The Lysosome–Cathepsin Axis in Pancreatic Cancer: Mechanisms of Stromal Remodeling, Immune Evasion, and Therapy Resistance

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors present a comprehensive review focusing on the lysosome–cathepsin axis in PDAC, with particular emphasis on stromal remodeling, immune evasion, autophagy-related adaptation, and therapy resistance. The topic is timely and clinically relevant, especially given the growing interest in lysosomal biology and tumor microenvironment–driven therapeutic resistance in PDAC. The manuscript is generally well organized, the figures are informative, and the authors successfully summarize a broad body of literature linking cathepsins to extracellular matrix remodeling, immune dysfunction, and metabolic stress adaptation.     However, the review would benefit from deeper critical synthesis, clearer differentiation between PDAC-specific findings and pan-cancer extrapolations, and a stronger translational perspective regarding therapeutic applicability.

Major concerns

1. The major limitation of the manuscript is that much of the discussion reads as a sequential summary of published findings rather than a critically integrated mechanistic review. Multiple sections separately discuss ECM remodeling, antigen presentation, autophagy, macrophage polarization, YAP1 regulation, and therapy resistance, yet the manuscript does not sufficiently unify these observations into a coherent PDAC-specific biological framework. In several areas, the review also does not clearly distinguish between mechanisms directly demonstrated in PDAC versus those extrapolated from other tumor models or based primarily on indirect evidence. This issue is particularly relevant in the discussions of CTSS-mediated antigen presentation, ferroptosis-associated CTSB signaling, lysosomal immune regulation, and mechanotransduction pathways involving YAP1. Some conclusions are presented in a manner suggesting established causality, whereas the available evidence remains associative, preclinical, or context-dependent. The manuscript would therefore be strengthened by introducing a clearer hierarchy of evidence, explicitly identifying which mechanisms are firmly supported in PDAC and which remain speculative or incompletely validated. A more critical discussion of conflicting findings and unresolved biological questions would substantially improve the scientific depth and authority of the review.
2. The translational and therapeutic discussion is underdeveloped relative to the biological complexity described. Although hydroxychloroquine, lysosomal inhibitors, and cathepsin inhibitors are discussed, the review does not adequately analyze why lysosome-targeting strategies have thus far demonstrated limited clinical efficacy in PDAC despite encouraging preclinical data. Important translational barriers—including desmoplastic stromal restriction of drug delivery, intratumoral heterogeneity of lysosomal dependency, compensatory TFEB-driven lysosomal adaptation, and the dual roles of lysosomal pathways in both tumor and immune cells—deserve more detailed evaluation. In addition, the manuscript insufficiently discusses the potential immunological consequences of broad cathepsin inhibition, particularly whether suppression of lysosomal proteolysis could impair antigen presentation or T-cell priming in dendritic cells and macrophages. The feasibility of combining lysosomal targeting with immunotherapy, chemotherapy, or stromal modulation strategies is also not critically addressed. Finally, the review lacks a clear perspective regarding which cathepsins are realistically druggable therapeutic targets versus those more suitable as prognostic or immune-context biomarkers. Adding a dedicated section discussing unresolved translational challenges, therapeutic limitations, and future research priorities would significantly enhance the clinical relevance of the manuscript.

Minor comments:

1. Several sections are excessively dense and would benefit from condensation to improve readability, particularly Sections 3.2–3.4.
2. A summary table categorizing individual cathepsins according to:

• cellular source,
• major biological function,
• prognostic significance,
• therapeutic relevance in PDAC

3. Some mechanistic statements rely heavily on preclinical or indirect evidence and should be more cautiously phrased.
4. The manuscript would benefit from a clearer discussion distinguishing:

• intracellular lysosomal functions,
• extracellular proteolytic activity,
• immune-regulatory effects of cathepsins.

5. Figure 3 is conceptually useful but could be strengthened by incorporating clinically relevant combination strategies (e.g., chemotherapy, immunotherapy, stromal targeting).

