Multiscale imaging on proton pump-driven acidity for assessing tumor progression and metastasis

Domain: physics
Published: 2026-01-16
Editorial outcome: major_revision

Abstract
An acidic tumor microenvironment (TME), a hallmark of cancer progression, promotes tumor growth, invasion, and metastasis. Detecting and targeting tumor acidity have emerged as key frontiers in early cancer diagnosis and treatment. However, current approaches lack sensitive and specific methods to visualize and quantify tumor acidity in vivo across different stages of tumor development. Here we show that ATP6V0C, a subunit of the V-ATPase responsible for proton transmembrane transport, is a critical mediator of TME acidification. High ATP6V0C expression correlates with hepatocellular carcinoma (HCC) progression and metastasis. To enable real-time assessment of tumor acidity, we develop a pH-responsive ratiometric photoacoustic sensor (PPS) that dynamically monitors TME acidity throughout HCC initiation, progression, and metastasis. Using PPS-assisted photoacoustic molecular imaging, we visualize the spatiotemporal evolution of TME acidity from early tumor foci to solid tumors. PPS exhibits enhanced photothermal effects under acidic conditions, and its combination with the proton pump inhibitor esomeprazole synergistically suppresses tumor growth. Overall, PPS-assisted photoacoustic molecular imaging provides a sensitive and specific approach to characterize tumor initiation and progression, supporting its potential application in early diagnosis and therapeutic strategies that target the acidic TME.

Introduction
The initiation and progression of cancer are closely associated with alterations in the tumor microenvironment (TME), particularly localized acidification arising from metabolic reprogramming and vascular perfusion abnormalities1. A major contributor to this acidification is enhanced glycolysis in tumor cells, commonly known as the “Warburg effect”, wherein cells preferentially rely on glycolysis for adenosine triphosphate production even under normoxic conditions2, resulting in the accumulation of lactic acid and protons. These acidic metabolites are exported via proton pumps such as V-ATPases and lactate transporters, thereby lowering extracellular pH3,4. In parallel, the structurally abnormal and poorly perfused tumor vasculature impairs proton (H+) clearance, producing a TME pH typically between 6.5 and 6.85. This acidic milieu is not only a hallmark of cancer progression but also actively promotes tumor growth, invasion, and metastasis4,6, making it an attractive target for diagnostic and therapeutic strategies. However, TME acidity is highly heterogeneous and dynamically evolves in early-stage tumors and metastatic lesions6,7. In these stages, low-level acidification and the complex structure of the extracellular matrix complicate both detection and therapeutic targeting8,9. Thus, there is a pressing need for highly sensitive, specific, and noninvasive imaging tools capable of detecting and characterizing TME acidity (i.e., H+ ions), particularly to improve early cancer diagnosis and therapeutic outcomes.

Biomedical imaging technologies are indispensable for tumor diagnosis and therapeutic evaluation10–12. Conventional modalities such as magnetic resonance imaging (MRI), ultrasound, and computed tomography are widely used to detect primary and metastatic tumors13–15, but often lack the spatial resolution or molecular sensitivity required to capture subtle changes within the TME, particularly in early-stage or small tumors. Molecular imaging provides greater sensitivity: fluorescence imaging enables responsive detection of acidity using acid-responsive probes16,17, yet limited optical penetration restricts its applicability in deeper tissues. MRI-based extracellular pH (pHe) measurement offers further biological insights18,19, but typically faces inherent trade-offs between spatial and temporal resolution that constrain real-time and high-resolution assessment of dynamic acidity. Therefore, beyond sensitivity and specificity, the ability to continuously monitor and image the spatial-temporal evolution of TME acidity remains crucial for advancing early cancer diagnosis and improving therapeutic outcomes.

Photoacoustic (PA) molecular imaging has emerged as a powerful modality that combines the advantages of PA imaging with molecular labeling20–23. Unlike conventional optical methods, which generally penetrate less than 1 mm, PA imaging, based on the “light-in, sound-out” principle, enables acoustic-resolution (~100 μm) imaging at centimeter-scale depths, while maintaining diffraction-limited optical resolution (~3 μm) imaging for superficial tissues (~1–2 mm)24–28. These capabilities allow PA imaging to detect acidic TMEs at multiple tumor depths with high resolution29–32. Furthermore, molecular labeling improves specificity and sensitivity by using imaging probes with specific properties to target tumor-associated molecules and enable quantitative pH mapping within the TME33–35. In recent years, several pH-activatable PA probes, including organic polymers and inorganic nanomaterials36–38, have demonstrated real-time intratumoral pH monitoring with high specificity and sensitivity39,40. While these advances address the critical challenge of characterizing tumor acidity, existing approaches remain limited in detecting TME acidity during cancer progression and metastasis, in which proton levels are low and fluctuating dynamically41. In this context, dynamic studies of TME acidity evolution across different stages of development are crucial for gaining a comprehensive understanding of tumor progression, metastasis, and therapeutic responses, ultimately guiding the development of more effective diagnostic and therapeutic strategies.

In this work, we develop a highly specific and sensitive PA molecular imaging strategy to detect TME acidity across tumor initiation, progression, and metastasis (Fig. 1a, b). We identify ATP6V0C, a key regulator of proton transport via V-ATPase, as a critical mediator of TME acidification through bioinformatics analyses and validation in clinical hepatocellular carcinoma (HCC) samples. Elevated ATP6V0C expression is strongly correlated with poor prognosis in HCC patients, suggesting that ATP6V0C-mediated TME acidification is a critical driver of HCC growth and metastasis. To monitor TME pH dynamics, we design a pH-responsive ratiometric PA sensor (PPS) with high sensitivity and specificity for H+ detection. Using PPS-assisted PA imaging, we visualize TME acidification in murine HCC models and capture pH dynamics throughout tumor progression and metastasis. We further apply pH-responsive ratiometric PA imaging to delineate tumor boundaries in resected clinic HCC tissues. Finally, we evaluate a therapeutic potential combining PPS-based H+-activated photothermal therapy (PTT) with proton pump inhibitors (PPIs), which effectively suppresses tumor growth (Fig. 1c). Overall, these results demonstrate that PPS-assisted ratiometric PA imaging provides a promising strategy for highly specific and sensitive detection and monitoring of TME acidity at different tumor stages, elucidate acidification mechanisms that drive HCC development, and support improved early tumor diagnosis and precision therapeutic strategies.

Results
Identification of ATP6V0C as a prognostic biomarker in human HCC
To investigate the clinical relevance of ATP6V0C in HCC, we conducted a comprehensive analysis of publicly available datasets in The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). ATP6V0C expression was significantly higher in HCC tissues than in adjacent nontumor liver tissues (Fig. 2a–d). Notably, ATP6V0C expression was elevated in advanced-stage HCC (stage III-IV) relative to early-stage disease (I-II) (Fig. 2e), suggesting its potential role in tumor progression. ATP6V0C, a key subunit of V-ATPase, mediates proton transport, contributing to extracellular acidification within the TME. This acidification promotes key oncogenic processes, including cell proliferation, invasion, and immune evasion42,43. Kaplan-Meier survival analysis revealed that higher ATP6V0C expression was significantly associated with poor prognosis in HCC patients (Fig. 2f), a finding that is consistent with the role of ATP6V0C in tumor aggressiveness. Further correlation analyses revealed positive associations between ATP6V0C expression and the expression of molecular markers of tumor proliferation, metastasis, and epithelial-mesenchymal transition (EMT) (Fig. 2g, h, Supplementary Figs. 1, 2). These findings were validated in paired tumor and adjacent normal tissues collected from HCC patients. Hematoxylin and eosin (H&E) staining and immunofluorescence analysis confirmed the marked overexpression of ATP6V0C in HCC tissues (Fig. 2i, j). This overexpression likely contributes to TME acidification through the role of ATP6V0C in regulating V-ATPase activity, thereby facilitating tumor progression. Collectively, these findings identify ATP6V0C as a critical mediator of HCC progression and a potential prognostic biomarker. Its role in promoting TME acidification highlights its therapeutic potential, suggesting that targeting V-ATPase-mediated acidification could represent a promising strategy to improve clinical outcomes in HCC patients.

