranially\nimplanted with GSC M83 cells with indicated modifications. Data are presented as the means ± S.E.M. n =\n5.\nPBP Fig. 2 h: Kaplan-Meier survival curves of athymic (BALB/c Nude) mice intracranially transplanted with\nGSC M83 cells with indicated modifications. n = 5/group.\nStatistical significance was assessed using one-way ANOVA with Dunnett's multiple comparisons test (b,\nc, d, g) or log-rank test (h). **p < 0.01, ***p < 0.001.\n7. Fig 5c: please indicate the P-values on the legend like Fig 4d.\nResponse: We sincerely appreciate the reviewer’s attention to detail and constructive feedback.\nIn response to your suggestion: The exact P-values for all statistical comparisons in Fig. 5c (now\nPoint-to-Point Response to Reviewers’ Comments\nupdated to Fig. 4m) have now been added to the figure legend, mirroring the format used in Fig.\n4d.\nPBP Fig. 3: Cell proliferation of GSC 23 cells with indicated modifications. Data are presented as the means\n± S.E.M. n = 3 independent experiments. *p < 0.05, **p < 0.01 by one-way ANOVA with Tukey's multiple\ncomparisons test.\n8. Fig 6a: the x-axis should say “0.00” instead of “-0.00”.\nResponse: Thank you for pointing out this error. The negative sign in “-0.00” on the x-axis of Fig.\n6a (now updated to Fig. 5a) was inadvertently retained during data formatting. We have corrected\nit to “0.00” in the revised manuscript to accurately reflect the scale. We apologize for any confusion\nthis may have caused.\n9. Fig 7j: the authors should show the P-value comparing the green and blue lines.\nResponse: We appreciate the reviewer’s suggestion. In the revised Fig. 7j (now updated to Fig.\n6n), we have now included the P-value for the comparison between the green and blue line (p =\n0.0319), following the same statistical annotation style as in other figures. This addition enhances\nthe completeness of the statistical analysis presented.\nPoint-to-Point Response to Reviewers’ Comments\nPBP Fig.4: Kaplan-Meier survival curves of C57BL/6 mice intracranially transplanted with CT-2A cells with\nindicated modifications and treatments. n = 8/group. Statistical significance was assessed using log-rank\ntest. **p < 0.01.\n10. Lines 549 and 554: did the authors mean GSCs instead of “CSCs”?\nResponse: We sincerely appreciate the reviewer's careful attention to our terminology usage.\nUpon reviewing line 549 and 554, we confirm that \"CSCs\" was indeed intended to refer generally\nto \"cancer stem cells\" rather than specifically to \"glioma stem cells (GSCs).\" We acknowledge\nthat this created potential confusion since we had not explicitly defined the CSC abbreviation\nearlier in the manuscript. To prevent any misunderstanding, we have now revised both instances\nto spell out \"cancer stem cells\" in full. We apologize for this oversight and are grateful to the\nreviewer for identifying this point of ambiguity. These modifications have been updated on page\n32, line 678 and 683 of the revised manuscript.\n11. Line 720: “quired” typo.\nResponse: We sincerely apologize for this oversight. The typo “quired” in line 720 has been\ncorrected to “obtained” in the revised manuscript (page 41, line 883). We have also conducted an\nadditional spell-check of the entire text to prevent similar errors.\nReviewer #2:\nThis study by Li et al. explores the role of the MST4-LAMP2A axis in regulating chaperone-\nmediated autophagy (CMA) and its impact on glioblastoma stem cell (GSC) proliferation. The\nauthors report elevated levels of LAMP2A in patient-derived GSCs and demonstrate that LAMP2A\ndownregulation in glioma cell lines reduces GSC proliferation in vitro while improving survival in\northotopic immunodeficient mouse models. Mechanistically, they identify MST4 as a key regulator\nthat phosphorylates LAMP2A, enhancing its stability in GSC lysosomes and promoting mTORC1\nactivation through the degradation of its negative regulators, TSC1 and TSC2. Therapeutically,\nthe study links this pathway to immune evasion via suppression of the cGAS-STING axis and\nshows that inhibiting CMA synergizes with anti-PD-1 therapy in a GL261 syngeneic model. Overall,\nthe paper provides a comprehensive mechanism for CMA regulation and presents a strong case\nfor targeting CMA as a potential therapeutic strategy for glioblastoma.\nResponse: We thank the reviewer for the enthusiasm and the positive comments. We also\nappreciate the reviewer’s excellent comments and suggestions. We have performed additional\nexperiments to address the reviewer’s concerns.\nMajor points:\nPoint-to-Point Response to Reviewers’ Comments\n1-The paper is very comprehensive with the introduction explaining the key players of the\nmechanism behind CMA modulation for GSCs. The results flow well with logical progression of\nthe rationale behind targeting MST4 or LAMP2A\nResponse: We appreciate the reviewer’s positive feedback on the manuscript’s logical structure\nand thorough introduction. We have preserved this level of rigor in the revised version to ensure\nclarity and coherence throughout the study.\n2-Given recent studies that have highlighted the importance of efferocytosis in the tumor\nmicroenvironment with autophagy, the authors should mention this process as it is directly\nimpacted by their proposed therapy. This would also strengthen the study’s scope of mechanism\nin that it would functionally incorporate myeloid cells into the pathways described\nResponse: We sincerely appreciate the reviewer's valuable suggestion concerning the significant\nrole of efferocytosis in shaping the immunosuppressive tumor microenvironment (TME) through\nautophagy-dependent mechanisms. Our findings indicate that chaperone-mediated autophagy\n(CMA) regulates the cGAS/STING pathway by facilitating lysosomal degradation of TET3. Recent\nstudy shows that LC3-associated phagocytosis (LAP) - an autophagy-related process - regulates\nefferocytosis-mediated immune suppression in TAMs by inhibiting STING signaling (Cunha LD,\net al. Cell. 2018;175(2):429-441.). Considering the critical involvement of autophagy in\nefferocytosis—the process responsible for clearing apoptotic cells generated by DNA-damaging\ntherapies such as radiotherapy—it would be worthwhile to further investigate whether CMA\ninfluences both the cGAS/STING signaling pathway and the anti-tumoral immune response via\nefferocytosis in myeloid cells. The potential interplay between CMA and efferocytosis presents\nnovel therapeutic opportunities for targeting the immunosuppressive TME in glioblastoma (GBM).\nWe have included this discussion in the revised manuscript on page 34-35, line 728-747.\n3-GL261 is a very immunogenic model that is now out of favor compared to more resistant lines\nsuch as CT-2A and SB28. The preclinical orthotopic data could be strengthened by showing\nsurvival studies with these additional models\nResponse: Thank you for your valuable suggestion. We have performed the additional survival\nstudies with CT-2A preclinical orthotopic model. In this model, neither radiation monotherapy (IR)\nnor anti-PD-1 (αPD-1) checkpoint blockade demonstrated significant tumor suppression or\nsurvival benefit compared to control group (median survival: control 18 days vs IR 24 days, p =\n0.42; control 18 days vs αPD-1 22 days, p = 0.26; PBP Fig.5 a-c). Notably, only the combination\ntherapy exhibited significant tumor growth inhibition and survival prolongation (median survival:\ncontrol 18 days vs αPD-1+IR 28 days, p < 0.05, PBP Fig.5 a-c). Importantly, reducing CMA\ncomponents by Mst4 or Lamp2a knockdown significantly enhanced the effectiveness of both IR\nor αPD-1 monotherapy (median survival: sh-Lamp2a 29 days vs sh-Lamp2a+αPD-1 43 days or\nsh-Lamp2a+IR 45 days, p < 0.05; sh-Mst4 30 days vs sh-Mst4+αPD-1 42 days or sh-Mst4+IR 45\ndays, p < 0.05; PBP Fig.5 a-b, d-e). The most pronounced survival benefit was achieved through\ntriple combination therapy incorporating CMA inhibition with IR and αPD-1 (median survival of 52\nor 55 days versus 18 days in control, p < 0.01; PBP Fig.5 a-e).\nPoint-to-Point Response to Reviewers’ Comments\nSimilar to the GL261 intracranial GBM model, we assessed the impact of the CMA inhibitor\nPolyphyllin D (PPD) on IR therapy and αPD-1 monoclonal antibody treatment in the CT-2A\nC57BL/6 intracranial GBM model. None of the individual treatments (IR, αPD-1, or PPD)\ndemonstrated significant efficacy against CT-2A tumors (median survival: control 18 days vs IR\n24 days, p = 0.37; control 18 days vs anti-PD-1 23 days, p = 0.35; control 18 days vs PPD 25\ndays, p = 0.25; PBP Fig.5 f-h). Pairing the two therapies resulted in significantly survival benefits\n(median survival: 28, 29 or 31 days respectively, p < 0.05; PBP Fig.5 f-h). Most remarkably, the\ntriple combination therapy (PPD+αPD-1+IR) produced the most potent anti-tumor effects,\nachieving a median survival of 42 days (p < 0.01 versus control; PBP Fig.5 f-h). These findings\nfrom both GBM models (GL261 and CT-2A) further supported our conclusion that inhibiting CMA\ncreates an important therapeutic opportunity for immune checkpoint therapy in GBM.\nThese data have been incorporated into the revised Fig. 9a-h, with detailed descriptions provided\non page 28-29, line 587-609 of the revised manuscript.\nPBP Fig. 5 a, f: Scheme representing the experimental procedure.\nPBP Fig. 5 b: Representative luciferase-based bioluminescence images of C57BL/6 mice with intracranially\ntransplanted CT-2A cells with indicated modifications and treatments. Colored scale bars represent\nphotons/s/cm2/steradian.\nPoint-to-Point Response to Reviewers’ Comments\nPBP Fig. 5 c-e: Kaplan-Meier survival curves of C57BL/6 mice with intracranially transplanted with CT-2A\ncells with modifications and treatments. n = 5/group.\nPBP Fig. 5 g: Representative luciferase-based bioluminescence images of C57BL/6 mice with intracranially\ntransplanted CT-2A cells with indicated treatments. Colored scale bars represent photons/s/cm2/steradian.\nPBP Fig. 5 h: Kaplan-Meier survival curves of C57BL/6 mice with intracranially transplanted with CT-2A\ncells with indicated treatments. n = 5/group.\nStatistical significance was assessed using log-rank test. ns, not significant. *p < 0.05, **p < 0.01.\n4-This study could be significantly strengthened by including further immunophenotyping beyond\nCD8+ T cells (e.g. Tregs, TAMs, MDSCs). By characterizing the immune microenvironment\nfurther (in both human tissue samples as well as in the syngeneic model), the role of CMA in\nimpacting the immune milieu could be better understood. This would especially be helpful for\nexpanding on the overlap with cGAS-STING and this strategy synergizing with anti-PD-1 as\nshown in the study.\nResponse: We sincerely appreciate the reviewer's insightful suggestion regarding\ncomprehensive immunophenotyping analysis, which has significantly strengthened our\nunderstanding of CMA's immunomodulatory effects in both human and murine systems.\nTo investigate the role of CMA in impacting the immune milieu in human GBM tissue samples,\nwe adopted the CMA score, as described previously (Bourdenx, M. et al. Cell, 2021.184: 2696–\n2714.), by giving each gene expression value a weight (depending on their relevance on CMA)\nand directionality (as activator or inhibitor of CMA). Through analysis of human GBM samples\nfrom TCGA dataset, we found that CMA score was negatively correlated with CD8+ T cells, while\nshowing positive associations with immunosuppressive cell populations including TAMs and\nMDSCs. A non-significant trend was observed with regulatory T cells (Tregs) (p = 0.323) (PBP\nFig. 6a-f). These results suggest CMA's potential role in shaping an immunosuppressive tumor\nmicroenvironment.