 

Overall, this review addresses an important and emerging area in pancreatic cancer biology and contains a substantial amount of useful information. Nevertheless, the current version remains predominantly descriptive and would benefit from deeper mechanistic integration, more balanced interpretation of the evidence, and stronger translational insight.

Comments for author File: Comments.pdf

Author Response

We sincerely thank the reviewer for the positive evaluation of our manuscript and for the constructive and insightful suggestions. We are grateful for the reviewer’s recognition that the topic is timely and clinically relevant and that the manuscript is generally well organized and informative. We have carefully revised the manuscript to address all major and minor concerns. In particular, we have strengthened the critical synthesis, clarified the hierarchy of evidence, better distinguished PDAC-specific findings from broader cancer or lysosomal biology, expanded the translational discussion, and improved the readability and conceptual structure of several sections.

Major concern 1

Reviewer comment:
The reviewer noted that the manuscript reads in places as a sequential summary of published findings rather than as a critically integrated mechanistic review. The reviewer also requested a clearer PDAC-specific biological framework and a more explicit distinction between mechanisms directly demonstrated in PDAC and those extrapolated from other tumor models or indirect evidence, especially regarding CTSS-mediated antigen presentation, ferroptosis-associated CTSB signaling, lysosomal immune regulation, and YAP1-related mechanotransduction.

Response:
We thank the reviewer for this important comment. We agree that the original version did not sufficiently emphasize an integrated mechanistic framework or consistently distinguish between levels of evidence. In the revised manuscript, we have substantially reframed the review around the concept of the lysosome–cathepsin axis as a PDAC-specific adaptive stress network rather than as a collection of isolated cathepsin functions.

Specifically, we now define the lysosome–cathepsin axis as an integrated system involving lysosomal biogenesis, vesicular trafficking, acidification, cathepsin maturation and activation, substrate degradation, extracellular secretion, and downstream signaling pathways. We explain how oncogenic KRAS signaling, nutrient limitation, hypoxia, stromal stiffness, inflammatory signaling, and therapy-induced stress converge on this system to regulate autophagy, macropinocytosis, ECM remodeling, antigen presentation, macrophage polarization, immune exclusion, and therapy resistance.

To address the reviewer’s concern about the hierarchy of evidence, we added explicit statements distinguishing:

1. mechanisms directly demonstrated in PDAC models or patient-derived PDAC material,
2. mechanisms supported by PDAC-associated observations but not yet fully causally validated, and
3. mechanisms inferred from broader cancer or lysosomal biology.

This distinction is now incorporated particularly in the sections discussing CTSS-mediated antigen presentation, CTSB-associated ferroptosis, YAP1-related mechanotransduction, extracellular cathepsin activity, and lysosomal immune regulation. We have also revised the wording of several mechanistic statements to avoid implying established causality where the evidence remains associative, preclinical, or context-dependent.

For example, we now clarify that CTSB/CTSL-associated ECM remodeling, KRAS/TFEB-linked lysosomal remodeling, CTSD-associated gemcitabine resistance, and autophagy-dependent MHC-I degradation are among the better-supported PDAC-specific mechanisms. In contrast, CTSS-driven immune modulation, CTSZ/X-associated integrin signaling, CTSB-related ferroptosis, and stromal-mechanics-dependent YAP1 regulation are now presented more cautiously as promising but incompletely validated mechanisms. We also expanded the discussion of unresolved questions and emphasized the need for spatial transcriptomics, activity-based protease profiling, lysosomal flux assays, and single-cell approaches to define compartment-specific cathepsin functions in human PDAC.

Major concern 2

Reviewer comment:
The reviewer noted that the translational and therapeutic discussion was underdeveloped relative to the biological complexity described. The reviewer requested a more detailed analysis of why lysosome-targeting strategies have shown limited clinical efficacy in PDAC, including stromal drug-delivery barriers, heterogeneity of lysosomal dependency, compensatory TFEB-driven lysosomal adaptation, and the dual roles of lysosomal pathways in tumor and immune cells. The reviewer also requested discussion of the immunological consequences of broad cathepsin inhibition, feasibility of combination strategies, and distinction between therapeutically druggable cathepsins and those better suited as biomarkers.