Synthesis and characterization of PPS
To investigate the role of V-ATPase-mediated tumor microenvironment acidification in cancer progression, we developed a pH-responsive ratiometric photoacoustic sensor (PPS) by integrating polyaniline (PANI) with DSPE-PEG-COOH to improve water stability (Fig. 2k). PANI was selected because of its distinct protonation behavior: under neutral or basic conditions, deprotonated PANI (emeraldine base, EB) loses conductivity and near-infrared (NIR) absorption, whereas in acidic environments, protonated PANI (emeraldine salt, ES) has high conductivity and strong NIR absorption44–46. Optimization of the synthesis conditions further increased the H+ responsiveness of PPS (Table S1 and Supplementary Fig. 3a–c). The ratio of the absorbance values at 780 nm and 1064 nm (Abs780/Abs1064) demonstrated a strong linear correlation with pH (Supplementary Fig. 3d–f). Transmission electron microscopy (TEM) revealed uniform PPS nanoparticles with an average diameter of approximately 80 nm (Fig. 2l), which remained stable in aqueous solution for 7 days (Supplementary Fig. 4). As the pH decreased from 7.0 to 3.0, PPS exhibited a progressive increase in absorbance at 780 nm, whereas the absorbance at 1064 nm remained relatively constant (Fig. 2m and Supplementary Fig. 5). This shift reflects the proton-responsive transition of PANI from the EB to ES state47. Correspondingly, PA imaging confirmed a significant increase in the PA signal at 780 nm under mildly acidic conditions (pH 6.5), whereas the signal at 1064 nm remained unchanged (Fig. 2n). Compared with that at pH 7.4 (80 nm), the particle size of PPS at pH 6.5 (130 nm) was greater (Supplementary Fig. 6). This may be attributed to the protonation of functional groups on the particle surface, as well as increased ionic interactions and potential aggregation between particles in the mildly acidic environment, which could facilitate accumulation and retention in the acidic TME.

PPS demonstrated excellent photostability after exposure to 2000 laser pulses at 780 nm under both neutral and acidic conditions (Supplementary Fig. 7). Using our customized PA imaging system (Supplementary Fig. 8)48, we detected PPS signals at depths of up to 15 mm in chicken tissues at both pH 6.5 and 7.4 (Supplementary Fig. 9), highlighting its strong sensitivity for deep-tissue pH sensing.

To confirm the specificity of PPS for acidic pH detection, we evaluated its performance in solutions containing PBS, KCl, NaCl, MgSO4, glucose, H2O2, and proteases, which represent different metabolites and components commonly present within the TME. PPS exhibited no significant absorbance or PA signal intensity at pH 7.4 in any solution; however, at pH 6.5, both the absorbance at 780 nm and the corresponding PA signal intensity increased significantly (Fig. 2o, p and Supplementary Figs. 10, 11). Ratiometric PA analysis further demonstrated a consistent increase in signal intensity as the pH decreased (Supplementary Fig. 12).

To mimic the in vivo environment, we embedded a PPS-containing tube subcutaneously in the abdomen of mice (Fig. 2q). As the pH decreased, the PA signal at 780 nm increased, whereas the signal at 1064 nm remained unchanged, with a strong linear correlation between the PA signal ratio (780 nm/1064 nm) and pH values ranging from 6.0 to 8.0 (Fig. 2r, s and Supplementary Fig. 13). These results collectively demonstrate the high specificity, stability, and sensitivity of PPS for pH-responsive PA imaging, positioning PPS as a promising tool for detecting pH changes in the TME.

PPS-assisted PA imaging in an early HCC model
To evaluate the pH responsiveness of PPS in vivo, we first preincubated PPS in buffers at pH 6.5 and 7.4 and injected the probe into the bilateral muscles of healthy mice. PA imaging confirmed a significantly greater PA signal at 780 nm under acidic conditions (pH 6.5) than at neutral pH (pH 7.4) (Supplementary Fig. 14). Then, in a subcutaneous HCC model, PPS was intratumorally injected into both tumor and muscle tissues (Supplementary Fig. 15a). PA imaging at 0.5 h post injection revealed a significantly elevated PA780/PA1064 ratio in tumor tissue, indicating local acidification (Supplementary Fig. 15b–f). Further intravenous administration of PPS resulted in peak tumor accumulation at 9 h post injection (Supplementary Fig. 16). These results establish PPS as a promising ratiometric PA probe for monitoring TME pH values.

Next, we evaluated the sensitivity of the PA ratio imaging strategy using PPS for the early detection of HCC development. During the initial stages of HCC progression, we hypothesize that the pH of the local TME dynamically decreases as the tumor grows, accompanied by neovascularization and hypoxia (Fig. 3a). Ultrasound imaging (Fig. 3b) revealed a progressive increase in tumor size following HCC cell injection, with the tumor mass confirmed as a solid tumor through H&E staining (Supplementary Fig. 17). To monitor pH changes during tumor formation, PPS was intravenously administered on days 0, 1, 3, 5 and 7. Using acoustic-resolution PA microscopy (AR-PAM) (Supplementary Fig. 18) for longitudinal monitoring, we observed a significant increase in the PA780/PA1064 ratio starting on day 3, corresponding to a decrease in the local pH from neutral (pH 7.33) to acidic (pH 7.08) (Fig. 3d). By day 7, the TME pH further decreased to 6.42, which is consistent with previously reported TME values49,50. Notably, pretreatment with PPIs significantly attenuated the decrease in PA (pH 7.56 to pH 7.09) (Fig. 3c, e), suggesting that PPS is highly sensitive in detecting changes in the pH of the TME. The PPS-assisted PA imaging results were comparable to the pH measurements obtained using clinical PET imaging (Supplementary Fig. 19), indicating that an acidic TME emerges during the super-early stages of tumor development.

To explore the mechanisms underlying TME acidification, tumor vessel density and tortuosity were quantified during HCC progression. Both parameters increased over time (Fig. 3f, g, and Supplementary Fig. 20), which is consistent with the development of disorganized tumor vasculature that promotes local hypoxia and the accumulation of metabolic waste (such as lactate), thereby disrupting pH homeostasis in the TME. Immunofluorescence staining further demonstrated a progressive increase in hypoxia and angiogenesis markers following tumor cell injection (Fig. 3h–j). Correlation analysis using the Pearson method revealed a negative correlation between hypoxia and acidity (r = −0.9586) and a positive correlation between hypoxia and angiogenesis (r = 0.9949) (Fig. 3k). These findings demonstrate that an acidic TME, hypoxia, and angiogenesis appear at the super-early stages of tumor formation and are closely interconnected. Given their critical roles in tumor proliferation, metastasis, and therapeutic resistance51–53, these biomarkers represent promising targets for early therapeutic interventions. PPS-assisted PA imaging offers a highly sensitive, noninvasive approach for detecting TME acidification, with significant potential for super-early tumor diagnosis and monitoring.

PPS-assisted PA imaging in liver orthotopic and metastatic tumor models
Real-time imaging of H+ levels in liver tumor lesions is crucial for understanding pathological processes within the acidic TME. Before in vivo tumor imaging, we evaluated whether other pathological factors could interfere with pH monitoring. Using two non-tumor disease models that induce local inflammation or tissue acidosis (acute liver failure and hepatic ischemia-reperfusion injury), PA imaging showed that PPS exhibited minimal or negligible signal enhancement compared with tumor tissues (Supplementary Fig. 21), indicating excellent pH specificity and insensitivity to other metabolic components in the TME.

To extend our findings beyond subcutaneous tumor models, we evaluated PPS-assisted PA imaging in liver orthotopic and metastatic tumor models. Orthotopic HCC and pancreatic cancer liver metastasis models were established via the in situ injection of HCC cells into the liver and pancreatic cancer cells into the spleen, respectively (Fig. 4a). Successful model establishment was confirmed by fluorescence imaging, which revealed strong signals in the liver region (observed by luminescence due to the luciferase-catalyzed luciferin oxidation in tumor cells) (Fig. 4b).

Following model validation, PPS was intravenously administered, and PA imaging of the liver region was conducted using circular-array PA tomography (Fig. 4c and Supplementary Fig. 8). In both models, PA signals at 1064 nm remained relatively unchanged over 24 h, whereas signals at 780 nm increased significantly in tumor-bearing livers, indicating PPS accumulation and responsiveness to the acidic TME (Fig. 4d, e). However, the direct visualization of tumor regions on the basis of only acidic signals was challenging. To address this, PA images at 780 nm and 1064 nm were analyzed using signal ratios. At 9 h post injection, PA images revealed distinct acidic regions corresponding to orthotopic and metastatic tumors, differentiating them from normal liver tissue. Quantification of the PA signal ratios further confirmed a significant increase in tumor-bearing livers compared with that observed in normal controls (Fig. 4f, g). Additionally, in an advanced orthotopic HCC model with intrahepatic metastases, PPS-mediated PA imaging enabled clear visualization of intraparenchymal tumor lesions, showing diffuse and markedly enhanced PA signals (Supplementary Fig. 22). The PA imaging results were corroborated by H&E staining (Fig. 4h), which confirmed the presence of tumors and an associated acidic TME.

To further evaluate the performance of PPS in deep-seated liver tumors, we established a rat orthotopic HCC model and conducted comparative imaging with MRI and PET. PPS-enabled PA imaging precisely visualized intraparenchymal liver tumors, showing tumor localization consistent with MRI and PET results (Supplementary Fig. 23). These findings highlight the potential of PPS as a sensitive and specific imaging modality for detecting and characterizing acidic TMEs in orthotopic and metastatic liver tumors, including in deep-seated liver tumors comparable to clinical imaging standards such as MRI and PET.