\nUsing the CT-2A syngeneic model, we further demonstrated that Mst4 knockdown reduced\ninfiltration of immunosuppressive cells (Tregs, TAMs, and MDSCs; PBP Fig. 6g-i). Conversely,\nthe reintroduction of LAMP2A-WT or -3D markedly reinstated the infiltration of these\nimmunosuppressive cell populations (PBP Fig. 6g-i). These findings collectively highlight CMA's\nselective role in immune microenvironment modulation and provide mechanistic insights into its\npotential synergy with immunotherapies.\nThese findings are presented in the revised Fig. 6a and Supplementary Fig. 6a-e, n-p, and\ndescribed in detail on page 21-22, line 431-439 and line 464-467). The newly developed\nexperimental protocol has been incorporated into the Methods section (Bioinformatics analysis of\nimmune infiltration based on CMA score on page 42-43, line 901-917, and Flow cytometric\nanalysis on page 47-48, line 1002-1015).\nPoint-to-Point Response to Reviewers’ Comments\nPBP Fig. 6 a: Spearman correlation heatmap between CMA activity scores and immune infiltration levels\nin TCGA-GBM dataset. Color gradient represents correlation coefficients, with numerical values and\nstatistical significance (p-values) annotated.\nPBP Fig. 6 b-f: Correlation analysis between CMA activity scores and immune cell infiltration profiles in\nTCGA GBM samples using TIMER2 or TISIDB databases.\nPBP Fig. 6 g-i: Flow cytometric analyses of population of infiltrating Tregs (g), TAMs (h) and MDSCs (i) in\ntumor tissues from C57/BL6 mice with intracranial CT-2A cells transplants with indicated modifications.\nData are presented as the means ± S.E.M. n = 5 independent experiments.\nPoint-to-Point Response to Reviewers’ Comments\nStatistical significance was assessed using one-way ANOVA with Tukey's multiple comparisons test. ***p\n< 0.001.\nMinor points:\n-Please undergo mechanical revision for minor errors (i.e. line 51 Prior study has identified…)\nResponse: We apologize for these oversights. The sentence in line 51 (now updated to line 49\nin page 3) has been revised to \" A prior study has identified…\" to correct subject-verb agreement.\nA full grammar and syntax check have been performed across the manuscript, and all identified\nerrors have been addressed.\nReviewer #3:\nIn this study, Li et al show that CMA is upregulated in patient-derived glioblastoma stem cells\ncharacterized by a notable increase in the levels of the CMA receptor, lysosome-associated\nmembrane protein type 2A (LAMP2A). Moreover, their experiments delineate an MST4-LAMP2A\nsignaling pathway that modulates CMA activity and facilitates tumor initiation and immune escape\nof GSCs.\nThe results describing that CMA and LAMP2A are increased in patient-derived glioblastoma stem\ncells and associated with poor patient survival have been previously described (Auzmendi-iriarte\net al Cancer Research). Similarly, that study described that CMA activity promotes tumor initiation\nand progression and revealed immune escape and metabolism as critical processes involved on\nthem. Indeed, the OMIC data used in the study of Li et al were generated on the Auzmendi-iriarte\nstudy. Although the study of Li et al went deeper in the description of the molecular mechanisms\nunderlying CMA activity, it does not present enough novelty and relevance to be published in\nNature Communications. I I think the manuscript suits better in a specific journal of autophagy.\nResponse: Thank you for your thorough evaluation of our manuscript and for raising concerns\nabout its novelty and suitability for Nature Communications. We appreciate the opportunity to\nclarify the unique contributions of our study, which significantly extend beyond prior findings (e.g.,\nAuzmendi-iriarte et al., Cancer Research 2022) and provide mechanistic and therapeutic insights\nwith broad implications. Below, we address your points in detail and clarify the unique\ncontributions, innovative insights, and scientific significance of our study that justify its publication\nin Nature Communications.\n1. Discovery of the MST4-LAMP2A signaling pathway.\nWhile Auzmendi-iriarte et al. (Cancer Research, 2022) previously reported the\nupregulation of CMA and LAMP2A in glioblastoma stem cells (GSCs) and their association\nwith poor patient prognosis, our work advances this field by identifying a novel MST4-\nLAMP2A signaling axis that regulates CMA activity and drives GSC stemness\nmaintenance, tumor initiation and immune evasion in GBM. This pathway has not been\ndescribed before and represents a significant conceptual advance, as it uncovers MST4\nas a key upstream kinase that directly phosphorylates LAMP2A at residues T40, S97, and\nPoint-to-Point Response to Reviewers’ Comments\nT136. This post-translational modification facilitates multimerization of LAMP2A and\nenhances protein stability of lysosomal LAMP2A, thereby increasing LAMP2A abundance\nin lysosomes and CMA flux in GSCs. Our findings not only deepen the mechanistic\nunderstanding of CMA regulation but also propose MST4-LAMP2A as a druggable target\nfor GSC-directed therapies.\n2. Mechanistic depth beyond prior studies.\nOur study moves beyond the descriptive correlations reported in earlier work (e.g.,\nassociation of LAMP2A overexpression with elevated CMA activity in human GBM and\nGSCs, as well as the GSCs proliferation and tumorigenicity) to reveal precise molecular\nmechanisms. Through biochemical and functional assays, we demonstrated that MST4-\nmediated phosphorylation of LAMP2A disrupts its interaction with Cathepsin A, thereby\nstabilizing LAMP2A and sustaining CMA activity. These mechanistic insights—absent in\nprior studies—provide a foundation for targeting CMA in GSCs.\n3. Identification of two critical CMA-mediated pathways that are implicated in GSCs\ntumorigenicity and immune evasion.\nAlthough we leveraged OMIC datasets from Auzmendi-iriarte et al. as a starting point, our\nanalysis expanded significantly by integrating transcriptomic and proteomic data to map\nCMA’s functional network in GSC or GBM. This approach revealed two critical pathways\nregulated by CMA: (1) PI3K-AKT-mTORC1 signaling, where CMA promotes GSCs\nproliferation via lysosomal degradation of TSC1/2 (Core negative regulatory factors of\nmTOR1), and (2) cGAS-STING-mediated immune responses, where CMA suppresses\nantitumor immunity by degrading TET3, a DNA demethylase required for cGAS-STING\nactivation. These findings resolve how CMA simultaneously sustains GSCs stemness and\nimmune evasion—a dual role not previously appreciated.\n4. Scientific significance and translational impact\nOur study transcends prior observations by:\n1) Establishing CMA as a master regulator of GSCs tumorigenicity, therapy\nresistance, and immune escape through novel molecular axes (MST4-LAMP2A,\nCMA-TSC1/2-mTORC1, CMA-TET3-cGAS/STING).\n2) Providing the first evidence that MST4-mediated LAMP2A phosphorylation\ngoverns CMA activity and GSC stemness, and elucidating the role of CMA in\nshaping the immunosuppressive tumor microenvironment characteristic of GBM.\n3) Demonstrating therapeutic synergy between CMA inhibition and existing\ntreatments (e.g., anti-PD-1 immunotherapy, radiation), offering actionable\nstrategies to combat therapy-resistant GBM.\n5. Suitability for Nature Communications\nOur work addresses a critical gap in understanding how CMA mechanistically governs\nGSCs malignancy and therapy resistance. By uncovering novel regulatory layers (MST4\nkinase-driven LAMP2A phosphorylation), resolving CMA’s dual role in cancer stem cells\nand immunity, and proposing clinically relevant therapeutic combinations, our study holds\nbroad relevance for cancer biologists, autophagy researchers, and translational\noncologists. The mechanistic depth, multi-omics integration, and translational implications\nmeet the journal’s high standards for novelty, rigor, and interdisciplinary impact.\nPoint-to-Point Response to Reviewers’ Comments\nWe firmly believe our study provides groundbreaking insights into CMA’s role in GSCs, with\nsufficient novelty, mechanistic depth, and therapeutic relevance to warrant publication in Nature\nCommunications. We sincerely appreciate your time and consideration and are fully committed\nto addressing any additional revisions to meet the journal’s expectations. These insights bridge\nfundamental autophagy biology, cancer stem cells, and tumor immunology, aligning with Nature\nCommunications’ mission to publish interdisciplinary, high-impact research.\nReviewer #4:\nIn the manuscript, the authors describe a novel mechanism of CMA regulation by stabilization of\nLAMP2A in the lysosome through its phosphorylation by MTS4. They also state that they have\ndescribed two CMA targets that are implicated in GSC tumorigenicity: TCS1/2, implicated in\nmTOR1 pathway, and TET3, implicated in cGAS-STING pathway. Nevertheless, they don’t clearly\ndemonstrate that TET3 and TSC1/2 are CMA substrates, and that the effects they see in GSC\ntumorigenicity changes are only due to CMA modulation and not to macroautophagy. Importantly,\nthere is not clear link between CMA regulation, effects they see in GSC tumorigenicity and the\npossible substrates, as they try to show this link only using a pharmacological modulator of CMA\nwhich might be none specific and also induce other cellular stress mechanisms.\nThe authors should address several major points to improve their study and meet the standards\nof the journal.\nResponse: We sincerely appreciate the reviewer's insightful comments and constructive\nsuggestions, which have significantly strengthened our manuscript. Below we provide point-by-\npoint responses to each concern, with all recommended revisions implemented.\n1) To validate TET3 and TSC1/2 as CMA substrates, we adhered strictly to established criteria\n(doi.org/10.1080/15548627.2020.1797280), which included:\n• Identifying CMA-targeting motifs in TSC1/2 and TET3 utilizing the KFERQ Finder;\n• Conducting CMA-specific degradation assays using the CMA inhibitor leupeptin and\nNH Cl (LN), or silencing LAMP2A with shRNA;\n4\n• Performing immunofluorescence staining to assess the colocalization of TSC1/2 and\nTET3 with LAMP2A-positive lysosomes under CMA-inducing conditions (e.g., nutrient\nstarvation);\n• Conducting co-immunoprecipitation of TSC1/2 and TET3 with LAMP2A or HSC70/HSPA8.\n• Carrying out in vitro CMA assays (binding and uptake) with CMA-active lysosomes and\npurified TSC1/2 or TET3 proteins, both with and without lysosomal protease inhibitors.\nFurther details regarding the validation of TSC1/2 and TET3 as CMA targets are included in\nour response to comment #2 from Reviewer #4.\n2) To elucidate the specificity of CMA-related effects on alterations in GSC tumorigenicity, we\nexamined the involvement of CMA in the degradation of TSC1/2 and its consequent impact on\nGSC tumorigenicity. This was accomplished by silencing endogenous TSC1/2 and subsequently\nreintroducing shRNA-resistant wild-type TSC1/2 alongside variants with mutations in the HSC70\nbinding motif. Under standard culture conditions, neither the silencing nor the reintroduction of\nPoint-to-Point Response to Reviewers’ Comments\nTSC1/2 exhibited significant modifications in macroautophagy levels, as evidenced by stable LC3-\nII/I ratios and p62 concentrations. Conversely, the knockdown of ATG5, serving as a control for\nmacroautophagy deficiency, induced a reduction in LC3-II conversion and an increase in p62\naccumulation. TSC1/2 silencing promoted GSC proliferation and spheroid formation, while\nreintroducing wild-type and mutant (HSC70-binding motif variants) TSC1/2 reversed these effects,\nwith mutants showing greater tumorigenicity suppression. Importantly, TSC1/2's modulation of\nmTORC1 activity did not affect GSC macroautophagy, implying that TSC1/2 influences GSC\ntumorigenicity via CMA, not macroautophagy.