Response:
We fully agree with the reviewer and have substantially expanded the translational and therapeutic sections. In the revised manuscript, we now provide a more critical analysis of the limited clinical efficacy of lysosome-targeting approaches in PDAC despite strong preclinical rationale.

We discuss several major barriers, including dense desmoplastic stroma, poor vascularization, elevated interstitial fluid pressure, heterogeneous perfusion, extracellular acidosis, incomplete intratumoral accumulation of chloroquine/hydroxychloroquine, metabolic plasticity, and compensatory TFEB-driven lysosomal biogenesis. We also added discussion of how lysosomal stress can paradoxically activate adaptive lysosomal transcriptional programs, allowing PDAC cells to restore degradative capacity and survive therapeutic pressure.

We further expanded the discussion of clinical and translational studies involving hydroxychloroquine/chloroquine, gemcitabine/nab-paclitaxel combinations, ERK/autophagy inhibition strategies, next-generation lysosomal inhibitors, cathepsin inhibitors, and cathepsin-responsive drug-delivery systems. This revision now emphasizes that the limited efficacy of lysosome-targeting therapy likely reflects not failure of the pathway as a pathogenic driver, but rather the difficulty of therapeutically suppressing a highly adaptive, spatially heterogeneous stress-response system within the PDAC tumor microenvironment.

In response to the reviewer’s concern regarding immune consequences, we added a more balanced discussion of broad cathepsin inhibition. We now explicitly state that although inhibition of tumor-associated CTSB, CTSL, or CTSS may reduce invasion, stromal remodeling, and immune exclusion in some contexts, broad suppression of lysosomal proteolysis could also impair antigen processing and T-cell priming in dendritic cells and macrophages. Therefore, we emphasize that future approaches should ideally use compartment-selective delivery, activity-based pharmacodynamic monitoring, intermittent dosing, or protease-activatable drug-delivery systems to avoid sustained suppression of antigen-presenting-cell function.

We also added a clearer perspective on therapeutic versus biomarker relevance of individual cathepsins. CTSB, CTSL, CTSS, and CTSD are now discussed as the most plausible therapeutic or combination targets, although each has important limitations related to redundancy, immune function, and compartment-specific activity. CTSE is presented primarily as a diagnostic or theranostic biomarker because of its disease-enriched expression in pancreatic precursor lesions and PDAC. CTSW is now described as an immune-context biomarker reflecting cytotoxic lymphocyte infiltration rather than as a tumor-cell drug target. CTSC, CTSK, CTSF, and CTSZ/X are discussed more cautiously as context-dependent or incompletely validated candidates requiring further PDAC-specific investigation.

Finally, we added a dedicated discussion of unresolved translational challenges and therapeutic limitations, including species differences between mouse models and human PDAC, stromal and immune heterogeneity, protease redundancy, insufficient pharmacodynamic validation, and the need for biomarker-guided patient stratification.

Minor comment 1

Reviewer comment:
Several sections are excessively dense and would benefit from condensation to improve readability, particularly Sections 3.2–3.4.

Response:
We thank the reviewer for this helpful suggestion. We revised Sections 3.2–3.4 to improve readability and reduce excessive density. In particular, we reorganized the text to better separate cathepsin trafficking and activation, molecular regulation, and extracellular/non-canonical functions. We also revised several long mechanistic passages and removed or condensed repetitive statements where appropriate.

Minor comment 2

Reviewer comment:
The reviewer requested a summary table categorizing individual cathepsins according to cellular source, major biological function, prognostic significance, and therapeutic relevance in PDAC.

Response:
We have added a new summary table entitled “Functional and Clinical Classification of Cathepsins in PDAC.” This table categorizes major PDAC-associated cathepsins according to their predominant cellular source, main biological function, prognostic significance, therapeutic relevance, and supporting references. The table includes CTSB, CTSL, CTSS, CTSD, CTSE, CTSZ/X, CTSC, CTSW, CTSK, and CTSF. We believe this addition improves clarity and helps distinguish cathepsins that are plausible therapeutic targets from those that are more appropriately viewed as diagnostic, prognostic, stromal, or immune-context biomarkers.