PPS-assisted PA imaging-guided lymphadenectomy in a lymph node metastatic tumor model
Lymph node metastasis is a major route for systemic tumor dissemination, and the complete removal of metastatic lymph nodes (MLNs) is critical for improving patient prognosis54–56. To explore the potential of PPS in detecting MLNs and guiding lymphadenectomy during navigational surgery, we established a lymph node metastatic tumor model (Fig. 5a). HCCLM3-Luc cells were injected into the mouse paw, and fluorescence imaging confirmed successful metastasis to the lymph nodes, with signals localized in the popliteal region (Fig. 5b). PPS was subsequently administered via paw injection, and PA imaging was performed 30 min later. Compared with that in benign lymph nodes (BLNs), a significantly greater PA signal was detected in metastatic lymph nodes, corresponding to an acidic microenvironment with an estimated pH of 6.5 (Fig. 5c–e). Guided by PA imaging, the high-signal regions indicative of MLNs were surgically identified and excised. Reimaging of the surgical area and excised tissues confirmed the successful removal of the high-signal regions (Fig. 5f–h). pH measurement results for the excised tissues were consistent with the PA imaging results (pH 6.5), and subsequent H&E and IHC staining confirmed the presence of tumor-invaded lymph nodes (Fig. 5i). These findings demonstrate that the PPS can be used to detect MLNs with high sensitivity and provide precise, image-guided navigation for lymphadenectomy, highlighting the potential of PPS to improve surgical outcomes in patients with metastatic disease.

PPS-assisted PA imaging of human HCC samples
To evaluate the clinical potential of PPS, human HCC specimens were obtained from patients who were histologically diagnosed with HCC (Table S2). These specimens were treated with PPS for PA imaging (Fig. 6a). PA signals at 780 nm were significantly stronger in the tumor regions of PPS-treated samples than in the surrounding paracancerous tissues (Fig. 6b, c). Ratio imaging analysis revealed that the tumor regions exhibited an acidic environment, with clear delineation of the tumor margins.

To further validate the applicability of PPS, additional liver cancer specimens from seven different patients were analyzed. All the specimens presented well-defined tumor boundaries. H&E staining corroborated the PA imaging findings by distinctly differentiating tumors from normal tissues (Fig. 6d). Quantitative analysis of the PA signal intensity in PPS-incubated samples indicated that the tumor regions had lower pH values than paracancerous tissues (Fig. 6e). Ex vivo imaging of clinical specimens offers a unique opportunity for molecular imaging studies of primary tumors and the TME in patients, as specimens from HCC patients preserve the molecular characteristics of the original TME and intratumoral heterogeneity. These findings highlight the potential of PPS to selectively identify tumor regions on the basis of their acid specificity, providing a promising approach for precise tumor margin delineation and for guiding surgical resection.

ATP6V0C promotes the production of an acidic TME
Inspired by the crucial role of an acidic TME in cancer progression and diagnosis, we investigated the molecular mechanisms underlying acidic TME formation. ATP6V0C, a subunit of V-ATPase, has been identified as a key regulator of proton transmembrane transport pathways, contributing to TME acidification57–59. Molecular docking analysis revealed that esomeprazole (ESOM), an FDA-approved PPI58,60,61, has a high binding affinity for ATP6V0C (Fig. 7a and Supplementary Fig. 24). Specifically, ESOM forms hydrogen bonds with SER98 and SER102, stabilizing the ligand‒protein complex. After 48 h of ESOM treatment, ATP6V0C expression was markedly lower in both HepG2 and HCCLM3 cells than in the PBS-treated controls (Fig. 7b). To assess the functional consequences of ATP6V0C inhibition, we measured intracellular pH using a fluorescent pH indicator. Compared with PBS-treated cells, ESOM-treated cells displayed weaker fluorescence, indicating a reduced intracellular pH, likely caused by impaired proton efflux and subsequent intracellular acidification (Fig. 7c). Correspondingly, shRNA-mediated knockdown of ATP6V0C led to blocked proton efflux and intracellular acidification, which in turn inhibited the proliferation and invasion capacity of liver cancer cells (Supplementary Fig. 25–27). Morphologically, ESOM-treated tumor cells presented nearly circular edges with fewer pseudopodia, suggesting that ESOM may inhibit actin polymerization and cytoskeletal remodeling through intracellular acidification, thereby suppressing tumor cell invasion and metastasis (Fig. 7d). Functional assays confirmed that ESOM significantly suppressed liver cancer cell proliferation and invasion (Fig. 7e–j), with additional in vivo evidence further supporting its tumor-suppressive effects (Supplementary Fig. 28).

To further assess PPS applicability across tumor types, we analyzed ATP6V0C expression in pan-cancer and found it highest in hepatocellular carcinoma (HCC), higher than in colon adenocarcinoma (COAD) and breast cancer (BC) (Supplementary Fig. 29). Consistently, PPS-mediated PA imaging in HCC, COAD, and BC xenografts showed the strongest signal in HCC, moderate in COAD, and weakest in BC (Supplementary Fig. 30), indicating that PPS signal intensity correlates with ATP6V0C-driven tumor acidification. We next evaluated the impact of ESOM on the acidic TME in vivo using PPS-assisted PA imaging (Fig. 7k). In PBS-treated tumor-bearing mice, the PA ratio signal showed minimal fluctuations over 96 h. In contrast, ESOM-treated mice exhibited a 36% reduction in the PA ratio at 48 h, followed by gradual recovery (Fig. 7l, m). The corresponding tumor pH measurements revealed transient alkalization in the ESOM-treated mice (Fig. 7n), indicating effective suppression of the acidic TME through V-ATPase inhibition.

A schematic overview of the pathways regulating acidic TME formation and its inhibition is provided in Fig. 7o. Overall, our findings suggest that ATP6V0C-driven proton efflux contributes to TME acidification and that its inhibition via ESOM induces cytotoxic effects, highlighting the potential of targeting proton transport pathways as a therapeutic strategy for cancer treatment.

PPS-based PTT and ATP6V0C inhibition synergistically suppress tumor growth
PPS not only exhibits pH-responsive PA properties but also enhances photothermal therapy (PTT) in acidic environments (Supplementary Figs. 31, 32), making it a promising candidate for tumor-specific PTT. To evaluate its efficacy, we first tested PPS under in vitro conditions. Compared with PPS at pH 7.4, PPS combined with 808 nm laser irradiation significantly reduced tumor cell viability at pH 6.5, confirming the acid-enhanced PTT effect (Supplementary Figs. 33, 34). The in vivo PTT efficacy of PPS was next evaluated.

We hypothesized that combining the acid-responsive PTT effect of PPS with the acid-suppressing properties of ESOM could synergistically inhibit tumor growth. To test this hypothesis, a subcutaneous xenograft model of liver cancer was established using HepG2 cells, and the mice were randomly assigned to six treatment groups: PBS, PBS + Laser, PPS, ESOM, PPS + Laser, and PPS + Laser + ESOM. Over the 21-day treatment period, no significant changes in body weight were observed across all groups (Fig. 8a–c). The tumor temperature increased significantly in the PPS + Laser and PPS + Laser + ESOM groups after 5 min of laser irradiation (Fig. 8b and Supplementary Fig. 35).

Treatment outcomes revealed substantial tumor suppression in the PPS + Laser group, whereas the PPS + Laser + ESOM group exhibited even greater inhibition (Fig. 8d, e and Supplementary Fig. 36). Pathological analyses corroborated these findings (Fig. 8f), with TUNEL and Ki-67 staining demonstrating significant apoptosis and reduced proliferation in the PPS + Laser + ESOM group (Fig. 8f–h). These results indicate that PPS-based PTT and ATP6V0C inhibition act synergistically to inhibit tumor growth by modulating the acidic TME. In addition, in vivo RNA-seq analysis was performed using tumors from mice one week after treatment with PPS + Laser + ESOM to comprehensively evaluate the complex changes induced in the TME. A hierarchical clustering heatmap based on gene expression data clearly illustrated the grouping of samples, with samples within the same cluster sharing similar expression profiles (Fig. 8i). As shown in Fig. 8j, k, a total of 602 differentially expressed genes (DEGs) were identified, including 398 upregulated and 204 downregulated genes. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these DEGs were enriched predominantly in core genes and pathways involved in regulating proliferation, metastasis, and metabolism (Fig. 8l, m). These findings underscore the potential role of the PPS + Laser + ESOM strategy in inhibiting tumor proliferation and metabolic reprogramming following treatment of the acidic TME.