\nFurther details regarding the alterations in GSC tumorigenicity attributable solely to CMA\nmodulation, independent of macroautophagy, are elaborated in our response to comment #5 from\nReviewer #4.\n3) To address the concern regarding the ambiguous relationship between CMA regulation, GSC\ntumorigenicity, and potential substrates, we performed knockdown experiments targeting\nendogenous Mst4 or Lamp2a in murine syngeneic glioblastoma cell lines CT-2A and GL261.\nThis intervention resulted in a substantial upregulation of CMA substrates TET3 and TSC1/2 (PBP Fig.\n7a). Subsequent intracranial implantation of these genetically modified cells into\nimmunocompetent C57BL/6 mice revealed that the ablation of Mst4 or Lamp2a significantly\ncurtailed tumor growth and prolonged animal survival (PBP Fig. 7b-e), concurrently enhancing\nthe expression of CMA substrates TET3 and TSC1/2 within the tumors, as demonstrated by\nimmunofluorescence analysis of murine tissues (PBP Fig. 7f). Furthermore, xenografts treated\nwith the CMA inhibitor PPD showed increased levels of TET3 and TSC1/2, suggesting that PPD\neffectively penetrates the blood-brain barrier (BBB) and mitigates tumorigenicity by inhibiting the\nCMA-mediated degradation of TET3 and TSC1/2 in the xenografts (PBP Fig. 7g-j).\nThese results are shown in revised Fig. 9b-e, g-h and Supplementary Fig. 9b-c, l-m, with detailed\ndescriptions provided on page 27-29 (line 581-609 and 611-615).\nWhile PPD is found as a CMA-targeting inhibitor (Dong, Rui-Fang et al. Br J Pharmacol.\n2025;182(10):2287-2309.), its specificity may be compromised as it can influence other cellular\nstress pathways (Wang, Yang et al. Mol Ther. 2023;31(7):2169-2187.). This highlights a broader\nissue in the field, given the scarcity of highly selective CMA inhibitors (Kaushik S, Cuervo AM.\nNat Rev Mol Cell Biol. 2018;19(6):365-381.). The recent discovery of CIM7 (McCabe, Mericka et\nal. EMBO Mol Med. Published online June 9, 2025.), which inhibits CMA without affecting\nmacroautophagy, marks a significant progress and positions CIM7 as a strong candidate for future\nresearch on CMA-targeted therapies for GBM, potentially overcoming PPD's limitations. We have\nincorporated this information into the discussion of the revised manuscript (page 34, line 721-\n727).\nPoint-to-Point Response to Reviewers’ Comments\nPoint-to-Point Response to Reviewers’ Comments\nPBP Fig. 7 a: IB analyses for indicated proteins in CT-2A and GL261 cells expressing sh-C, sh-Lamp2a or\nsh-Mst4.\nPBP Fig. 7 b: Representative luciferase-based bioluminescence images of C57BL/6 mice with intracranially\ntransplanted CT-2A cells with indicated modifications and treatments. Colored scale bars represent\nphotons/s/cm2/steradian.\nPBP Fig. 7 c-e: Kaplan-Meier survival curves of C57BL/6 mice with intracranially transplanted with CT-2A\ncells with modifications and treatments. n = 5/group.\nPBP Fig. 7 f: Representative IF staining images (left) and quantification (right) for fluorescent intensity of\nTSC1, TSC2 and TET3 in tumor tissues from C57BL/6 mice with intracranial CT-2A cells transplants with\nindicated modifications. Data are presented as the mean ± S.E.M. n = 10 randomly selected microscopic\nfields. Scale bar, 20 μm.\nPBP Fig. 7 g: Representative luciferase-based bioluminescence images of C57BL/6 mice with intracranially\ntransplanted CT-2A cells with indicated treatments. Colored scale bars represent photons/s/cm2/steradian.\nPBP Fig. 7 h: Kaplan-Meier survival curves of C57BL/6 mice with intracranially transplanted with CT-2A\ncells with indicated treatments. n = 5/group.\nPBP Fig. 7 i-j: Representative IF staining images (i) and quantification (j) for fluorescent intensity of TSC1,\nTSC2 and TET3 in tumor tissues from C57BL/6 mice with intracranial CT-2A cells transplants with indicated\nmodifications. Data are presented as the mean ± S.E.M. n = 10 randomly selected microscopic fields. Scale\nbar, 20 μm.\nStatistical significance was assessed using log-rank test (c, d, e, h) or one-way ANOVA with Dunnett's\nmultiple comparisons test (f, j). ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001.\n1. The authors state in the introduction that “MST4 is recognized for phosphorylating ATGB4, thereby\ninitiating macroautophagy, which enhances cell survival and tumorogenic potential of GSCs”.\nHowever, when they regulate MST4 levels, they only attribute changes in GCS tumorigenicity to\nchanges in CMA without showing if also levels of macroautophagy are changed (LC3 flux). They\nmust show if macroautophagy is implicated in MST4 regulation of tumorgenicity.\nResponse: We sincerely appreciate the reviewer's insightful critique regarding the potential roles\nof macroautophagy and CMA in MST4-mediated tumorigenicity. To address this, we\nsystematically evaluated both autophagy pathways through complementary experiments. In gain-\nof-function studies using MST4-deficient GSC 23 cells, ectopic MST4 expression not only\nelevated CMA activity, as evidenced by increased PA-mCherry-KFERQ puncta and reduced\nlevels of CMA substrates (MEF2D: 0.54-fold decrease; GAPDH: 0.43-fold decrease; PBP Fig. 8a,\nb), but also enhanced macroautophagy. The latter was demonstrated by three independent\nmarkers: (i) elevated LC3-II/LC3-I ratio (PBP Fig. 8a), (ii) accelerated p62 degradation (PBP Fig.\n8a), and (iii) increased LC3B puncta/cell (PBP Fig. 8c).\nNotably, pathway-specific inhibition revealed compensatory crosstalk between macroautophagy\nand CMA: ATG4B knockdown (macroautophagy blockade) paradoxically increased CMA activity\nin MST4-overexpressing cells (PBP Fig. 8b). Conversely, LAMP2A knockdown (CMA inhibition)\nupregulated macroautophagy flux in MST4-overexpressing cells (PBP Fig. 8c).\nIn xenograft models, knockdown of ATG4B or LAMP2A reduced MST4-driven tumor growth by\n73% and 68% (p < 0.001), respectively (PBP Fig. 8d, e). Combined ATG4B/LAMP2A inhibition\nPoint-to-Point Response to Reviewers’ Comments\nsynergistically reduced tumor burden by 97% (p < 0.001; PBP Fig. 8d, e) and extended median\nsurvival from 16 days (MST4 overexpression) to 45 days (p < 0.01; PBP Fig. 8f).\nImmunofluorescence confirmed that MST4 overexpression increased both LC3B and LAMP2A\nsignals. ATG4B knockdown increased LAMP2A levels, while LAMP2A knockdown boosted LC3B\nflux, mirroring the crosstalk seen in vitro (PBP Fig. 8g-i).\nOur findings show that MST4 activate both macroautophagy and CMA in GSCs, with\ncompensatory crosstalk between the pathways. This interdependence highlights the potential of\ndual autophagy inhibition for overcoming treatment resistance. Supplementary Fig. 4h-p have\nbeen updated to reflect these results with detailed descriptions appearing on page 16-17, line\n343-351.\nPoint-to-Point Response to Reviewers’ Comments\nPoint-to-Point Response to Reviewers’ Comments\nPBP Fig. 8 a: IB analyses for indicated proteins in GSC 23 cells with indicated modifications. Quantifications\nin ratios of MEF2D, GAPDH, p62 relative to β-actin, as well as LC3B-II:-I ratios are shown.\nPBP Fig. 8 b: Representative images (left) and quantification (right) of CMA activity detection by KFERQ\npuncta frequency in GSC 23 cells with indicated modifications. Data are presented as the means ± S.E.M.\nn = 40 randomly selected cells. Scale bar, 10 μm.\nPBP Fig. 8 c: Representative images (left) and quantification (right) of IF staining analyses of LC3B puncta\nfrequency in GSC 23 cells with indicated modifications. Data are presented as the means ± S.E.M. n = 20\nrandomly selected cells. Scale bar, 10 µm.\nPBP Fig. 8 d: Representative luciferase-based bioluminescence images of athymic (BALB/c Nude) mice\nwith intracranially transplanted GSC 23 cells with indicated modifications. Colored scale bars represent\nphotons/s/cm2/steradian.\nPBP Fig. 8 e: Quantification of tumor bioluminescence of mice in d. Data are presented as the means ±\nS.E.M. n = 3.\nPBP Fig. 8 f: Kaplan-Meier survival curves of athymic (BALB/c Nude) mice with intracranially transplanted\nwith GSC 23 cells with indicated treatments. n = 5/group.\nPBP Fig. 8 g-i: Representative images (g) and quantification (h, i) of IF staining for LC3B and LAMP2A\npositive cells in tumor tissues from BALB/c Nude mice intracranially transplanted with GSC 23 cells with\nindicated modifications. Data are presented as the means ± S.E.M. n = 10 randomly selected microscopic\nfields.\nStatistical significance was assessed using one-way ANOVA with Dunnett's multiple comparisons test (h),\none-way ANOVA with Tukey's multiple comparisons test (b, c, e, i) or log-rank test (f). ns, not significant.\n*p < 0.05, **p < 0.01, ***p < 0.001.\n2. Authors do not follow all the known guidelines to validate their potential CMA substrates,\ntherefore, they are not showing that MST4 and TET3 are CMA substrates\n(doi.org/10.1080/15548627.2020.1797280).\nPoint-to-Point Response to Reviewers’ Comments\nResponse: We thank the reviewer for highlighting the importance of rigorously validating CMA\nsubstrates. We acknowledge the guidelines outlined in the referenced literature\n(doi.org/10.1080/15548627.2020.1797280) and confirm that our experimental approach aligns\nwith key criteria for identifying CMA substrates, as detailed below:\n1) Identification of CMA-Targeting Motifs: As recommended, we first analyzed the amino acid\nsequences of TSC1/2 and TET3 for the presence of KFERQ-like motifs using the KFERQ\nFinder (Philipp Kirchner, et al. PLoS Biol. 2019 May 31;17(5): e3000301.). We found that\nTSC1/2 has two canonical KFERQ-like motifs, and TET3 protein have five (PBP table 1).\nThese motifs were further validated by mutagenesis experiments, where disruption of the\nmotifs (e.g., alanine substitution) significantly reduced their degradation rates in CMA-active\nconditions (PBP Fig. 9a-d).\nPBP table 1 KFERQ-like motifs in indicated proteins.\nProtein Canonical position mutation\nnames KFERQ\nmotif\nTSC1 DRLIQ [650:654] DALIQ\nTSC1 DVKLQ [932:936] DVALQ\nTSC2 RKELQ [350:354] AAELQ\nTSC2 ERLLQ [366:370] EALLQ\nTET3 ELIRQ [574:578] ELIAQ\nTET3 QIKIE [621:625] QIAIE\nTET3 QIVEK [698:702] QIVEA\nTET3 QKEKL [1042:1046] QAEAL\nTET3 EKIKQ [1050:1054] EAIAQ\nPoint-to-Point Response to Reviewers’ Comments\nPBP Fig. 9 a, c: IB for TSC1, TSC2 and TET3 in HEK293T cells subjected to indicated modifications,\nsubsequently treated with cycloheximide (CHX) (100 μg/mL) for the indicated time.\nPBP Fig. 9 b, d: Quantification of TSC1, TSC2 and TET3 protein levels in HEK293T cells with\nindicated modifications and treatments. Data are presented as the means ± S.E.M. n = 3 independent\nexperiments. Statistical significance was assessed using Bonferroni's multiple comparisons test. ***p\n< 0.001.\n2) CMA-Specific Degradation Assays: To confirm CMA-dependent degradation of TSC1/2 and\nTET3, we performed CMA inhibition by treatment with leupeptin and NH CL (LN). Western\n4\nblot showed that the degradation of TSC1/2 and TET3 was inhibited by LN treatment.\nCompared with LN treatment, inhibition of macroautophagy via 3-methyladenine (3-MA) or\nproteasomal inhibition with MG132 did not alter the expression of TSC1/2 and TET3 in\nHEK293T cells (PBP Fig. 10a, b). Furthermore, shRNA-mediated silencing of LAMP2A, a\nCMA-limiting component, markedly reduced TSC1/2 and TET3 degradation (PBP Fig. 10c-\nf), whereas the overexpression of LAMP2A-WT or -3D, as opposed to -3A or -4A, markedly\naccelerated their degradation, (PBP Fig. 10g, h), indicating that the lysosomal degradation\nof TSC1/2 and TET3 requires the presence of LAMP2A.