Minor comment 3

Reviewer comment:
Some mechanistic statements rely heavily on preclinical or indirect evidence and should be more cautiously phrased.

Response:
We agree and have revised the manuscript accordingly. Throughout the revised version, we now use more cautious wording when discussing mechanisms that are not yet firmly established in PDAC. For example, CTSS-mediated antigen-processing effects, CTSB-related ferroptosis, CTSZ/X-associated integrin signaling, inflammatory transcriptional regulation of cathepsins, and YAP1-associated mechanotransduction are now described as plausible, emerging, context-dependent, or incompletely validated, depending on the strength of the evidence. We also explicitly identify where evidence derives from non-PDAC systems, PanNET models, broader cancer biology, or preclinical studies rather than human PDAC.

Minor comment 4

Reviewer comment:
The manuscript would benefit from a clearer discussion distinguishing intracellular lysosomal functions, extracellular proteolytic activity, and immune-regulatory effects of cathepsins.

Response:
We thank the reviewer for this suggestion. We have revised the manuscript to more clearly distinguish these three functional categories. Intracellular lysosomal functions are now discussed in relation to autophagy, macropinocytosis, nutrient recycling, lysosomal biogenesis, cathepsin maturation, antigen processing, MHC-I degradation, and therapy-induced stress adaptation. Extracellular proteolytic activity is discussed separately in relation to lysosomal exocytosis, cathepsin secretion, pericellular matrix degradation, basement membrane disruption, invasion, and stromal remodeling. Immune-regulatory functions are now more clearly presented in the context of antigen presentation, macrophage polarization, cytokine signaling, immune exclusion, cytotoxic lymphocyte infiltration, and the potential consequences of cathepsin inhibition for APC function.

Minor comment 5

Reviewer comment:
Figure 3 is conceptually useful but could be strengthened by incorporating clinically relevant combination strategies such as chemotherapy, immunotherapy, and stromal targeting.

Response:
We agree with the reviewer and have revised the Figure 3 legend and conceptual framing to include clinically relevant combination strategies. The revised version now explicitly incorporates chemotherapy, immune checkpoint blockade, stromal modulation or vascular normalization, MAPK pathway inhibition, and cathepsin-activatable drug-delivery systems. We also modified the accompanying discussion to emphasize that cathepsin or lysosome targeting is unlikely to be effective as a stand-alone strategy in PDAC and will most likely require rational, biomarker-guided combination approaches that address adaptive lysosomal biogenesis, stromal drug-delivery barriers, immune exclusion, and metabolic rewiring.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The review by N. Mazej Jeram et al. analyzed an important problem in basic and clinical oncology, namely, the roles of proteolytic enzymes in the pathogenesis of pancreatic cancer. This tumor type is one of the most intractable, therefore every step forward in understanding its biology and advancing treatment strategies is valuable.

The authors presented a broad panorama of proteolytic pathways and their regulation as major mechanisms in pancreatic cancer. The material is detailed, well documented and presented at a high professional level. By and large, the review is a nicely written work expectedly appreciated by the readership of Biomolecules.

One concern before the manuscript can be recommended for publication: the authors rightly stated that, at present, the results of therapeutic targeting of lysosomal enzymes have not been encouraging. Would it be possible to address this issue by a detailed analysis of a couple of selected refs. in sections 5.1-5.3)?  What are the advantages and disadvantages of treatment approaches presented in the cited literature, I mean experimental design, drug concentrations, readouts, etc.? This anlaysis would clarify the reasons for failures. Yes, the proteolytic axis is  justified pathogenetically but is there a hope for its successful targeting or treatment of this tumor should involve different mechanism(s)? 

Author Response

We sincerely thank the reviewer for the positive evaluation of our manuscript and for recognizing the relevance of the lysosome–cathepsin/proteolytic axis in pancreatic cancer. We also appreciate the important suggestion to more critically discuss why therapeutic targeting of lysosomal enzymes has so far shown limited clinical success despite strong biological rationale.