Furthermore, the GSE149614 HCC scRNA-seq dataset was obtained and preprocessed to extract tumor cells, followed by Harmony-based normalization. UMAP analysis revealed significant differences in tumor cell distribution across cancer stages (Fig. 8n). Using Scissor, bulk RNA-seq data from the PPS + Laser + ESOM-treated and control groups were projected onto single-cell data, categorizing the cells into target, nontarget, and nonreaction groups (Fig. 8o). Cell type distribution analysis revealed that nontargeted cells predominated in stage I, whereas target cells were markedly elevated in stage IIIB (Fig. 8p). Pseudotime analysis (Fig. 8q) mapped target, nontargeted, and nonreactive cells along the tumor progression trajectory (Fig. 8r). The results indicated a gradual increase in target cell abundance as the tumor advanced, suggesting that the efficacy of the PPS + Laser + ESOM strategy may improve in advanced HCC stages. These findings suggest that the synergistic combination of PPS-based PTT and ATP6V0C inhibition effectively suppresses tumor growth by targeting the acidic TME and disrupting metabolic reprogramming.

Biosafety evaluation
To assess the potential for the clinical translation of PPS, its biosafety was evaluated both in vitro and in vivo. As shown in Supplementary Figs. 37, 38, PPS at concentrations up to 500 μg/mL had no significant cytotoxicity in normal liver cells (THLE-3) or the liver cancer cell lines HepG2 and HCCLM3. Hemocompatibility testing demonstrated minimal hemolysis following a 4-hour incubation with red blood cells (Supplementary Fig. 38). To further evaluate long-term safety, chronic toxicity studies were performed over a 28-day period following intravenous administration of PPS. Comprehensive hematological, histological, and physiological analyses showed no significant abnormalities in blood parameters, body weight, or major organ histopathology compared to control mice (Supplementary Figs. 39–42). Furthermore, histological examinations of major organs revealed no significant pathological changes compared with those in saline-treated controls (Supplementary Fig. 43). These findings demonstrate that PPS has excellent in vitro and in vivo biosafety, even under prolonged systemic exposure, supporting its potential for future clinical applications.

Methods
Ethical statement
This study adhered to all relevant ethical regulations. Animal experiments were conducted in compliance with the “Guide for the Care and Use of Animals” of the Animal Care and Use Committee of Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences (SIATACUC), with all protocols receiving prior approval from SIATACUC (SIAT-IACUC-240228-YGS-CJQ-A2486). According to the animal ethics of the institute and animal welfare, animals were euthanized by carbon dioxide inhalation if the tumor burden exceeded 10% of their own body weight, the body weight decreased by 20% of the normal body weight, or ulcers occurred at the tumor occurred. The maximal tumor size of 1500 mm3 was permitted by SIATACUC. The experimental animals in this study did not exceed the maximum tumor burden.

Materials
1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Carboxy(Polyethylene Glycol)2000] (DSPE-PEG-COOH) was purchased from Ruixi Biologic Tech (Xi’an, China). Iron (III) chloride hexahydrate (FeCl3·6H2O) was supplied by Sinopharm Chemical Reagent Co. (Shanghai, China). Aniline was provided by Adamas-beta (Shanghai, China). All other chemicals were bought from Aladdin (Shanghai, China). Dulbecco’s modified Eagle medium (DMEM with high glucose), penicillin-streptomycin solution, fetal bovine serum (FBS), trypsin EDTA solution (0.5% trypsin), and phosphate-buffered saline (PBS, PH 7.4) were procured from Gibco (AG, USA). Esomeprazole was purchased from APExbio (Shanghai, China). Antibodies used in this study included ATP-6V0C (1:200, Abcam, #ab230323), anti-CK8 (1:200, Servicebio, #GB15231-100), anti-CD31 (1:200, Servicebio, #GB153151-100), anti-HIF-1a (1:200, Servicebio, #GGB151339-100), and anti-Ki67 (1:200, Servicebio, #GB111499-100). Cell Counting Kit-8 (CCK-8) and Calcein-AM solution were obtained from Dojindo (Tokyo, Japan). BCECF-AM was procured from Beyotime (Shanghai, China). The ATP6V0C ELISA kit (RX103493H) was obtained from Ruixin bio (Fujian, China). Unless otherwise stated, all other materials and reagents were purchased from Beyotime (Shanghai, China).

Human liver cancer specimen analysis
The study involving human liver cancer specimens was approved by the Ethics Committee of Zhujiang Hospital, and informed consent was obtained from all tissue donors. Four pairs of fresh HCC tissue samples and their corresponding adjacent non-tumor tissues were collected for hematoxylin-eosin (H&E) staining and immunofluorescence analysis.

Preparation and characterization of PPS
PPS nanoparticles were synthesized through a two-step process. First, an aniline/HCl solution was prepared by mixing 95 μL of aniline with 95 μL of hydrochloric acid (36% by mass) in 1 mL of deionized water at room temperature. The solution was then subjected to ultrasonic treatment using a SONICS VCX150 ultrasonic processor (150 Hz, 35%, 30 min).

Next, 0.5 mL of FeCl3·6H2O aqueous solution (100 mg/mL) and 50 mg of DSPE-PEG-COOH were dissolved in 9 mL of deionized water and stirred for 30 min. The two solutions were then combined and stirred in an ice bath for 6 h, during which the color changed from light yellow to dark green. The mixture was dialyzed overnight at room temperature using a dialysis bag (MW=80000 Da), resulting in the formation of PPS.

The morphology of the nanoparticles was assessed using transmission electron microscopy (TEM, JEM-1200EX, 120 kV). Particle size distribution was determined by dynamic light scattering (DLS) using a Zetasizer Nano-ZS (Malvern Instruments, USA). The optical properties were characterized by recording the UV-Vis-NIR absorption spectra with a Shimadzu UV-3600 spectrophotometer (Japan).

pH-responsive PA detection of PPS
PA imaging was performed using customized acoustic-resolution photoacoustic microscopy (AR-PAM) and photoacoustic computed tomography (PACT) systems48,68. PA spectra of PPS (1 mg/mL) were recorded at pH 7.4 and 6.5 across a wavelength range of 700 to 1064 nm.

To assess the pH specificity of PPS, solutions of KCl (20 nM), NaCl (20 nM), MgSO4 (20 nM), and glucose (20 nM) were incubated with PPS (0.25 mL, 5 mg/mL) at pH 7.4, 7.0, and 6.5 for 5 min at 37 °C. UV-vis absorption and PA signals at 780 nm were measured.

In vivo pH responsiveness of PPS was further evaluated by embedding PPS solutions at different pH levels into mouse skin. PA images were acquired at both 780 nm and 1064 nm. The pH sensitivity of PPS was quantified by calculating the ratio of PA signals at 780 nm and 1064 nm.

Photothermal properties of PPS
PPS solutions at pH levels 6.5, 7.0, and 7.4 were irradiated with a continuous 808 nm laser at power densities of 0.5, 0.75, and 1.0 W cm−2. Temperature changes were monitored every 30 seconds using a thermal imager (Fluke), and thermal images were captured accordingly. Photothermal conversion efficiency (PCE) was calculated using established protocols from the literature69.

Cell lines and cell culture
Human liver cancer HepG2 cell lines, human THLE-3 liver cell line and murine pancreatic tumor PAN02 cells lines were purchased from the American Type Culture Collection (ATCC). Human liver cancer HCCLM3 cells lines were purchased from the China Center for Type Culture Collection (CCTCC). All cell lines were recently authenticated by STR profiling. Each was also tested and found to be negative for mycoplasma. HepG2 and HCCLM3, along with THLE-3, were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in a 5% CO2 atmosphere. Pan02 were cultured in RPMI 1640 medium with 10% FBS and 1% penicillin-streptomycin under the same conditions. The construct expressing luciferase was introduced into Pan02 cells by lentiviral transduction for bioluminescence imaging.

In vitro PTT
Cytotoxicity of PPS was first evaluated using the CCK-8 assay. HepG2 cells were treated with increasing concentrations of PPS (0, 50, 100, 200, 300, 400, 500 μg/mL) for 24 h, and cell viability was measured.

For PTT evaluation, HepG2 and HCCLM3 cells were incubated with PPS at varying concentrations (0, 50, 100, 200, 300, 400, 500 μg/mL) at either pH 6.5 or pH 7.4 for 4 h. After incubation, cells were irradiated with an 808 nm laser (1 W/cm2, 5 min). Cell viability was assessed 24 h post-irradiation using the CCK-8 assay, and results were validated by calcium-AM/PI double-staining to assess cell death.

Animal models
All animal experiments were conducted in strict compliance with protocols approved by the Animal Care and Use Committee of SIATACUC. Male BALB/c nude mice (5-8 weeks old, 18−20 g) were sourced from the Laboratory Animal Center of Guangdong Province (Guangzhou, China). All mice were housed and maintained under specific pathogen-free (SPF) conditions in groups of 4-5 mice per cage, and maintained at a temperature of 25 °C in a humidity-controlled environment with a 12 h light/dark cycle, with free access to standard food and water. Four tumor models were established as follows:
(i) Subcutaneous tumor model: HepG2 cells (1×106 cells/mL in 100 μL) were injected subcutaneously into the abdominal region of each mouse.
(ii) Orthotopic tumor model: HepG2-luc cells (1×106 cells/mL in 50 μL) were directly injected into the liver lobe of the mouse.
(iii) Liver metastasis model: Pan02-luc cells were injected into the splenic parenchyma, allowing for implantation into the liver parenchyma via the blood circulation.
(iv) Lymph node metastasis model: HCCLM3-luc cells (1×106 cells/mL in 50 μL PBS) were injected into the hind footpad of each mouse.