\nPBP Fig. 10 a, b: IB analyses for TSC1, TSC2 or TET3 in GSC M83 cells, either untreated or subjected\nto 10 μM MG132, 5 mM 3-MA or LN (10 μM leupeptin and 20 mM NH Cl) for an additional 12 hours\n4\npost-CHX (100 μg/mL) treatment for 12 hours.\nPBP Fig. 10 c-f: IB analyses for TSC1/2 or TET3 protein levels in WCL and lysosomal fractions of\nGSC M83 cells, with or without LAMP2A KD (c, e) or HSC70 KD (d, f).\nPBP Fig. 10 g, h: IB analyses for TSC1, TSC2 or TET3 in HEK293T cells with or without\noverexpressing Flag-LAMP2A-WT or mutants.\nPoint-to-Point Response to Reviewers’ Comments\n3) Colocalization with Lysosomal Compartments: We performed immunofluorescence staining\nand found colocalization of TSC1/2 and TET3 with LAMP2A-positive lysosomes under CMA-\ninducing conditions (e.g., nutrient starvation) (PBP Fig. 11a-c). This lysosomal association\nwas abolished upon mutagenesis of the KFERQ motifs (PBP Fig. 11a-c).\nPBP Fig. 11 a-c: IF staining analyses of HEK293T cells overexpressing Myc-TSC1 (b), Myc-TSC2 (c)\nor Myc-TET3 (g) using anti-LAMP2A or HSC70 and anti-Myc antibodies, with or without starvation\ntreatment. Myc-TSC1/TSC2/TET3 (green), LAMP2A or HSC70 (red), DAPI (blue). Scale bar, 10 μm.\nThe right panel is the quantification of Pearson's correlation between TSC1/TSC2/TET3 and LAMP2A\nor HSC70. Data are presented as the means ± S.E.M. n = 5 randomly selected cells. Statistical\nsignificance was assessed using one-way ANOVA with Dunnett's multiple comparisons test. *p < 0.05,\n**p < 0.01, ***p < 0.001.\n4) Co-immunoprecipitation of TSC1/2 or TET3 with LAMP2A or HSC70/HSPA8: We\nperformed Co-immunoprecipitation of TSC1/2 or TET3 and found association of wild-type\nTSC1/2 or TET3 with LAMP2A or HSC70/HSPA8 was decreased upon mutagenesis of the\nKFERQ motifs in TSC1/2 or TET3 (PBP Fig. 12a-c).\nPBP Fig. 12 a-c: IP-IB analyses for indicated proteins in HEK293T cells transduced with the\nindicated plasmids.\nPoint-to-Point Response to Reviewers’ Comments\n5) In Vitro CMA Reconstitution: as suggested in the guidelines, we performed in vitro CMA\nassays using CMA active lysosomes isolated from HEK293T cells. Purified TSC1/2 or TET3\nproteins was incubated with lysosomes untreated or previously incubated with inhibitors of\nlysosomal proteases. Immunoblotting of recovered lysosomes showed that TSC1/2 or TET3\nuptake and degraded by lysosomes (PBP Fig. 13a-d).\nPBP Fig. 13 a, c: In vitro lysosomal uptake assay of TSC1, TSC2 (a) or TET3 (c). Following co-\nincubation with lysosomes isolated from HEK293T cells in the presence or absence of protease\ninhibitors (PI), Myc-TSC1/TSC2/TET3 were internalized and degraded by lysosomes. An empty\nlysosome sample served as the negative control, while substrate-only reactions (without lysosomes)\nwere included as input controls. LAMP1 was used to demonstrate equal lysosomal loading.\nPBP Fig. 13 b, d: Quantification of TSC1, TSC2 (b) or TET3 (c) levels. Data are presented as the\nmeans ± S.E.M. n = 3 independent experiments. Statistical significance was assessed using two-tailed\nStudent's t-test. *p < 0.05, **p < 0.01.\nOur experimental workflow rigorously adheres to established criteria for CMA substrate validation,\nincluding motif identification, degradation assays, lysosomal colocalization, and functional\nreconstitution. Collectively, these lines of evidence supported TSC1/2 or TET3 as the targets of\nCMA. We appreciate the reviewer’s emphasis on comprehensive validation.\nAdditional supporting Data were presented in revised Supplementary Table 1, Figures 5, 7 and\nSupplementary Figures 5, 7, with detailed explanations provided on page 18-19, line 378-402 and\npage 24-25, line 507-529. The newly developed experimental protocol has been incorporated into\nthe Methods section (In vitro lysosome binding and uptake assay) on page 52, line 1104-1109.\n3. Authors must show the quality of lysosome preparations. They should show enrichment of\nLAMP1 or LAMP2A in the lysosome lysates vs whole cell lysates by WesternBlot. Moreover, they\nmust show the quality of the lysosomal preparations, showing absence of mitochondrial and\ncytosolic markers in their Western-blot with lysosomal preparations.\nResponse: We sincerely appreciate the reviewer's valuable suggestion regarding the quality\ncontrol of lysosomal preparations. We performed validation of our lysosome isolation protocol\nusing GSC 23, M83, and 456 cells, following established methods from the previous publication\n(Caballero et al., Nat Commun 2021). Western blot analysis demonstrated a marked enrichment\nof the lysosomal marker LAMP1 in purified lysosomal fractions relative to whole-cell lysates, along\nwith concomitant enrichment of HSC70 - a critical chaperone for chaperone-mediated autophagy\n(CMA) (PBP Fig. 14a-e). Importantly, our quality control assays confirmed the absence of\ndetectable mitochondrial (COX IV) and cytosolic (β-actin) contaminants in the lysosomal\npreparations, confirming preparation purity (PBP Fig. 14a-e).\nPoint-to-Point Response to Reviewers’ Comments\nThese Data were included in Fig. 4a, i and Supplementary Fig. 4a, b, e, along with page 15, line\n303-316 and page 16, line 324-327, and Lysosomes isolation in Methods section (page 51, line\n1086-1087) of the revised manuscript.\nPBP Fig.14 a, d: IB analyses for indicated proteins in GSC M83, GSC 456, and GSC 23 cells with indicated\nmodifications.\nPBP Fig.14 b, c, e: IB analyses for indicated proteins in WCL and lysosomal fractions of GSC M83, GSC\n456, and GSC 23 cells with indicated modifications.\n4. Authors must clearly demonstrate that lysosomes are CMA competent: they should see an\nincrease in HSC70 and not only in LAMP2A levels in WB to conclude there’s an activation of CMA.\nThey should corroborate that those CMA competent lysosomes have increased phosphorylated\nLAMP2A when modifying MST4 levels.\nResponse: We appreciate the reviewer for pointing out this important issue. We isolated\nlysosomes from GSC 23, M83, and 456 cells using established methods as described in the\nprevious publication (Caballero et al., Nat Commun 2021). Western blot analysis revealed a\nsubstantial enrichment of HSC70 in the purified lysosomal fractions compared to whole-cell\nlysates (PBP Fig.15 a-e). Notably, MST4 overexpression in GSC 23 resulted in a significant\nincrease in both p-LAMP2A and total LAMP2A levels within the lysosomes. Furthermore,\nLAMP2A knockdown effectively abolished the MST4-induced accumulation of p-LAMP2A (PBP\nFig.15 a-b). In contrast, MST4 knockdown in M83 and 456 cells led to decreased levels of p-\nLAMP2A and reductions in lysosomal LAMP2A; however, HSC70 levels remained unchanged\n(PBP Fig.15 c-e). Interestingly, the phosphomimetic variant of LAMP2A (3D) was able to restore\nthe diminished lysosomal levels of LAMP2A resulting from MST4 deletion (PBP Fig.15 c-e). These\nPoint-to-Point Response to Reviewers’ Comments\nfindings suggest that MST4-mediated phosphorylation of LAMP2A enhances its enrichment within\nlysosomes and activates CMA.\nThe new results were presented as Fig. 4a, i and Supplementary Fig. 4a, b, e, with detailed\nmethodological information provided in page 15, line 303-316 and page 16, line 324-327, of the\nrevised manuscript.\nPBP Fig. 15 a, c: IB analyses for indicated proteins in WCL of GSC 23, GSC 83 and GSC 456 cells with\nindicated modifications.\nPBP Fig. 15 b, d, e: IB analyses for indicated proteins in WCL and lysosomal fractions of GSC 23, GSC\n83 and GSC 456 cells with indicated modifications.\n5. TCS1/2 is a known regulator of macroautophagy. Authors should prove that regulation of\nTCS1/2 does not alter macroautophagy levels, so that the effects on GCS that they see in\ntumorigenicity are caused only by CMA regulation.\nResponse: We appreciate the reviewer's insightful comment regarding TSC1/2's potential roles\nin regulation of macroautophagy. To address this, we systematically evaluated macroautophagy\nactivity under three experimental conditions: (1) TSC1/2 knockdown, (2) rescue with shRNA-\nresistant WT or CMA-resistant (KFERQ-mutant) TSC1/2, and (3) ATG5 knockdown as a\nmacroautophagy-deficient control.\nKey findings demonstrate that neither TSC1/2 manipulation (knockdown or rescue) altered LC3-\nII/I ratios or p62 levels, while ATG5 knockdown showed expected macroautophagy impairment\n(PBP Fig. 16a). Intriguingly, intracranial xenografts revealed: (1) Rescue of CMA-resistant TSC1/2\nPoint-to-Point Response to Reviewers’ Comments\nmutants significantly decreased tumorigenicity compared to TSC1/2 wild-type (PBP Fig. 16b);\n(2)\nEquivalent LC3B expression in TSC1/2-modulated tumors versus controls, while significant LC3B\nreduction in ATG5-knockdown tumors (p < 0.001; PBP Fig. 16c). Taken together, these results\nsuggest that modulation of TCS1/2 by CMA does not alter macroautophagy levels.\nMechanistically, TSC1/2 knockdown enhanced mTORC1 signaling (increased p-P70S6K/p-\n4EBP1) and tumor growth, effects reversed by TSC1/2 re-expression with mutant superiority\n(PBP Fig. 16a-c). Crucially, ATG5 knockdown did not affect mTORC1 activity, confirming CMA-\nspecific regulation of the mTORC pathway (PBP Fig. 16a-c).\nThese comprehensive data establish that TSC1/2's tumorigenic effects in GSCs operate\nexclusively through CMA-mediated mTORC1 regulation, independent of macroautophagy\nmodulation.\nThese results are now included in the updated Fig. 5 k and Supplementary Fig. 5 m-o, as\ndescribed on page 19-20, line 409-425.\nPoint-to-Point Response to Reviewers’ Comments\nPBP Fig. 16 a: IB analyses for indicated proteins in GSC M83 and 456 cells with indicated modifications.\nQuantifications in ratios of p-4EBP1, p-P70S6K, p62 relative to β-actin, as well as LC3B-II:-I ratios are\nshown.\nPBP Fig. 16 b: Representative H&E images of mouse brain sections (left) and quantification of tumor\nvolume (right) from athymic (BALB/c Nude) mice intracranially implanted with GSC M83 cells with indicated\nmodifications. Data are presented as the means ± S.E.M. n = 3. Scale bar, 1.0 mm.\nPBP Fig. 16 c: Representative images (left) and quantification (right) of IF staining for LC3B and p-P70S6K\nexpression in tumor tissues from BALB/c Nude mice intracranially transplanted with GSC M83 cells with\nindicated modifications. Data are presented as the means ± S.E.M. n = 10 randomly selected microscopic\nfields.\nStatistical significance was assessed using one-way ANOVA with Dunnett's multiple comparisons test (b,\nc). ns, not significant. **p < 0.01, ***p < 0.001.\n6. Besides all the above recommendations to validate CMA substrates, authors also should\nvalidate that TET3 expression they see is due to changes in protein levels and not due to gene\nexpression (measuring mRNA levels).\nResponse: We thank the reviewer for this insightful suggestion. To address whether TET3\nregulation occurs at the protein versus transcriptional level, we performed qPCR analysis under\nCMA inhibition conditions. As shown in PBP Fig. 17a and b, neither LAMP2A knockdown nor\nHSC70 depletion induced significant changes in TET3 mRNA levels (p > 0.05 by one-way ANOVA\nwith Dunnett's multiple comparisons test). This demonstrates that the altered TET3 protein\nexpression observed following CMA modulation results from post-translational regulation rather\nthan transcriptional changes, consistent with CMA's established role in protein degradation.\nThese findings further support our conclusion that TET3 is a bona fide CMA substrate.