Reviewer comment:
The reviewer asked us to expand Sections 5.1–5.3 by analyzing selected therapeutic studies in more detail, including the advantages and disadvantages of the approaches, experimental design, readouts, and possible reasons for limited efficacy. The reviewer also asked whether the proteolytic axis remains a realistic therapeutic target or whether alternative mechanisms should be prioritized.

Response:
We thank the reviewer for this valuable comment. In the revised manuscript, we have expanded Sections 5.1–5.3 to provide a more critical translational analysis of lysosome- and cathepsin-targeted approaches in PDAC.

We now discuss representative clinical and preclinical studies in more detail, including hydroxychloroquine/chloroquine-based lysosomal inhibition, HCQ combined with gemcitabine/nab-paclitaxel, ERK/autophagy inhibition strategies, direct cathepsin inhibitors, and cathepsin-responsive prodrug or nanoparticle systems. We emphasize that these approaches are biologically well justified and, in some cases, show pharmacodynamic activity or improved response-related endpoints. However, their clinical efficacy is limited by several factors, including weak and nonspecific lysosomal inhibition by HCQ/CQ, poor drug penetration through the dense desmoplastic stroma, heterogeneous tumor perfusion, incomplete intratumoral target inhibition, extracellular acidosis, compensatory TFEB-driven lysosomal biogenesis, metabolic plasticity, and protease redundancy.

We also added a new summary table comparing representative lysosome–cathepsin-targeting strategies. This table outlines the experimental or clinical design, main readouts, advantages, limitations, and translational lessons of selected approaches. We believe this directly addresses the reviewer’s request for clearer analysis of why several promising preclinical strategies have not translated into durable clinical benefit.

In addition, we expanded the discussion of direct cathepsin inhibition, noting that inhibition of CTSB, CTSL, CTSS, or broad cathepsin activity can reduce invasion, ECM remodeling, and tumor growth in experimental models. However, we now also emphasize the limitations of this strategy, including overlapping substrate specificity, compensatory protease activity, limited inhibitor selectivity, and the possibility of impairing physiological lysosomal and immune functions such as antigen processing.

Finally, we clarified our conclusion regarding therapeutic potential. We agree that the proteolytic axis remains pathogenetically justified, but the revised manuscript now states more clearly that it is unlikely to be successfully targeted as a stand-alone strategy. Instead, future approaches will likely require biomarker-guided combinations with chemotherapy, MAPK pathway inhibition, stromal modulation or vascular normalization, metabolic targeting, immunotherapy, or cathepsin-activatable drug delivery systems. Thus, the lysosome–cathepsin axis should be viewed as an important component of a broader adaptive resistance network rather than as an isolated therapeutic target.

Reviewer 3 Report

Comments and Suggestions for Authors

This review article focused the lysosome-cathepsin axis in pancreatic cancer. This review is interesting and can provide important and comprehensive information the role of lysosome in pancreatic cancer. 

1. Detailed explanation in the normal function and disfunction in pancreatic cancer of antigen presentation of MHC Class I by lysosome is desirable.  
2. The explanation of the molecular mechanism of cathepsins expression changes in pancreatic cancer is needed. 
3. Significance of cathepsin expression change in prognosis was described. The description of its molecular mechanism and therapeutic implication are required. 

Author Response

We thank the Reviewer for the constructive comments and for identifying areas where additional mechanistic explanation would improve the manuscript.

Reviewer comment: A detailed explanation of the normal function and dysfunction in pancreatic cancer of antigen presentation of MHC class I by lysosome is desirable.

Response: We expanded Section 4.2 to describe the normal MHC-I antigen-presentation pathway and to clarify the specific role of lysosomes in MHC-I turnover rather than classical peptide generation. The revised text explains that endogenous proteins are processed by the proteasome, peptides are transported into the ER by TAP1/TAP2, loaded onto MHC-I with beta-2 microglobulin and peptide-loading machinery, and transported to the plasma membrane for CD8+ T-cell recognition. We then explain how PDAC disrupts this process: MHC-I complexes can be diverted from the cell surface into autophagosomal and lysosomal compartments through NBR1-dependent pathways, leading to lysosomal degradation, reduced surface MHC-I, impaired CD8+ T-cell recognition, and immune evasion. We also discuss evidence that autophagy or lysosomal inhibition can restore surface MHC-I and improve CD8+ T-cell-mediated tumor control in PDAC models. Figure 2 and Sections 2.3 and 4.2 were revised to support this explanation.