Tumor formation and model establishment were confirmed by visual inspection and bioluminescence imaging. According to the animal ethics of the institute and animal welfare, animals were euthanized by carbon dioxide inhalation if the tumor burden exceeded 10% of their own body weight, the body weight decreased by 20% of the normal body weight, or ulcers at the tumor occurred. The experimental animals in this study did not exceed the maximum tumor burden. Our research results are not exclusive to a particular sex, but are universally applicable.

PET imaging
HepG2 tumor-bearing mice were anesthetized with 2% isoflurane and intravenously injected with 18F-FDG. PET scans were performed using a small animal PET system, and the mean standardized uptake values (SUVmean) was recorded. Maximum Intensity Projection (MIP) images were reconstructed for visualization.

In vivo PA imaging
In vivo PA imaging was performed on HepG2 tumor-bearing mice, which were anesthetized with 2% isoflurane. PPS (1 mg/mL, 200 μL) was administered intravenously, and both photoacoustic and ultrasound imaging were performed using an AR photoacoustic microscopy system. Imaging was carried out at baseline (pre-injection) and at 3, 6, 9, 12, and 24 h post-injection, with a focus on the tumor area at a wavelength of 780 nm.

For the subcutaneous tumor model, HepG2 cells (1×106 cells/mL) were injected subcutaneously into the abdominal region of the mice. Tumor-bearing mice were intravenously administered PPS (1 mg/mL, 200 μL) before imaging, and at 1, 3, 5, and 7 days post-tumor induction. PA imaging was performed at 780 nm and 1064 nm wavelengths, and the ratio of PA signals at these two wavelengths was calculated. Vessel density within the tumor region was quantified using PA imaging and analyzed with MATLAB (Mathworks Inc.).

To assess tumor hypoxia and neovascularization, immunofluorescence staining for HIF1-α and CD31 was performed at various stages of tumor development. Pearson correlation analysis was conducted to investigate the relationships between pH, hypoxia, and neovascularization. The Pearson product-moment correlation coefficient (r) was calculated, where values range from −1 (strong negative correlation) to 1 (strong positive correlation), with 0 indicating no correlation. The equation for Pearson’s correlation coefficient is as follows:

r = [Σ(xi − x)(yi − y)] / [sqrt(Σ(xi − x)2) * sqrt(Σ(yi − y)2)]

where x and y represent the two variables corresponding to pH, hypoxia and neovascularization expression.

For PA imaging in the orthotopic liver metastasis and lymph node metastasis models, PPS (1 mg/mL, 200 μL) was intravenously injected into tumor-bearing mice at different time points. PA imaging was performed at both 780 nm and 1064 nm wavelengths, followed by detailed analysis of the tumor regions. Tumor tissues were further examined using H&E staining and immunohistochemistry.

In vivo PA imaging of tumors after acid inhibition
HepG2 tumor-bearing mice were randomly divided into two groups: one group received an intravenous injection of esomeprazole (ESOM, 2.5 mg/kg, APExBIO Technology LLC, USA), while the control group was administered PBS. PA imaging was conducted at 0, 24, 48, 72, and 96 h post-ESOM or PBS administration. PPS was intravenously injected 9 h prior to each scanning session. PA signal intensity and pH values were measured at each time point and compared between the ESOM-treated and PBS-treated groups.

PA imaging of human liver cancer specimens
To assess the effects of PPS in human HCC tissues, surgical specimens from both tumor and adjacent normal liver tissues were collected. A cohort of eight HCC patients, scheduled for hepatectomy, was selected, and informed consent was obtained from each participant. Immediately after hepatectomy, liver tumor specimens (approximately 1×2 cm2 in size and 2~3 mm thick) were excised. Specimens containing both tumor and adjacent normal liver tissue were rinsed with 0.9% normal saline and soaked in either PBS or PPS (1 mg/mL) solution at 4 °C for 2 h. PA imaging was then performed on the treated specimens, followed by pathological examination to assess the effects of PPS on tissue characteristics.

Molecular docking of ATP6V0C with ESOM
To investigate the interaction between ESOM and ATP6V0C, the 3D structure of ESOM was retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov) and converted into mol2 format using Chem3D software. Structural optimization was performed using SYBYL software with the Tripos force field, and Gasteiger-Huckel charges were assigned to the ligand. The optimized structure was then saved in mol2 format for subsequent molecular docking simulations.

The crystal structure of ATP6V0C (PDB ID: 6WLW) was obtained from the Protein Data Bank (PDB, https://www.rcsb.org). The protein structure was preprocessed in SYBYL’s Docking module, which involved removing water molecules, adding hydrogen atoms, and assigning charges. The active binding site was identified based on the co-crystallized ligand in the original complex structure. The prepared protein was then saved for docking simulations.

Molecular docking was carried out using the Surflex-Dock module in SYBYL, with esomeprazole as the ligand and ATP6V0C (6WLW) as the receptor. Binding affinity was quantified using the Total Score function, where higher scores indicate stronger binding interactions. A Total Score ≥ 7 was considered indicative of significant binding. Post-docking analysis included the generation of 2D interaction diagrams to visualize and analyze the molecular interactions between ESOM and ATP6V0C.

ESOM inhibition profiles
ATP6V0C activity in HepG2 and HCCLM3 tumor cells (1×107 cells/mL) was quantified using ELISA (ATP6V0C ELISA kit, Ruixin Biotech, China) after 24-hour incubation with 160 μM ESOM. Absorbance at 450 nm was measured. HepG2 and HCCLM3 cells were cultured in fresh medium containing either PBS or ESOM for an additional 24 h. After treatment, cells were stained with the pH-sensitive fluorescent probe BCECF-AM (Beyotime Biotechnology, China) and imaged using confocal microscopy.

To evaluate the effects of ESOM on tumor invasion and metastasis, the actin cytoskeleton was stained with Actin-Tracker Red-Rhodamine (Beyotime Biotechnology, China). After 24 h of incubation with PBS or ESOM, cells were further stained with Actin-Tracker Red-Rhodamine and 4′,6-diamidino-2-phenylindole (DAPI) to visualize the cell nucleus. Confocal microscopy was used for imaging.

Silencing of ATP6V0C
To reduce the expression of the ATP6V0C, two specific shRNA sequences were designed: shRNA1 (TGACGACATCAGCCTCTACAA) and shRNA2 (GCTCGGCCTCTACGGTCTCAT). These shRNAs were synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China) and subcloned into the pLKO.1-PURO vector. The resulting shRNA-expressing plasmids were co-transfected with packaging plasmids into HEK293T cells to produce lentiviral particles. The collected viral supernatants were used to transduce human hepatocellular carcinoma cell lines HepG2 and HCCLM3. After infection, cells were cultured in medium containing 2 μg/mL puromycin to select for stably transduced clones. The selected stable cell lines were used for subsequent experiments, and knockdown efficiency was assessed by Western blot analysis.

Antitumor treatment studies of ESOM in subcutaneous xenograft tumor models
To establish HCC subcutaneous xenografts, HepG2 cells (1×106) were implanted subcutaneously into the right hind limb of BALB/c nude mice. On day 10 post-implantation, when tumor volumes reached approximately 50 mm3, mice were randomly divided into treatment groups. ESOM treatment (2.5 mg/kg) was initiated on day 10 and administered intravenously every other day for a total of three doses. On day 21, mice were euthanized, and tumors were excised for weight analysis to assess the antitumor effects of ESOM.

Acid inhibition-enhanced PTT
Subcutaneous tumor-bearing mice (n=5 per group) were randomly assigned to six treatment groups: (i) Group 1: PBS (control); (ii) Group 2: PBS + laser; (iii) Group 3: PPS; (iv) Group 4: ESOM; (v) Group 5: PPS + laser; (vi) Group 6: PPS + laser + ESOM.

Mice were intravenously administered PPS (200 μL, 1 mg/mL) and ESOM (0.2 mg/kg). Nine hours post-injection, tumors were irradiated with a continuous 808 nm laser (1 W/cm2) for 5 min, and thermal images were captured during irradiation to monitor temperature changes. Tumor volume was measured at regular intervals throughout the treatment period using the formula: volume = (tumor length) × (tumor width)2/2. Body weight was also monitored every three days.