\nSupplementary Fig. 7d and e have been updated to reflect these results, and detailed descriptions\nare provided on page 24, line 516-517 of the manuscript.\nPBP Fig. 17 a, b: qPCR assessment of mRNA level of TET3 in HEK293T cells with or without LAMP2A (a)\nor HSC70 (b) knock down. Data are presented as the means ± S.E.M. n = 3 independent experiments.\nPoint-to-Point Response to Reviewers’ Comments\nStatistical significance was assessed using one-way ANOVA with Dunnett's multiple comparisons test. ns,\nnot significant.\n7. If authors are able to demonstrate that TET3 and TCS1/2 are CMA substrates, they should\nshow if the increased stability of the phosphorylated LAMP2A at the lysosomal membrane is a\ncommon mechanism among other types of cells, not only in GSCs. They should include a model\nwhere CMA is downregulated (for example an aging model) and test whether they can\nrestore CMA by introducing the phosphomimic version they use.\nResponse: Thank you for this insightful suggestion. To address whether the increased stability\nof phosphorylated LAMP2A at the lysosomal membrane represents a common mechanism across\ncell types, we extended our analysis to human MRC-5 fibroblasts. Specifically, we compared the\nhalf-life of LAMP2A and the levels of phosphorylated LAMP2A in lysosomal membranes between\nearly-passage (P18) and late-passage (P45) fibroblasts (PBP Fig. 18a-c). Although cell passage\ndoes not recapitulate all aspects of aging, senescent fibroblasts in culture have been shown to\nmirror CMA alterations observed in aged rodents, including reduced CMA activity and lower\nlysosomal LAMP2A levels (A M Cuervo & J F Dice. J Biol Chem. 2000;275(40):31505-13.). While\nlate-passage fibroblasts exhibited significantly faster LAMP2A degradation rates compared to\nearly-passage cells, we found that lysosomal membranes from late-passage fibroblasts also\nshowed markedly lower levels of phosphorylated LAMP2A than those from early-passage cells\n(PBP Fig. 18a-c).\nTo further test whether CMA dysfunction in aging-like models can be rescued by enhancing\nLAMP2A phosphorylation, we introduced LAMP2A-WT, a non-phosphorylatable mutant\n(LAMP2A-3A), or a phosphorylation-mimic mutant (LAMP2A-3D) into MRC-5 fibroblasts. \nStrikingly, expression of LAMP2A-WT or LAMP2A-3D, but not LAMP2A-3A, significantly reduced\nthe senescence-associated β-galactosidase (SA-β-gal) positive cells burden in late-passage\nfibroblasts (PBP Fig. 18d, e). Critically, this rescue was accompanied by a decrease in known\nCMA substrates (MEF2D and GAPDH) in whole-cell lysates and their concurrent accumulation in\nlysosomal fractions, demonstrating enhanced CMA-mediated substrate degradation (PBP Fig.\n18f-h). These results suggest that phosphorylation-dependent stabilization of LAMP2A is a\nconserved mechanism across cell types and that introducing the phosphomimic LAMP2A version\n(LAMP2A-3D) can ameliorate CMA dysfunction in aging-like models.\nWe acknowledge that further validation in vivo and in other cell types will strengthen these\nconclusions. However, our data provide evidence for the broad relevance of LAMP2A\nphosphorylation in CMA regulation.\nThese Data were included in the updated Supplementary Fig. 3k-p, with detailed descriptions\nprovided on page 14, line 285-300 of the revised manuscript. The newly developed experimental\nprotocol has been incorporated into the Methods section (SA-β-gal staining) on page 52, line\n1110-1116).\nPoint-to-Point Response to Reviewers’ Comments\nPoint-to-Point Response to Reviewers’ Comments\nPBP Fig. 18 a, b: Assessment of LAMP2A stability in lysosomes isolated from MRC-5 of early (P18) and\nlate (P45) passage. Lysosomes were incubated at 0℃ for 10 minutes with or without a protease inhibitor\n(PI), followed by pelleting at indicated time points. LAMP2A and LAMP1 levels were analyzed through\nimmunoblotting, with densitometric quantification of LAMP2A shown in b. Data are presented as the means\n± S.E.M. n = 3 independent experiments.\nPBP Fig. 18 c: IB analyses for indicated proteins in MRC-5 cells of early (P18) and late (P45) passages.\nPDL, population doubling level.\nPBP Fig. 18 d: Representative images of senescence-associated beta-galactosidase (SA-β-gal) staining\nof MRC-5 cells at early (P18) and late (P45) passage with or without overexpressing Flag-LAMP2A-\nWT/3D/3A. Scale bar, 50 μm.\nPBP Fig. 18 e: Quantification analyses of the proportion of SA-β-gal positive MRC-5 cells with indicated\nmodifications. Data are presented as the means ± S.E.M. n = 10 randomly selected microscopic fields.\nPBP Fig. 18 f: IB analyses for indicated proteins in WCL and lysosomal fractions from MRC-5 cells at early\n(P18) and late (P45) passage with or without overexpressing Flag-LAMP2A-WT/3D/3A.\nPBP Fig. 18 g, h: Band intensities of indicated proteins were quantified using Image J software, normalized\nto β-actin levels. Data are presented as the means ± S.E.M. n = 3 independent experiments.\nStatistical significance was assessed using two-way ANOVA with Bonferroni's multiple comparisons test\n(b) or one-way ANOVA with Dunnett's multiple comparisons test (e, g, h). ns, not significant. *p < 0.05, **p\n< 0.01, ***p < 0.001.\n8. During the whole manuscript, authors are trying to point out the relevance of CMA for GSC\ntumorigenicity and proliferative potential. They are trying to prove that by performing several in\nvitro and in vivo analysis with different lines of human GSC. Nevertheless, when they test in the\nin vivo model if LAMP2A or MTS4 regulation in tumor cells changes the immune response to the\ntumor (Figure 7), they use GB mouse cells, even if they have the tools to do it with the modified\nGSC. They also use GB mouse cells when studying the effects of IR therapy and anti-PD1 therapy\nin the in vivo model (Figure 10). Authors should show the results of immune regulation effects\nwith the human GCS lines with the corresponding genetic modifications.\nResponse: Thank you for this insightful suggestion. We fully recognize the significance of\nvalidating immune-modulatory effects in human glioma stem cells (GSCs) and have addressed\nthis through additional experiments. Human GSCs are unable to engraft in immunocompetent\nC57BL/6 mice due to species-specific barriers, such as a lack of HLA compatibility. Consequently,\nwe employed syngeneic murine GL261 and CT-2A glioma cell lines for our in vivo immunotherapy\nstudies (revised Figures 6, 8, and 9).\nTo directly investigate CMA-mediated immunomodulation within human systems, we conducted\nin vitro co-cultures of modified GSC cell lines (MST4-KD and LAMP2A-KD) with allogeneic human\nCD8+ T cells. After 48-72 hours of activation using anti-CD3/CD28 beads, T cells are co-cultured\nwith GSC cells at a 1:5 ratio for 24 hours. Flow cytometry analysis revealed that depletion of\nLAMP2A or MST4 significantly enhanced cytotoxic T-cell responses, as evidenced by increased\npolyfunctional cytokine production (TNF-α MFI: 0.42×10³ or 0.44×10³ vs. 0.25×10³ in sh-C; p <\n0.001; IFN-γ MFI: 3.76×10³ or 4.86×10³ vs. 1.39×10³ in sh-C; p < 0.001), elevated effector\nPoint-to-Point Response to Reviewers’ Comments\ndegranulation production (Granzyme B mean fluorescence intensity: MFI = 0.83×10² or 0.81×10²\nvs. 0.62 ×102 in sh-C; p < 0.01), and increased apoptosis among GSCs (Annexin V+PI+ cells:\n67.10% or 75 .13% vs 52 .70 % in sh-C; p < 0.01) (PBP Fig. 19a-d). These findings from human\ncell data align with observations made from murine tumor models (Supplementary Figure 6g-l),\ndemonstrating an evolutionary conservation of CMA's role in immune evasion.\nAlthough the validation through humanized models remains an important direction for future\nresearch, our in vitro co-culture experiments indicate that CMA contributes to the functional\nimpairment of CD8+ T lymphocytes.\nThe updated Fig. 6b-d and Supplementary Fig. 6f presents these findings, which are further\nexplained on page 21, line 439-444 and Methods section (page 48, line 1016-1035).\nPBP Fig. 19 a-d: GSC M83 cells with indicated modification were co-cultured for 24 hours with or without\nCD8+ T cells isolated from GBM patients' peripheral blood. Flow cytometry quantified T cell-derived: TNF-\nα (a), IFN-γ (b) and GZMB (c) production，as well as Apoptosis analysis of GSC M83 cells (d).Data are\npresented as the mean ± S.E.M. n = 3 independent experiments. MFI, mean fluorescence intensity. PI,\npropidium iodide.\nStatistical significance was assessed using one-way ANOVA with Dunnett's multiple comparisons test (a-\nc) or one-way ANOVA with Tukey's multiple comparisons test (d). **p < 0.01, ***p < 0.001.\n9. The inhibitor PPD is used arbitrarily and without context. To improve the quality of the work,\nauthors should show the link, if any, between the phosphorylation of LAMP2A and the modulation\nof the CMA activity by PPD.\nResponse: We sincerely appreciate the reviewer's insightful suggestion regarding the\nmechanistic relationship between LAMP2A phosphorylation and PPD-mediated CMA modulation.\nIn response to this valuable comment, we have conducted additional experiments to elucidate\nthis connection.\nAs shown in a recent study (Dong RF, et al. Br J Pharmacol. 2025), Polyphyllin D (PPD) functions\nas a selective CMA inhibitor through dual mechanisms: (1) interference with HSC70-LAMP2A\ninteraction and (2) suppression of LAMP2A multimerization. Given that MST4-mediated\nphosphorylation of LAMP2A promotes its multimerization—an essential step for CMA activation—\nwe explored the impact of PPD on LAMP2A phosphorylation, multimerization, HSC70-LAMP2A\ninteraction, and overall CMA activity.\nPoint-to-Point Response to Reviewers’ Comments\nOur additional experimental results demonstrate:\n1) Co-immunoprecipitation and western blot assays revealed that while PPD effectively\ndisrupts the binding between HSC70 and LAMP2A, leading to a decrease in lysosomal\nassociation of CMA substrates (TSC1/2, TET3, MEP2D) and an increase in their\nexpression in whole cell lysate (PBP Fig. 20a).\n2) PPD does not appear to affect the phosphorylation of LAMP2A (PBP Fig. 20a).\n3) Native PAGE analysis provided direct evidence that PPD significantly inhibits LAMP2A\nmultimerization (PBP Fig. 20b)\n4) Quantitative photoactivatable CMA reporter assays consistently showed PPD-induced\nreduction in cellular CMA activity (PBP Fig. 20c, d)\nIn light of PPD's inhibitory impact on the multimerization of LAMP2A, a downstream consequence\nof LAMP2A phosphorylation, we employed PPD as a potential inhibitor to interfere with LAMP2A\nhomo-multimerization and subsequently CMA activity.\nThese Data were shown in revised Supplementary Fig. 8d-g, with detailed descriptions provided\non page 26, line 539-550, as well as page 28, line 601-604.\nPPD has been identified as a CMA-targeting inhibitor (Dong, Rui-Fang et al. Br J Pharmacol.\n2025;182(10):2287-2309.); however, its selectivity may be compromised due to its potential\nimpact on alternative cellular stress pathways (Wang, Yang et al. Mol Ther. 2023;31(7):2169-\n2187.). These limitations of PPD have been discussed in the revised manuscript (page 34, line\n721-727).\nPBP Fig.20 a: IP-IB analyses for indicated proteins in WCL and IB analyses for indicated proteins in\nlysosomal fractions of HEK293T and HeLa cells with or without 3.2 μM PPD treatment.\nPBP Fig.20 b: Purified lysosomes from the indicated HEK293T cells with or without 3.2 μM PPD treatment\nwere analyzed using native continuous gel electrophoresis and subjected to IB detection for LAMP2A.