Reviewer comment: The molecular mechanism of cathepsin expression changes in pancreatic cancer is needed.

Response: We added a new Section 3.3, “Molecular Regulation of Cathepsin Expression in PDAC.” This section explains that cathepsin dysregulation in PDAC results from coordinated oncogenic, metabolic, inflammatory, and lysosomal stress programs rather than one isolated transcriptional event. We discuss KRAS–MAPK signaling, TFEB/MiT-TFE-mediated lysosomal biogenesis, therapy-induced autophagy, hypoxia and extracellular acidosis, inflammatory cytokine pathways, epigenetic mechanisms such as CTSK hypomethylation, post-transcriptional regulation, altered lysosomal trafficking, zymogen maturation, secretion, lysosomal pH, and cystatin/cathepsin balance. We also clarify that increased expression does not necessarily equal increased enzymatic activity, because proteolytic output depends on localization, activation state, pH, substrate accessibility, secretion, and endogenous inhibitors.

Reviewer comment: The significance of cathepsin expression change in prognosis was described, but the molecular mechanism and therapeutic implication are required.

Response: We expanded Section 3.5 to connect prognostic associations with mechanistic and therapeutic interpretation. We now explain that high CTSB and CTSL expression likely reflects increased pericellular proteolysis, ECM degradation, basement membrane disruption, release of matrix-bound growth factors, invasive signaling, and stem-like or stress-adaptive phenotypes, thereby linking these cathepsins to lymphatic invasion, recurrence, and poor survival. We clarify that high CTSD expression is associated with gemcitabine resistance and lysosomal stress tolerance, whereas CTSS may reflect both ECM remodeling and immune regulation. Conversely, low CTSW likely reflects reduced cytotoxic lymphocyte infiltration rather than tumor-cell-intrinsic protease activity. Therapeutically, we distinguish cathepsins that may be targeted by selective or multi-cathepsin inhibitors or lysosome-directed combinations (CTSB, CTSL, CTSS, CTSD) from cathepsins better suited as diagnostic, theranostic, prognostic, or immune-context biomarkers (CTSE, CTSW, CTSF). These points are summarized in the newly added Table 1 and further discussed in Sections 5.2–5.5.

Reviewer 4 Report

Comments and Suggestions for Authors

The review entitled “The Lysosome–Cathepsin Axis in Pancreatic Cancer: Mechanisms of Stromal Remodeling, Immune Evasion, and Therapy Resistance” systematically summarizes the roles of the lysosome–cathepsin axis in pancreatic ductal adenocarcinoma (PDAC), covering stromal remodeling, immune evasion, and therapeutic resistance. The topic is highly relevant to current PDAC research and clinical translation. The manuscript is well-structured, with a clear logical flow and comprehensive literature coverage. However, several aspects require revision to enhance scientific rigor, clarity, and translational depth prior to acceptance for publication. 1. The term of the “lysosome–cathepsin axis” is used throughout the manuscript but is not formally defined. A clear mechanistic definition of this axis，including its signaling, trafficking, proteolytic, and crosstalk modules，should be provided. 2. In the Section 2.2.1，the authors describe the link between hypoxia/low pH and extracellular cathepsin activation. To strengthen causality, I suggest adding quantitative or mechanistic evidence, such as pH–activity kinetics of CTSB/CTSL within the PDAC tumor microenvironment (TME). 3. In the Section 5.1, the clinical data for hydroxychloroquine (HCQ) and chloroquine (CQ) are outdated. I recommend incorporating recent phase II/III trial results, along with explanations for their limited clinical efficacy (e.g., poor tissue penetration, TFEB-driven compensatory mechanisms). 4. In Section 6, the authors repeat some content from earlier sections. This section should be condensed to emphasize unanswered questions and future research priorities. 5. A brief note on species differences between mouse models and human PDAC regarding cathepsin expression and function should be added.