At the end of the treatment, mice were euthanized, and tumors were excised, washed with PBS, fixed in 4% paraformaldehyde, and embedded in paraffin. Tumor sections were sliced using a microtome (Leica RM2235, Germany) and mounted on slides for histological analysis. H&E staining, TUNEL staining, and anti-Ki67 immunostaining were performed on separate sections. Histological analysis was conducted using a virtual slide microscope (Olympus VS120, Japan).

Histological analyses
Mice were euthanized at different time points (days 1, 3, 5, and 7) after tumor implantation. Subcutaneous tumors were excised, rinsed with PBS, and fixed in 4% paraformaldehyde, then embedded in paraffin. Paraffin-embedded tumor sections were sliced using a microtome (Leica RM2235, Germany) and mounted onto slides. H&E-staining was performed according to standard histological protocols.

For immunofluorescence analyses, tumor sections were incubated with primary antibodies: anti-HIF-1α (1:200, Servicebio) and anti-CD31 (1:300, Servicebio). Immunofluorescence images were captured using a laser scanning confocal microscope. Lymph node involvement was evaluated through immunohistochemistry for cytokeratin 8 (CK8) expression as per the manufacturer’s protocol (Servicebio, China).

Bulk RNA sequencing of tumor samples
Subcutaneous xenografts of HCC were established by subcutaneously injecting 1×106 HepG2 cells into the right hind limb of mice. When tumors reached a volume of approximately 50 mm3, mice were randomly divided into two groups. Treatment was initiated on day 7 post-implantation, and after one week, mice were euthanized, and tumors were dissected. Tumor samples were snap-frozen in liquid nitrogen for RNA extraction, quality control, and subsequent bulk RNA sequencing analysis.

Bulk RNA-seq analysis
The GSE62232 dataset, consisting of 81 HCC samples and 10 adjacent non-tumor samples, was obtained from the GEO database to analyze the differential expression of ATP6V0C in HCC. By comparing expression levels between tumor and normal tissues, the potential role of ATP6V0C in HCC development was assessed. The GSE203024 dataset was additionally used to evaluate ATP6V0C expression across multiple cancer types, including BLCA, BC, CESC, COAD, EC, PRAD, and STAD. To further explore the clinical relevance of ATP6V0C, transcriptomic and clinical data from 370 HCC patients were downloaded from the TCGA database. The association between ATP6V0C expression and patient prognosis was analyzed to determine its potential as a prognostic biomarker.

Single-cell Transcriptomic analysis
The single-cell RNA-seq dataset GSE149614 (10 HCC patients) was retrieved from GEO. Data preprocessing was performed using R (version 4.2.2) and the Seurat package, including gene filtering (removal of zero-expression genes), normalization, scaling, identification of highly variable genes, and batch correction with Harmony. Cell clustering and dimensionality reduction were conducted using FindNeighbors, FindClusters, and UMAP, and tumor cells were identified based on the expression of KRT9 and KRT19. Gene–gene correlations were calculated and visualized using the ggstatsplot package to uncover potential biological relationships.

The Scissor algorithm was applied to integrate bulk RNA-seq (Treatment vs. Control) data with single-cell data, classifying cells into Scissor+ (treatment-associated/responsive), Scissor− (negatively correlated/non-responsive), and background cells based on bulk-derived signals. Monocle was used to reconstruct pseudotime trajectories of tumor cell development, and the abundance of drug-targeted cells across developmental stages was analyzed to reveal dynamic changes in drug sensitivity during tumor progression.

Additionally, single-cell datasets GSE290925, GSE29282, and GSE277905 were included to analyze ATP6V0C expression patterns in HCC, BC, and COAD. UMAP projections were visualized based on cell-type annotations. The analysis primarily utilized the GEOquery, Seurat, dplyr, patchwork, ggplot2, and ggpubr R packages.

Biosafety evaluation
The biosafety of PPS was evaluated through hemolysis testing using solutions at various concentrations (0.1, 0.2, 0.4, 0.6, 0.8, and 1 mg/mL). PBS was used as the negative control, and distilled water served as the positive control. The hemolytic percentage (HP) was calculated according to the following formula:

Hemolytic percentage (HP) (%) = (Dt − Dnc) / (Dpc − Dnc) × 100%

where Dt, Dpc, and Dnc represent the absorbance values of the tested samples, the positive control (DI water), and the negative control (PBS), respectively.

In vivo biosafety evaluation: Healthy BALB/c nude mice were randomly divided into three groups: PBS control, PPS (0.5 mg/kg), and PPS (1 mg/kg). Body weight was recorded every four days, as a general indicator of systemic toxicity and overall health status.

For hematological and histopathological analyses, healthy BALB/c nude mice were intravenously injected with either PBS or PPS (1 mg/kg). At 7, 14, and 28 days post-injection, the mice were euthanized, and blood samples as well as major organs—including the heart, liver, kidneys, lungs, and spleen—were collected. Complete blood count (CBC) analyses were performed using an automated hematology analyzer (BC-2800vet, Mindray). The collected organs were sectioned and stained with hematoxylin and eosin (H&E), followed by imaging and histopathological evaluation using a digital microscope.