\nPoint-to-Point Response to Reviewers’ Comments\nPBP Fig.20 c, d: Representative images (c) and quantification (d) of CMA activity detection by KFERQ\npuncta numbers in GSC M83 cells with or without 3.2 μM PPD treatment. Data are presented as the\nmeans ± S.E.M. n = 40 randomly selected cells. Scale bar, 10 μm. Statistical significance was assessed\nusing two-tailed Student's t-test. ***p < 0.001.\n10. Supplementary Fig. 1E-H- Authors show some redundant analyses, previously published by\nother authors (Auzmendi-Iriarte et al., 2022) DOI: 10.1158/0008-5472.CAN-21-2161\nResponse: Thank you for your suggestion. We have removed Supplementary Figure S1E-H\nfrom the revised manuscript, as some redundant analyses previously reported by Auzmendi-\nIriarte et al. (2022) (DOI: 10.1158/0008-5472.CAN-21-2161).\n11.Introduction. Authors should enhance the quality of the documentation for the work since\ntheir claims are poorly substantiated. Fox example, authors state in line 72 that “the regulatory\nmechanisms and 73 physiological roles of CMA remain poorly understood”, which shows a lack\nof documentation for the study. To improve the introduction section, they could add different\nworks and reviews on the physiological significance of CMA (doi:10.1038/s41580-018-0001-6;\ndoi:10.1007/978-1-59745-157-4_15)\nResponse: To improve the introduction, we have added key literature on page 4, line 72,\nciting important reviews that discuss the physiological roles of CMA (doi:10.1038/s41580-018-0001-6;\ndoi:10.1007/978-1-59745-157-4_15). These additions clarify the regulatory mechanisms and\nphysiological functions of CMA, strengthening the relevance of our study.\n12· Results. Authors claim to have significant differences in several results that are not\nquantified and are not clear. They also do not explain why they treat lysosomes with just\nprotease inhibitors and they do not add also lysosomal inhibitors such as NH4CL. They also do\nnot really check the glycosylation status of LAMP2A despite having the tools to do so. In\naddition, all the brain sections shown are at different depths, so they are not comparable.\nResponse: We sincerely appreciate the reviewer’s insightful critiques, which have significantly\nstrengthened our study. Below we provide a point-by-point response to the concerns raised:\n1) We apologize for the lack of statistical clarity in the original manuscript. To address this issue,\nwe have reviewed the text and all figures, and provided p-values for all instances of\n\"significant differences\" in the figure legends (e.g., Fig. 4b-d).\n2) We sincerely appreciate the reviewer's insightful suggestion regarding lysosomal protease\ninhibition. In our investigation of lysosomal LAMP2A stability and CMA substrate\nbinding/uptake, we employed a protease inhibitor cocktail (10 μM leupeptin, 10 mM AEBSF,\n1 mM pepstatin, 100 mM EDTA) to specifically block lysosomal proteolytic activity. This\napproach effectively prevented luminal degradation of LAMP2A or CMA substrates while\nmaintaining lysosomal integrity, following established CMA research protocols (Juste &\nCuervo, Methods Mol Biol. 2019). Notably, we excluded NH Cl from our purified lysosome\n4\nPoint-to-Point Response to Reviewers’ Comments\nexperiments despite its known lysosomal pH-neutralizing effects, as recent evidence\nsuggests it may cause lysosomal membrane damage (Zhang H., et al. Nat Cell Biol.\n2024;26(11):1892-1902.).\nThese clarifications have been added to the updated manuscript (page 8, line 162-163), and\nthe revised methods for Immunoblot (IB) analysis (page 45, line 958-964) and in vitro\nlysosome binding and uptake assays (page 51, line 1093-1095).\n3) We thank the reviewer for their valuable suggestion. We evaluated the glycosylation status\nof LAMP2A through PNGase F treatment and Concanavalin A (ConA) blotting, as detailed in\nthe Methods section, followed by immunoblot analysis. LAMP2A was immunoprecipitated\nusing an anti-LAMP2A antibody. After PNGase F treatment, the molecular weight of LAMP2A\nmonomers in both GSC and DGC decreased from about 100 kDa to approximately 50 kDa,\nindicating complete deglycosylation. The similar sizes of the LAMP2A monomers post-\ntreatment suggest that GSCs and DGCs have comparable glycosylation patterns.\nAdditionally, ConA blotting results showed similar glycosylation levels of LAMP2A in both\nGSCs and DGCs (PBP Fig. 21 a).\nThese findings were presented in revised Supplementary Fig. 2a and discussed on page 8-\n9, line 168-174 of the updated manuscript. PNGase F treatment and Lectin blotting have\nbeen added to the revised methods on page 54, line 1144-1158.\nPBP Fig. 21 a: IP-IB analyses for LAMP2A and Concanavalin A (ConA) in GSC and DGC M83 cells\nwith or without PNGase F treatment.\n4) We thank the reviewer for their detailed assessment and acknowledge the variability in\nhistology sampling. In our tumorigenicity studies, we stereotactically injected different cell\nlines—5 × 104 GSC M83 cells, 5 × 105 GSC 23 cells, or 1 × 105 GL261 cells—into BALB/c\nNude or C57BL/6 mice using standardized coordinates: 2.5 mm lateral, 1.5 mm anterior, and\n3.0 mm deep from the skull surface. Coronal sections of the entire brains were prepared at\na thickness of 10 μm with a cryostat (Leica), covering the entire rostral-caudal axis of each\ntumor. We conducted serial section analysis to identify the slice with the largest tumor area\nfor measurement. Due to the invasive nature of the xenografted GBM tumors, they were\nfound at varying depths in the brain sections. To ensure consistency in tumor volume\ncomparisons, we reviewed all tumor sections within each group and selected a comparable\nPoint-to-Point Response to Reviewers’ Comments\ndepth for representative H&E-stained sections, then recalculated the tumor volumes of the\nGBM brain xenografts (PBP Fig. 22 a-f). These data have been updated to the revised\nFigures 3g-h, 4o-p, and 6h-i.\nPBP Fig. 22 a-f: Representative H&E images and quantitative analysis of tumor volume from athymic\n(BALB/c Nude) mice or C57BL/6 mice intracranially implanted with GSC M83 cells (a, c) or GL261\ncells (e) with indicated modifications. Data are presented as the mean ± S.E.M. n = 3. Scale bar, 1.0\nmm. Statistical significance was assessed using one-way ANOVA with Dunnett's multiple comparisons\ntest. ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001.\n13· Results, line 106. The explanation of the CMA photo-activable reporter is not correct.\nResponse: We sincerely apologize for the imprecise description of the CMA reporter system.\nLine 106 in the Results section (now updated to line 103-108 on page 5-6) has been revised to\naccurately describe the CMA photo-activatable reporter (KFERQ-PA-mCherry). We used this\nreporter to visualize and quantify CMA activity by counting the fluorescent puncta per cell. This\noccurs when the fluorescent CMA substrate protein, fused with a KFERQ-like motif, is delivered\nto CMA-active lysosomes by HSC70, resulting in a transition from a diffuse to a punctate\ndistribution (Nat Commun. 2011; 2:386. doi: 10.1038/ncomms1393).\n14· Results, line 112. Authors mention CMA substrates that are not addressed in the text.\nResponse: We appreciate the reviewer's insightful comment. In the page 6, line 113 of the\nrevised manuscript, we have now provided explicit citations in the revised manuscript (Page 6,\nLine 114) to substantiate that both MEF2D (Science. 2009;323(5910):124-7.) and GAPDH (J\nBiol Chem. 1993;268(14):10463-70.) are well-established CMA substrates in the literature.\nPoint-to-Point Response to Reviewers’ Comments\n15· Results, line 117. The description of the results obtained in Fig 1c does not match what is\nobserved in the figure. The expression of MEF2D and GAPDH seems to be altered with the\ndifferent inhibitors.\nResponse: We appreciate the reviewer's careful observation. In the revised Fig. 1c (PBP Fig.\n23a), we have repeated the experiments and included all Western blot replicates for clarity.\nThe results demonstrate that in GSCs, lysosomal inhibition using lysosomal protease inhibitors\n(leupeptin and ammonium chloride (NH Cl), LN) significantly increased the levels of CMA\n4\nsubstrates (MEF2D and GAPDH), whereas this effect was not observed in conventional GBM\ncells. In contrast, neither macroautophagy inhibition (3-MA) nor proteasomal inhibition (MG132)\naltered MEF2D or GAPDH expression. We have corrected the Fig. 1c (page 6, line 111-118) to\naccurately reflect these findings.\nPoint-to-Point Response to Reviewers’ Comments\nPBP Fig. 23 a: IB analyses for indicated proteins after treating with 10 μM MG132, 5 mM 3-MA or, LN (10\nμM leupeptin and 20 mM NH Cl) in GSC cell lines (M83 and 23) and non-GSC cell lines (LN229 and\nactin. n = 3 independent experiments.\n16· Results, line 294. Authors cannot state with only an Immunoprecipitation, what effects there\nmay be on CMA activity.\nPoint-to-Point Response to Reviewers’ Comments\nResponse: We sincerely appreciate the reviewer's insightful comment regarding the need for\nadditional evidence to support the effects on CMA activity. In response to this concern, we have\nnow conducted additional experiments to further validate our findings. Specifically, we have\nincluded: (1) quantification of KFERQ-positive puncta as a direct measure of CMA activity (PBP\nFig. 24 a); (2) analysis of lysosomal binding and uptake of GAPDH, a well-established CMA\nsubstrate (PBP Fig. 24 b); and (3) examination of MEF2D lysosomal association (PBP Fig. 24 c).\nThese new data collectively provide multiple lines of evidence demonstrating that MST4 indeed\nenhances CMA activity through LAMP2A. We believe these additional experiments have\nadequately addressed the reviewer's concern and strengthened our conclusions.\nThese data were included in the updated Fig. 4i-l, and the revised text now more accurately\nreflects these experimental validations (page 16, line 324-331 in the revised manuscript).\nPBP Fig. 24 a: Representative images (left) and quantification (right) of CMA activity detection by KFERQ\npuncta numbers in GSC 23 cells with indicated modifications. Data are presented as the means ± S.E.M.\nn = 40 randomly selected cells. Scale bar, 10 μm.\nPBP Fig. 24 b: In vitro binding and uptake assay of GAPDH by the lysosome in GSC 23 cells with indicated\nmodifications. Data are presented as the means ± S.E.M. n = 3 independent experiments.\nPBP Fig. 24 c: IP-IB analyses for indicated proteins in WCL and IB analyses for indicated proteins in\nlysosomal fractions of GSC 23 cells with indicated modifications.\nStatistical significance was assessed using one-way ANOVA with Tukey's multiple comparisons test. ns,\nnot significant. *p < 0.05, *p < 0.01, ***p < 0.001.\nMinor points:\n17· Results, line 413. There is a typo.\nResponse: Thank you for catching the typo. We have corrected this in original line 413 (now\nupdated to page 26, line 544) (\"However\" → \"however\") and conducted a full-text review to ensure\nconsistency in capitalization and grammar throughout the manuscript. No additional issues were\nidentified.\nPoint-to-Point Response to Reviewers’ Comments\n18· Results, line 429. The text and the figures mentioned do not match.\nResponse: We thank the reviewer for their detailed observations. Upon reviewing the text and\nfigures, we realized that the description of Interferon Beta 1 expression in the original manuscript\n(line 429), as it was not included in Supplementary Fig. 7e, f (now updated to Supplementary Fig.\n8b, c). To address this, we have removed the related text on page 25, line 534-537 of the revised\nmanuscript. Additionally, we have meticulously checked the entire manuscript to ensure the\nconsistency of all cited results and figures.\n19· Results, line 466. Authors do not mention PDL-2 in the text even though it is included in\nsupplementary figure 8a.