Author Response

We thank the Reviewer for the positive assessment and for the specific suggestions that helped us improve scientific rigor, mechanistic clarity, and translational relevance.

Reviewer comment: The term “lysosome–cathepsin axis” is used throughout the manuscript but is not formally defined. A clear mechanistic definition including signaling, trafficking, proteolytic, and crosstalk modules should be provided.

Response: We added a formal definition of the lysosome–cathepsin axis in Section 3.2. The revised definition describes the axis as an integrated network connecting lysosomal biogenesis, vesicular trafficking, acidification, cathepsin maturation and activation, substrate degradation, extracellular secretion, and downstream signaling. We also clarify crosstalk with mTORC1, TFEB/TFE3, autophagy, inflammasomes, mitochondrial quality-control pathways, ECM remodeling, immune regulation, apoptosis, and lysosomal stress signaling. This definition is then used consistently throughout the revised manuscript.

Reviewer comment: In Section 2.2.1, the link between hypoxia/low pH and extracellular cathepsin activation should be strengthened with quantitative or mechanistic evidence, such as pH–activity kinetics of CTSB/CTSL within the PDAC TME.

Response: We revised Section 2.2.1 to provide a more mechanistic and quantitative explanation. The text now states that CTSB and CTSL achieve optimal catalytic activity in acidic endolysosomal compartments, approximately pH 4.5–5.5, while remaining functionally active in the moderately acidic extracellular PDAC TME, where pH commonly decreases to approximately 6.5–6.8 in hypoxic and poorly perfused regions. We also added explanation that localized pericellular acidification, interactions with cell-surface or ECM components, and sustained secretion of active enzymes can support persistent extracellular CTSB/CTSL-mediated proteolysis even though the extracellular pH is less acidic than the lysosomal lumen.

Reviewer comment: The clinical data for HCQ/CQ are outdated. Please incorporate recent phase II/III trial results and explain limited efficacy, including poor tissue penetration and TFEB-driven compensatory mechanisms.

Response: We updated Section 5.1 with more recent clinical and translational findings on HCQ/CQ-based strategies, including phase II metastatic and neoadjuvant studies and the ERK inhibitor LY3214996 plus HCQ trial. We now explain that HCQ combinations can show biological activity, CA19-9 or response-rate effects, pathological responses, and pharmacodynamic evidence of autophagy inhibition, but have generally failed to produce robust survival benefit in advanced PDAC. We added mechanistic explanations for limited efficacy, including poor intratumoral accumulation, dense stromal barriers, hypovascularity, elevated interstitial pressure, extracellular pH effects on lysosomotropic drug distribution, incomplete autophagy suppression, compensatory TFEB-driven lysosomal biogenesis, and metabolic rewiring. These points are also summarized in Table 2 and discussed in Section 5.5.

Reviewer comment: Section 6 repeats content from earlier sections and should be condensed to emphasize unanswered questions and future research priorities.

Response: We revised the Conclusions and Future Perspectives section to reduce repetition and focus on unresolved questions. The revised section emphasizes compartment-specific roles of cathepsins in tumor cells, CAFs, TAMs, and other immune populations; functional redundancy; temporal and spatial dynamics of lysosomal adaptation during therapy; the need for spatial transcriptomics, single-cell multi-omics, functional imaging, activity-based profiling, and human-relevant models; and the importance of biomarker-guided combination strategies rather than stand-alone lysosome/cathepsin inhibition.

Reviewer comment: A brief note on species differences between mouse models and human PDAC regarding cathepsin expression and function should be added.

Response: We added this point in Section 5.2 and expanded it in Section 5.5. The revised text notes that murine and human tissues can differ in cathepsin expression patterns, substrate specificity, functional redundancy, stromal architecture, immune-cell infiltration, ECM composition, vascularity, and drug penetration. We therefore caution that preclinical efficacy data for cathepsin- or lysosome-targeting strategies should be interpreted carefully, especially when conclusions depend on stromal or immune-cell proteolysis.

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

The author’s revision reply meets my all requirements.