Statistical analysis
All data are presented as mean ± SEM, with sample sizes selected to provide sufficient statistical power. For comparisons involving two groups, the Student’s t-test was applied, while for multiple group comparisons, ANOVA followed by Tukey’s post-hoc test was used. Statistical significance was defined as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Statistical analysis was performed using GraphPad Prism (version 10.2.1). The number of experimental replicates is indicated in each figure legend, where applicable.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "General Assessment\nThis study presents a novel pH-responsive ratiometric photoacoustic sensor (PPS) for real-time monitoring of tumour\nmicroenvironment (TME) acidification in hepatocellular carcinoma (HCC). The authors integrate bioinformatics, molecular\nimaging, and therapeutic intervention to demonstrate the role of ATP6V0C in TME acidification and its implications for\ntumour progression and metastasis. The work is well-structured, methodologically rigorous, and clinically relevant. However,\ncertain aspects require clarification and additional validation to strengthen the claims.\nStrengths\n1. Innovative Imaging Tool:\nThe development of PPS for pH-sensitive photoacoustic imaging is a significant technical advance, enabling non-invasive,\nhigh-resolution monitoring of TME acidification.\nThe dual-wavelength ratio-metric approach enhances specificity and minimizes background interference with potential to\ninfluence HCC diagnosis and targeted therapy.\n2. Mechanistic Insights:\nStrong bioinformatics and clinical validation linking ATP6V0C overexpression to HCC progression and poor prognosis.\nMolecular docking and functional assays convincingly demonstrate the interaction between esomeprazole (ESOM) and\nATP6V0C, supporting the therapeutic rationale.\n3. Multimodal Validation:\nThe study employs multiple models (subcutaneous, orthotopic, metastatic) and human specimens to validate PPS utility.\nCorrelative analyses (hypoxia, angiogenesis) strengthen the biological relevance of TME acidification.\n4. Therapeutic Implications:\nThe combination of PPS-mediated photothermal therapy (PTT) and ESOM provides a synergistic anti-tumour effect,\nsupported by RNA-seq and single-cell transcriptomics.\nMajor Concerns and Recommendations\n1. Specificity and Sensitivity of PPS\nInterference from Other Metabolites:\nWhile Supplementary Fig. 10 shows minimal interference from ions (K⁺, Na⁺, Mg²⁺), tumour TME contains lactate, ROS, and\nproteases. PPS response might not\nRequest: Test PPS response in the presence of lactate (due to Warburg effect) or H₂O₂ (ROS from metabolic stress and\nimmune activity) to rule out other possibilities of false positives or signal modulation.\n2. Biological Validation of ATP6V0C-Driven Acidification\nFunctional Rescue Experiments:\nWhile the study associates ATP6V0C with tumor acidification, direct functional evidence establishing causality is lacking.\nThe cited reviews (Stransky et al., 2016; Luciani et al., 2004; Neri & Supuran, 2011) broadly implicate V-ATPase activity in\nTME acidification but do not dissect the specific role of the ATP6V0C subunit. To strengthen the mechanistic claims, the\nfollowing experiments are essential\nRequest: Include siRNA/CRISPR-mediated ATP6V0C modulation to validate its role in pH regulation and tumour\nprogression.\n3. Therapeutic Efficacy and Mechanisms\nPTT-ESOM Synergy:\nThe combinatorial effect is promising, but the mechanism remains speculative. Does ESOM directly sensitize cells to PTT\nby decreasing intracellular pH levels?\nRequest: Provide intracellular pH measurements post-PTT+ESOM to clarify the mechanism.\nIn Vivo Toxicity:\nBiosafety data (Supplementary Figs. 28–33) are reassuring, but long-term toxicity (e.g., 28 days) is not assessed.\nRequest: Include chronic toxicity studies, especially given PPS’s potential clinical translation.\n4. Technical Considerations\nPA Imaging Depth and Resolution:\nThe study claims deep-tissue imaging (15 mm, Supplementary Fig. 9), but tumour models (subcutaneous, orthotopic) may\nnot fully recapitulate deep HCC lesions.\nRequest: Compare PPS performance with current standards of clinical imaging (e.g., MRI or PET) in deeper tumours.\nMinor Concerns\nFigure Clarity:\nFig. 8 (RNA-seq heatmap) lacks gene labels—highlight key DEGs (e.g., metabolic regulators).\nOverall Recommendation\nThis study presents a compelling advance in TME imaging and therapy. The findings are well-supported but would benefit\nfrom additional mechanistic and translational validation.\nAcceptance Decision:\nMinor Revision (address major concerns before publication).", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_3": "The study by Zeng et al. reports the development of a real-time imaging protocol to monitor acidity in time in the context of\nhepatocellular carcinoma (HCC). In particular, the authors identified ATP6V0C as an important factor for acidification and\nlevels of ATP6V0C correlated with acidification and poor prognosis. Therefore they designed a sensor for tumor detection\nand targeted HCC therapy.\nThe manuscript is well written, the results convincing and the conclusions are supported by the numerous data shown in the\nmanuscript figures and in the supplemental material.\nMajor point:\nDiscussion is quite short and it seems more a summary of the data presented in the results section. Indeed, data are not\ndiscussed critically comparing them with literature data (in the discussion there is not even a single citation). Moreover, even\nsuggestions about the use of these sensors together with other therapeutical means remain general and superficial without\ndetails.\nI believe that the authors should discuss their data throughly, comparing their sensors with others and underlining the\nadvantages of their sensors together with their limitations. In addition, they should discuss throughy their applications in\ncombination with defined therapeutical means.\nMinor points\nFigure 1 is not cited in the text. I believe that it is a sort of graphical abstract of the entire manuscript but it is named figure 1,\nand should be cited in the text. Moreover, the figure legend of this figure should be more informative giving details on the\nproposed ways to use the sensor developed, in particular because it is the first figure that one encounters even before\nreading results.", "reviewer_4": "The article presents a highly innovative approach to detecting and monitoring the acidity of the tumor microenvironment\n(TME) during tumor progression and metastasis using a pH-responsive ratiometric photoacoustic sensor (PPS). The study\ncombines cutting-edge photoacoustic imaging technology with the molecular targeting of ATP6V0C, a subunit of V-ATPase,\nwhich plays a crucial role in TME acidification. The results show that the sensor can effectively visualize spatiotemporal\nchanges in tumor acidity, providing real-time monitoring of TME dynamics in vivo. Additionally, the article explores the\npotential therapeutic implications of combining the PPS with proton pump inhibitors, offering a novel approach to cancer\ntherapy. Overall, the paper is well-written, presenting significant advancements in both diagnostic and therapeutic strategies\nin cancer research. The experimental design is robust, and the use of in vitro, in vivo, and clinical samples strengthens the\nfindings. However, there are a few areas where clarification and further explanation are needed.\n1. i) Clarification on the Sensitivity Range of PPS: The manuscript indicates that PPS is sensitive to pH changes within the\nphysiological range (pH 8-6). Could you provide more detailed information about the sensor's performance outside this\nrange, particularly in more extreme acidic conditions that may occur in advanced tumors?\nii) Specificity and Sensitivity of PPS. The study shows that PPS exhibits significant photoacoustic signal enhancement\nunder acidic conditions. However, in complex in vivo environments, how do the authors rule out interference from other\nfactors (e.g., inflammation or local ischemia) that may also alter pH? Are there experimental data supporting PPS's\nspecificity in non-tumor acidic conditions?\n2. PPS Application in Different Cancer Types: While the study focuses on hepatocellular carcinoma (HCC), it would be\nvaluable to discuss the potential applicability of PPS in other types of cancers with different TME characteristics. Are there\nany limitations in the current approach for other cancer types, such as those with lower acidity or different metabolic profiles?\n3. In Vivo Imaging Depth: The study demonstrates the ability to detect PPS signals at depths of up to 15 mm in tissues.\nHowever, this depth is quite limited in clinical settings, particularly for larger tumors. how does imaging quality and\nquantitative accuracy perform in even deeper tissues (e.g., visceral tumors)? Are there supporting data?\n4. Mechanism of Synergistic Photothermal Therapy. The study suggests that combining PPS with the proton pump inhibitor\n(PPI) esomeprazole (ESOM) synergistically suppresses tumor growth, but the underlying molecular mechanism is not\nthoroughly explored. Are there data showing whether ESOM directly enhances PPS's photothermal effect by inhibiting\nATP6V0C, or do the two act independently?\n5. Reproducibility and Generalizability. The study employs multiple tumor models (e.g., subcutaneous, orthotopic, and\nmetastatic), but are there performance differences in PPS across these models? Do the authors plan to validate PPS's\ngeneralizability in other cancer types? Additionally, some experiments have small sample sizes (e.g., n=3)—could this affect\nstatistical power?"}, "author_response": "Response to the Editorial Comments\nEditorialComment:\nIn particular, we would expect you to further explore the specificity and sensitivity\nof PPS, as well as the potential interference from other metabolites; biologically\nvalidate the claimed ATP6V0C-driven acidification; support PTT-ESOM synergistic\neffects and related mechanism with additional in vitro/vivo analysis; perform in vivo\ntoxicity assessment; included appropriate model evaluating PA imaging\ndepth/resolution;benchmark against current standards ofclinical imaging.\nIf you feel that you are able to comprehensively address the reviewers’ concerns,\nplease provide a point-by-point response to these comments along with your revision.\nPlease show all changes in the manuscript text file with track changes or colour\nhighlighting. If you are unable to address specific reviewer requests or find any points\ninvalid,please explain why in thepoint-by-point response.\nResponse: We sincerely thank you for the valuable and constructive guidance. In this\nrevised version of our manuscript, we have comprehensively addressed all of the\nissues raised, as summarized below:\n1. Specificity and sensitivity of PPS: We have conducted additional experiments to\nvalidate the specificity and sensitivity of PPS both in vivo and in vitro. Moreover,\ncontrol assays were conducted to exclude potential interference from other\nmetabolites,includingROS and proteases.\n2. Biological validation of ATP6V0C-driven acidification: To biologically validate\nthe ATP6V0C-driven acidification, we have added functional rescue experiments,\nwhich demonstrate that ATP6V0C promotes tumor progression by modulating\nacidification ofthetumormicroenvironment.