\nResponse: We appreciate the reviewer's careful attention to the detail. We apologize for the\noversight and have now included a detailed description of the PD-L2 results in the Results section\n(page 27, line 578-581) to align with the data presented in Supplementary Figure 9a.\n20· Methods. The brain coordinates where the stereotactic procedure was performed should be\nindicated.\nResponse: Thank you for your important comment. We have indicated the brain coordinates at\nwhich the stereotactic procedure was conducted in the revised Methods section of the Animal\nStudies (page 40, line 851-853). Tumor cells were injected into the brain of individual mice, using\nthe following coordinates from bregma: 2.5 mm lateral, 1.5 mm anterior, and 3.0 mm deep from\nthe skull.\n21· Figure 5E. They show the images and quantification of tumor volume in mice with shC or\nshLAMP2A GSC 23, and how the volume increases upon MTS4 upregulation. In the text,\ndescribing the results, they indicate that the knock down of LAMP2A in GSC23 (a GSC line with\nlow MTS4 expression) had minimal effects on tumorigenicity of GSCs. However, in the figure, it\nis shown a clear reduction of the tumor in shLAMP2A with a significant statistical analysis.\nResponse: We apologize for this confusion. We have revised the text to clarify that sh-LAMP2A\nsignificantly reduced tumorigenicity in GSC 23 (page 16, line 331-336 of the revised manuscript),\nconsistent with the data in updated Fig. 4o-p.\n22· Supplementary Figure 3d. In order to verify the antibody T136A specificity, it should be\ntested in the single and double mutants generated for the study, not only in the 3A mutant.\nPoint-to-Point Response to Reviewers’ Comments\nResponse: We sincerely appreciate the reviewer’s insightful suggestion. To rigorously validate\nthe specificity of the phospho-T136-LAMP2A antibody, we have now performed additional\nexperiments using single and double mutants, alongside the triple mutant (T136A/T40A/S97A,\n3A). As shown in PBP Fig. 25a, the antibody specifically detected phosphorylation in LAMP2A-\nWT, T40A, S97A, and T40A/S97A under exogenous overexpression conditions. However, it\ncompletely failed to recognize the T136A phospho-null mutant (T136A), as well as all mutants\ncontaining the T136A substitution (T136A/T40A, T136A/S97A, and 3A). These data conclusively\ndemonstrated the antibody’s exclusive specificity for the phosphorylated T136 residue.\nThe updated results are presented in Supplementary Fig. 3d and described in the Results section\non page 13, line 262-266 of the revised manuscripts.\nPBP Fig. 25 a: IP-IB assessments were conducted for Flag-LAMP2A, HA-MST4, and LAMP2A pT136 in\nHEK293T cells, both under conditions of MST4 and LAMP2A-WT or -Mut overexpression and in their\nabsence.\n23· Figure 7 and supplementary figure 6. The cytotoxic effect of CD8 T cells should be\nconfirmed with a double staining of CD8 and GZMB to confirm the colocalization. They could\nperform further analysis to confirm the functionality. In addition, authors could check the\nreduction of immunosuppressive T cell populations by IHC and by flow cytometry.\nResponse: We sincerely appreciate the reviewer’s insightful suggestion. As recommended, we\nconducted dual immunofluorescence staining for CD8 (membrane marker) and granzyme B\n(GZMB, cytotoxic granule marker) in tumor tissues. We found a notable increase in the\ncolocalization of CD8 and GZMB in the tumor tissues of the sh-Mst4 and sh-Lamp2a groups,\nindicating enhanced cytotoxic activity of CD8+ T cells. (PBP Fig. 26a). In addition, flow cytometry\nanalysis further evaluated the function of infiltrating CD8+ T cells in tumor tissues. In line with the\nimmunofluorescence findings, we observed a significant increase in functionally active CD8+ T\ncell infiltration within tumors from the sh-Mst4 and sh-Lamp2a groups (p < 0.001, PBP Fig. 26b-\nd). These results reinforce the involvement of CMA in facilitating tumor immune evasion.\nRepresentative images and quantitative analysis are provided in revised Supplementary Fig. 6i-l.\nThe updated data were described on page 22, line 456-464 of the revised manuscript.\nFor assessment of immunosuppressive T cells populations, we performed double staining for\nCD4 (membrane, red) and FoxP3 (nuclear, green) in tumor sections. Quantitative analysis of\nFoxP3+ CD4+ cells within tumor regions revealed decreased Tregs infiltration in the sh-Mst4 and\nsh-Lamp2a groups (p < 0.001, PBP Fig. 26e). In addition, we analyzed FoxP3+ CD4+ regulatory\nPoint-to-Point Response to Reviewers’ Comments\nT cells (Tregs) in tumor single-cell suspensions using flow cytometry, and we observed diminished\ninfiltration of Tregs within the tumor microenvironment in the sh-Mst4 and sh-Lamp2a groups (p\n< 0.001, PBP Fig. 26f). These data were included in revised Supplementary Fig. 6h, m, and are\ndescribed on page 22, line 456-464 of the revised manuscript.\nPBP Fig. 26 a, e: Representative IF staining images (left) and quantification (right) for CD8+ GZMB+ cells\n(a) and CD4+ FoxP3+ Tregs (e) in tumor tissues from C57/BL6 mice with intracranial CT-2A cells transplants\nwith indicated modifications. Data are presented as the means ± S.E.M. n = 10 randomly selected\nmicroscopic fields. Scale bar, 20 μm.\nPBP Fig. 26 b-d: Flow cytometric analysis of population of TNF+CD8+ T cells (b), IFN+CD8+ T cells (c) and\nGZMB+CD8+ T cells (d) in infiltrating CD8+ T cells from tumor tissues of C57/BL6 mice with intracranial CT-\n2A cells transplants with indicated modifications. Data are presented as the means ± S.E.M. n = 5\nindependent experiments.\nPBP Fig. 26 f: Flow cytometric analyses of population of infiltrating Tregs in tumor tissues from C57/BL6\nmice with intracranial CT-2A cells transplants with indicated modifications. Data are presented as the\nmeans ± S.E.M. n = 5 independent experiments.\nStatistical significance was assessed using one-way ANOVA with Dunnett's multiple comparisons test (a-\nf). **p < 0.01, ***p < 0.001.\n24· Figure 11. An IHC analysis of GB patient samples is shown as well as a correlation analysis\nbetween the studied markers. In material and methods, authors describe that they have defined\na score in order to perform the correlation analysis between the high and low expression of the\nPoint-to-Point Response to Reviewers’ Comments\nmarkers. Nevertheless, the ranges the use for high and low expression are not well stablished.\nFor instance, a sample with a score of 3 or 4 could be high and low at the same time according\nto their criteria. In addition, it is not mentioned in the text.\nResponse: We re-evaluated the quantitative scoring of tissue section staining based on the\npercentage of positively stained cells and staining intensity (PBP Fig. 27a, b), adhering to\nestablished protocols (Nat Cell Biol. 2022;24(11):1655-1665; Nature. 2011;480(7375):118-22.).\nThe scoring was as follows: 0 for 0% positive tumor cells, 1 for 0–1%, 2 for 2–10%, 3 for 11–30%,\n4 for 31–70%, and 5 for 71–100%. Staining intensity was rated from 0 to 3, with 0 indicating\nnegative, 1 weak, 2 moderate, and 3 strong. The total score, ranging from 0 to 8, was calculated\nby combining the proportion and intensity scores. We compared these scores with overall survival\nduration, defined as the time from diagnosis to death or last follow-up. Kaplan-Meier plots were\ngenerated for overall survival rates of GBM patients, categorized into high (staining score 4–8)\nand low (staining score 0–3) expression groups of the indicated protein. These results are\nincluded in the updated Fig. 10 b-c and detailed on page 53, line 1124-1134 of the revised\nmethods for IHC analysis of clinical glioma specimens.\nPBP Fig. 27 a: Scoring of IHC staining of human glioma samples using the specified antibodies, followed\nby correlation analysis utilizing a two-tailed Pearson correlation test. n = 83. Note that some sample scores\nmay overlap.\nPBP Fig. 27 b: Kaplan-Meier survival analyses for glioma patients categorized by high or low expression\nlevels of LAMP2A pT136, MST4, TET3, TSC1 and TSC2. n = 83. IHC score ≥ 4 was considered as high\nexpression samples, IHC score ≤ 3 was considered as low expression samples. Statistical significance was\nassessed using log-rank test. ***p < 0.001.\nPoint-to-Point Response to Reviewers’ Comments\nManuscript Number: NCOMMS-25-06239A-Z-R1\nPoint-to-Point Responses to Reviewer’s Comments\nWe sincerely appreciate the enthusiasm with which the editor and reviewers have engaged with\nour revised submission. We have thoroughly addressed all of their concerns and questions in this\nupdated manuscript. We believe that the revised version offers a more comprehensive and\ninformative presentation of our research, and we are grateful to the reviewers for their valuable\nsuggestions. Therefore, we are pleased to submit this revised manuscript for further consideration.\nBelow, we provide point-by-point responses to each comment:\nReviewer #1:\nThe authors have addressed my comments, except the code availability as per Nature\ncommunications' guidelines.\nResponse: We sincerely appreciate the reviewer’s constructive feedback and valuable\nsuggestions, which have significantly strengthened the clarity and reproducibility of our\nmanuscript. We regret the oversight regarding code availability in our resubmission and confirm\nthat all relevant code and data access details have been successfully made public via the PRIDE\ndatabase (https://www.ebi.ac.uk/pride/archive/projects/PXD060464).\nWe sincerely apologize for any inconvenience caused and deeply appreciate the reviewer’s\ndiligence in highlighting this critical requirement. Your feedback has been instrumental in\nenhancing the rigor and accessibility of our work.\nReviewer #2:\nWe appreciate and applaud the authors’ thoughtful and substantial revisions in response to our\nprior feedback. The manuscript is now suitable for publication, pending resolution of a few minor\ncomments outlined below.\nResponse: We thank the reviewer for the enthusiasm and the positive comments. We also\nappreciate the reviewer’s excellent comments and suggestions. We have included further\nclarification and discussion of limitations to address the reviewer’s concerns in our revised\nmanuscript.\nMinor Comments:\n1- Clarification of CMA Marker Interpretation:\nPage 6, Results, Line: 104-116:\nThe authors quite effectively demonstrate increased CMA activity in GSCs compared to non-\nGSCs with the PA-mCherry-KFERQ reporter. For clarity, however, it would be helpful to include\nPoint-to-Point Response to Reviewers’ Comments\na brief description of how puncta per cell reflects CMA activity and to differentiate this\nmeasurement from general lysosomal activity, given the centrality of this assay\nResponse: We sincerely appreciate the reviewer’s insightful feedback regarding the\ninterpretation of CMA activity in our study. We have added a brief description of how puncta per\ncell reflects CMA activity and to differentiate this measurement from general lysosomal activity in\nthe revised manuscript (Page 7, Lines 106–108).\n2- Expanded Discussion on CMA Regulation:\nPage 9-10, Line: 180-194:\nThe authors well demonstrate MST4-LAMP2A interactions in conditions of stress (IR and\nstarvation). Perhaps mention in brief the overall implications of this stress-induced interaction\nwithin the tumor microenvironment, such as how it may regulate GSC behavior in vivo under\nconditions of hypoxia or starvation commonly encountered within GBM tumors.\nResponse: We sincerely appreciate the reviewer’s insightful suggestion regarding the broader\nimplications of MST4-LAMP2A interactions within the tumor microenvironment. In response to\nthis valuable feedback, we have incorporated a brief discussion on the overall implications of\nstress-induced MST4-LAMP2A interactions in the tumor microenvironment in the revised\nmanuscript (Page 11, Lines 194-197).\n3- Lysosomal Protease Inhibition Consistency:\nPage 8, Lines: 156-163 (Methods in Rebuttal response, Minor Comment 5):\nAuthors detailed in the rebuttal the explicit use of a protease inhibitor cocktail other than the LN\n(leupeptin/NH4Cl). However, a transitional mention in main manuscript methods or figure legends\nwould raise transparency and methodological clarity in the light of the nuanced difference between\nLN and the more general PI cocktail.\nResponse: We sincerely appreciate the reviewer's suggestion regarding methodological\ntransparency. To address this concern, we have taken the following steps:\nMethodological Clarification: We have now added a transitional statement in the Methods section (Page\n47, Lines 981-984; Page 54, Lines 1117-1118) explicitly stating: \"For CMA activity assays, a protease\ninhibitor cocktail (containing 10 μM leupeptin (targeting serine/cysteine proteases), 10 μM AEBSF (serine\nprotease inhibitor), 1 μM pepstatin A (aspartic protease inhibitor), and 100 μM EDTA (metalloprotease\ninhibitor)) was used instead of leupeptin/NH Cl to specifically inhibit lysosomal proteases while minimizing\n4\ninterference with CMA machinery.\"\nFigure Legend Update: In relevant figure legends, we have included: \"protease inhibitor cocktail (PI) to\ninhibit lysosomal protease activity\" (Page 9, Line 163; Page 12, Line 215; Page 20, Line 396; Page 69,\nLines 1629-1633; Line 8, Supplementary Figure 7 legend; Line 11, Supplementary Figure 10 legend).\nPoint-to-Point Response to Reviewers’ Comments\nWe appreciate this suggestion as it improves the methodological rigor of our study. The changes have been\nmade consistently across all relevant experimental descriptions.\n4- Depth of Immunophenotyping in Syngeneic Models:\nThe authors have been able to address previous reviewer recommendations regarding the\nimmunological mechanisms of CMA modulation. An inclusion of a short discussion sentence in\nthe revised manuscript acknowledging the potential role of other significant immune cells beyond\nCD8+ T cells (Tregs, MDSCs, TAMs), although briefly, would provide a complete immunological\nperspective, especially since potential future research might venture into exploring these avenues.\nResponse: We sincerely appreciate the insightful suggestions provided by the reviewer. In\nresponse to this significant point, we have incorporated a brief discussion in the revised\nmanuscript (Page 35, Lines 725-728), which states: \"While our study primarily focuses on CD8+\nT cells, we acknowledge that other immune cell populations, including Tregs, MDSCs, and TAMs,\nmay also participate in CMA-mediated immune modulation within the tumor microenvironment.\"\nReviewer #4:\nIn the revised version, the new experiments added, provide evidence that TCS1/2 and TET3 are\nCMA substrates in human GSCs and could be used as therapeutic targets. However, there is not\nyet a clear link between the described mechanism and the inhibitor used. Additionally, several\nmajor points needs to be addressed so that the work meets the standards of the journal.\nResponse: We sincerely appreciate the reviewer's insightful comments and constructive\nsuggestions, which have significantly strengthened our manuscript. Below we provide point-by-\npoint responses to each concern, with all recommended revisions implemented.\n1. The authors state in the introduction that “the specific role of CMA in these cells [GSCs] is yet\nto be elucidated”, and in the first results section, they conclude that “these result//s indicated that\nCMA is required for GSC stemness and tumorigenicity”. However, these results are just a\nvalidation of the study already published in 2022 by Auzmendi-Iriarte et al. (doi.org/10.1158/0008-\n5472.CAN-21-2161). Although they could include these results as supplementary material to give\ncontext to the subsequent analysis of LAMP2A stability in the lysosomes of GSCs, the authors of\nthis paper cannot include the first section of results as novel. In addition, they should elaborate\nfurther in the introduction and discussion on the work of Auzmendi-Iriarte et al. to expand the\ninformation. Overall, the authors should restructure the article in detail to actually report the\nmechanism they are describing.\nResponse: We sincerely appreciate the reviewer's valuable feedback. To address these\ncomments, we have substantially expanded the discussion of Auzmendi-Iriarte et al.'s important\nwork in both the Introduction (Page 5, Lines 68-72) and Discussion (Pages 33-34, Lines 681-686)\nsections. Concurrently, we have adjusted the strength of our claims regarding CMA's role in GSC\nstemness and tumorigenicity to more accurately reflect the current evidence.\nPoint-to-Point Response to Reviewers’ Comments\n2. With the new experiments provided, the authors show that the inhibitor PPD does not affect the\nphosphorylation state of LAMP2A and that it inhibits its multimerization (Supplementary Fig. 8d-\ng). However, these results increase the lack of connection between the MST4-LAMP2A regulation\nand the use of PPD in the study. The results obtained with the inhibitor PPD both in vitro and in\nvivo are very promising, but do not justify why the authors have included this inhibitor in their\ntherapeutical strategies when, apparently, it has no relevance on the described mechanism. It\nwould be more appropriate for authors to submit the work to an autophagy journal, as without a\nspecific inhibitor with effect on the in vivo models, the described regulatory mechanism lacks\ntranslational impact.\nResponse: We sincerely appreciate the reviewer's insightful critique regarding the mechanistic\nconnection between MST4-LAMP2A regulation and our use of PPD inhibitor. In response to these\nimportant comments, we would like to clarify the following points:\n1. PPD is identified as an inhibitor of chaperone-mediated autophagy (CMA) that acts by\npreventing the multimerization of LAMP2A (Dong, Rui-Fang et al. Br J Pharmacol.\n2025;182(10):2287-2309). Although PPD does not directly influence LAMP2A phosphorylation\n(Supplementary Fig. 11d-g), it inhibits LAMP2A multimerization, a downstream consequence of\nMST4-LAMP2A signaling in CMA. Our data show that knocking down MST4 or using a phospho-\nnull LAMP2A mutant (3A) significantly blocks LAMP2A multimerization (Fig. 2k, Supplementary\nFig. 3g), demonstrating that PPD functions similarly to MST4 inhibition or preventing LAMP2A\nphosphorylation.\n2. Given our in vitro findings that radiation significantly increases MST4 expression and enhances\nthe MST4-LAMP2A interaction (Supplementary Fig. 2i), radiation therapy should similarly elevate\nMST4 expression and strengthen the MST4-LAMP2A interaction in vivo. This enhanced\ninteraction would promote LAMP2A phosphorylation, multimerization, and ultimately increase\nCMA activity in in vivo models. Since PPD effectively inhibits LAMP2A multimerization—a\ndownstream consequence of LAMP2A phosphorylation—we investigated the effects of this CMA\ninhibitor when combined with radiation therapy and anti-PD-1 monoclonal antibody (mAb)\ntreatment in the GL261 and CT-2A C57BL/6 intracranial glioblastoma (GBM) models. Further\nstudies are required to determine whether PPD suppresses CMA activity by inhibiting radiation-\ninduced LAMP2A multimerization in in vivo models.\n3. We have revised the manuscript to include a discussion on the limitations of PPD as a CMA\ninhibitor. Additionally, we have proposed future investigations into more selective CMA-targeting\ninhibitors, such as CIM7, to enhance our therapeutic strategies and translational impact (Page 36,\nLines 732-740).\nWe are grateful for the opportunity to strengthen these aspects of our work and appreciate the\nreviewer's constructive feedback.\n3. In the response to reviewers’ comments, the authors state that they have not been able to\nperform the in vivo model with human GSC lines because “human GSCs are unable to engraft in\nimmunocompetent C57BL/6 mice due to species-specific barriers”. However, they have also not\ntried the different therapeutic strategies in BALB/c nude mice that could perfectly well develop\nhuman GSCs tumors. In addition, there are other authors who have used human glioblastoma\ncells lines to develop a tumor in immunocompetent mice (Molina et al., 2022,\ndoi.org/10.3389/fcell.2022.797945; Garcia et al., 2014,\ndoi.org/10.1186/1471-2407-14-923).\nPoint-to-Point Response to Reviewers’ Comments\nTherefore, without the in vivo experiments using human glioma cells the study lacks the\ntranslational impact necessary to get the standars of Nature Communications.\nResponse: We are deeply grateful for the reviewer's thoughtful comments regarding the\nutilization of human GSC models in immunocompetent murine systems. Our preliminary\ninvestigations with multiple human GSC lines in C57BL/6 mice demonstrated an inability to\nestablish tumors in immunocompetent hosts, consistent with established species-specific barriers.\nTo address this technical constraint, we developed sophisticated in vitro co-culture platforms\ninterrogating human GSC-CD8+ T cell interactions. Quantitative flow cytometric analyses revealed\nthat genetic perturbation of either LAMP2A or MST4 significantly augmented cytotoxic T\nlymphocyte responses, as demonstrated by: (1) enhanced polyfunctional cytokine production\n(TNF-α, Fig. 6b; IFN-γ, Fig. 6c), (2) elevated effector molecule expression (Granzyme B, Fig. 6d),\nand (3) increased GSC apoptosis (Supplementary Fig. 9f).\nThese limitations and future directions have been addressed in the revised Discussion section\n(Page 35-36, Lines 728-732). The reviewer's constructive critique has substantially strengthened\nour experimental framework and will undoubtedly inform subsequent translational studies.\n4. Although the authors have followed the request to include papers on the physiological\nsignificance of CMA, they have not updated the introduction in accordance with the new works\ncited.\nResponse: We sincerely appreciate the reviewer’s valuable feedback regarding the inclusion of\nrecent literature on CMA’s physiological significance. In response to this constructive suggestion,\nwe have now cited two of the most recent studies on CMA’s physiological significance (Reynolds\nCA et al., PNAS, 2024; Khawaja RR et al., Nature Aging, 2025) in the Introduction section.\n5. Fig. 5A- Authors show some redundant analyses, previously published by Auzmendi-Iriarte et\nal., 2022 (doi.org/10.1158/0008-5472.CAN-21-2161), which are the original source of the\nRNAseq study used.\nResponse: Thank you for your insightful feedback regarding the analyses presented in Fig. 5A.\nWe acquired the GSE181556 dataset (Auzmendi-Iriarte et al., 2022) and utilized the normalized\nexpression matrix to analyze LAMP2A-associated signaling pathways using Gene Set\nEnrichment Analysis (GSEA). The original study by Auzmendi-Iriarte et al. (2022, doi:\n10.1158/0008-5472.CAN-21-2161) employed RNAseq analysis based on the Kyoto Encyclopedia\nof Genes and Genomes (KEGG) pathway framework. We have now explicitly cited this\nfoundational work in the revised manuscript (Page 18, Lines 356-358) and clearly specified the\nspecified the\nRNAseq data source in the Methods section (Page 44, Line 902)."}]

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