\n3. PTT-ESOM synergistic effects and mechanistic analysis: We have further\nelucidated the synergistic anti-tumor effects of PTT-ESOM and its underlying\nmechanisms.\n1\n4. In vivo toxicity assessment: We have enhanced the long-term toxicity assessment\nof PPS in vivo by supplementing hematological, histological, and physiological\nevaluations within 28days after intravenous administration inmice.\n5. Photoacoustic imaging depth and resolution: Using a rat orthotopic liver cancer\nmodel, we evaluated the depth-resolved imaging capability of PPS-based\nphotoacoustic imaging (PAI). The imaging depth and resolution were analyzed and\nbenchmarked against two clinical imaging modalities:MRIand PET.\nAdditionally, all newly added or modified sections have been highlighted in red\nwithin the revised manuscript and supplementary materials.Adetailed, point-by-point\nresponse toeach reviewercomment is provided inthe followingsection.\nWe sincerely appreciate the opportunity for revision and believe that the\nmodifications have significantly improved the quality of our study. Once again, we\nthank you for your valuablesuggestions.\nReviewer#1(HCC, livercancertherapy):\nGeneralAssessment\nThis study presents a novel pH-responsive ratiometric photoacoustic sensor (PPS) for\nreal-time monitoring of tumor microenvironment (TME) acidification in\nhepatocellular carcinoma (HCC). The authors integrate bioinformatics, molecular\nimaging, and therapeutic intervention to demonstrate the role of ATP6V0C in TME\nacidification and its implications for tumor progression and metastasis. The work is\nwell-structured, methodologically rigorous, and clinically relevant. However, certain\naspects require clarification and additional validation to strengthen theclaims.\nStrengths\n1. Innovative Imaging Tool: The development of PPS for pH-sensitive photoacoustic\nimaging is a significant technical advance, enabling non-invasive, high-resolution\nmonitoringofTME acidification.\nThe dual-wavelength ratio-metric approach enhances specificity and minimizes\nbackground interference with potential to influence HCC diagnosis and targeted\ntherapy.\n2. Mechanistic Insights: Strong bioinformatics and clinical validation linking\nATP6V0C overexpression to HCCprogression and poor prognosis.\nMolecular docking and functional assays convincingly demonstrate the interaction\nbetween esomeprazole(ESOM) andATP6V0C,supporting thetherapeutic rationale.\n3. Multimodal Validation: The study employs multiple models (subcutaneous,\northotopic, metastatic) and human specimens to validate PPSutility.\nCorrelative analyses (hypoxia, angiogenesis) strengthen the biological relevance of\nTME acidification.\n4. Therapeutic Implications: The combination of PPS-mediated photothermal therapy\n(PTT) and ESOM provides a synergistic anti-tumor effect, supported byRNA-seq and\nsingle-cell transcriptomics.\nWe are particularly grateful for the recognition that “the work is well-structured,\nmethodologically rigorous, and clinically relevant”. We have carefully considered all\nyour comments and have performed additional experiments and analyses to address\neachpointcomprehensively,as detailed below.\nMajorConcerns and Recommendations\nComment1:Specificity and Sensitivity ofPPS\nInterference from Other Metabolites: While Supplementary Fig. 10 shows minimal\ninterference from ions (K⁺, Na⁺, Mg²⁺), tumor TME contains lactate, ROS, and\nproteases.\nRequest: Test PPS response in the presence of lactate (due to Warburg effect) or H₂O₂\n(ROS from metabolic stress and immune activity) to rule out other possibilities of\nfalse positivesorsignal modulation.\nResponse: We thank you for this insightful comment regarding the specificity and\nsensitivity of PPS under complex TME conditions. To address this concern, we\nperformed validation experiments to exclude potential interference from reactive\noxygen species (ROS) and proteases (matrix metalloproteinases, MMPs) on the\npH-responsivebehavior ofPPS.\nAs presented in Supplementary Fig. 11, the absorbance and photoacoustic signals\nof PPS remained unaffected across a wide pH range in the presence of H O and\n2 2\nproteases. These data confirm that ROS/proteases do not interfere with the accuracy\nof the PPS response. Based on the unique material properties of polyaniline (PANI)\nwithin PPS and the supporting experimental evidence, we confirm that the\nphotoacoustic signal enhancement of PPS arises exclusively from proton-induced\nchanges in acidity rather than false-positiveeffects induced byROS orproteases.\nThe reasons why PPS remains unaffected by ROS and proteases within the TME\nare summarizedas follows:\n4\n1. Independence from redox activity: ROS are characteristic by-products of tumor\nmetabolism and cell death, primarily generated through lipid peroxidation and\nmitochondrial dysfunction1, 2. The conductive form of PANI (emeraldine salt，ES)\nwithin PPS is produced through protonic acid doping, a proton-dependent but\nredox-inert process. Thus, ROS do not participate in or influence the proton-doping\nmechanism of PANI, ensuring that PPS selectively responds to changes in pH rather\nthan oxidativestress.\n2. Resistance to proteolytic degradation: PANI lacks peptide bonds or enzymatically\ncleavage sites, and is therefore theoretically resistant to interference from proteases in\nthe TME3. Proteases in the TME are not expected to alter the structural integrity or\nfunctional specificity of PPS. Moreover, since proteases do not participate in protonic\nacid doping, any protease-mediated artefacts or false-positive photoacoustic signals\ncan beexcluded.\n3. Relevance of lactate-driven acidification: The primary source of H⁺ in the TME is\nlactate accumulation resulting from Warburg effect, a hallmark of most solid tumors4.\nCancer cells preferentially rely on glycolysis for rapid ATP generation, converting\npyruvate to lactate, which dissociates into lactate ions and protons (H⁺)5. These are\nsubsequently exported, leading to TME acidification. Impaired perfusion and reduced\nclearancefurther exacerbate local H⁺ accumulation. Therefore, PPSaccurately reflects\nlactate-associated pH alterations without interference from other metabolites. The\nrevisions are as follows:\n“To confirm the specificity of PPS for acidic pH detection, we evaluated its\nperformance in solutions containing PBS, KCl, NaCl, MgSO , glucose, H O and\n4 2 2\nproteases, which represent different metabolites and components commonly present\nwithin the TME. PPS exhibited no significant absorbance or PAsignal intensity at pH\n7.4 in any solution; however, at pH 6.5, both the absorbance at 780 nm and the\ncorresponding PA signal intensity increased significantly (Fig. 2o-p and\nSupplementary Fig. 10,11)......”\nSupplementary Fig. 11Anti-interference properties of PPS. a Photographs of PPS\nincubated with various substances at pH 6.5, 7.0 and 7.4. b PAimages of PPS at 780\nnm incubated with different substances at pH 6.5, 7.0 and 7.4. c Absorbance\nmeasurements of PPS at 780 nm incubated with different substances. d Quantitative\nanalysis ofphotoacoustic intensityin(b).\nReferences\n1. Liang, X. et al. Oxidative stress in cancer: from tumor and microenvironment\nremodeling to therapeuticfrontiers. Mol.Cancer.24,219(2025).\n2. Wu, K., El, Z. A., Sayin, V. I. & Papagiannakopoulos, T. The pleiotropic functions\nofreactive oxygen species in cancer.Nat Cancer.5,384-399 (2024).\n3. Linderman, S. W. et al. Enhancing immunotherapy with tumour-responsive\nnanomaterials. Nat. Rev.Clin. Oncol. 22,262-282 (2025).\n4. Swietach, P., Boedtkjer, E. & Pedersen, S. F. How protons pave the way to\naggressivecancers. Nat. Rev.Cancer.23,825-841 (2023).\n5. Chen, J. et al. Lactate and lactylation in cancer. Signal Transduction and Targeted\nTherapy.10,38(2025).\nFunctional Rescue Experiments: While the study associates ATP6V0C with tumor\nacidification, direct functional evidence establishing causality is lacking. The cited\nreviews (Stransky et al., 2016; Luciani et al., 2004; Neri & Supuran, 2011) broadly\nimplicate V-ATPase activity in TME acidification but do not dissect the specific role\nof the ATP6V0C subunit. To strengthen the mechanistic claims, the following\nexperimentsare essential.\nRequest: Include siRNA/CRISPR-mediated ATP6V0C modulation to validate its role\ninpH regulation and tumorprogression.\nResponse: We thank you for highlighting the request for direct biological validation\nof ATP6V0C-driven acidification. In accordance with this suggestion, we conducted\nadditional functional rescue experiments to validate the causal relationship between\nATP6V0C and tumoracidification (Supplementary Fig. 25-27).\nSpecifically, two independent shRNAs targeting ATP6V0C were designed and\ntransfected into HCC cells. The functional consequences of ATP6V0C silencing on\ncellular behaviour were then systematically assessed.\nFunctional assays assessing cell migration, colony formation, and invasion\ncollectively demonstrated the role of shRNA-mediated ATP6V0C regulation in tumor\nacidification and progression. Compared with the negative control (NC) group,\nATP6V0C knockdown markedly suppressed the migratory ability of HCC cells. The\ncolony formation assay revealed a pronounced reduction in both colony number and\nsize, indicating impaired proliferative capacity. Furthermore, in Matrigel-coated\nTranswell chambers, the number of invading cells was significantly decreased upon\nATP6V0C knockdown, confirming its critical role in promoting HCC cell\ninvasiveness (Supplementary Fig. 25-26).\nTo directly validate the role of ATP6V0C in regulating intracellular pH (pHi), we\nemployed the pH-sensitive fluorescent probe BCECF-AM to stain and perform\nimaging analysis on liver cancer cells from different treatment groups. Compared to\nthe NC cells, liver cancer cells transfected with sh1-ATP6V0C or sh2-ATP6V0C\n7\nexhibited significantly lower fluorescence signals. Quantitative analysis of\nfluorescence intensity further confirmed that the intracellular pH of ATP6V0C\nknockdown cells was significantly lower than that of the control group\n(Supplementary Fig. 27).\nThese results provide direct mechanistic evidence that ATP6V0C is a critical\nsubunit required for maintaining proton pump function and pH homeostasis in tumor\ncells; its depletion leads to defective proton export, intracellular acidification, and\nsuppression of key malignant behaviors, including proliferation, migration, and\ninvasion.Therevisions are as follows:\nChanges in themanuscript on page20:(Results section)\n“To assess the functional consequences of ATP6V0C inhibition, we measured\nintracellular pH using a fluorescent pH indicator. Compared with PBS-treated cells,\nESOM-treated cells displayed weaker fluorescence, indicating a reduced intracellular\npH, likely caused by impaired proton efflux and subsequent intracellular acidification\n(Fig. 7c). Correspondingly, shRNA-mediated knockdown ofATP6V0C led to blocked\nproton efflux and intracellular acidification, which in turn inhibited the proliferation\nand invasion capacity of liver cancer cells (Supplementary Fig. 25-27).\nMorphologically, ESOM-treated tumor cells presented nearly circular edges with\nfewer pseudopodia, suggesting that ESOM may inhibit actin polymerization and\ncytoskeletal remodeling through intracellular acidification, thereby suppressing tumor\ncell invasion and metastasis (Fig.7d).....”\nChanges intheSupporting Information on page11:(Methodssection)\n“Silencing of ATP6V0C: To reduce the expression of the ATP6V0C, two specific\nshRNA sequences were designed: shRNA1 (TGACGACATCAGCCTCTACAA) and\nshRNA2 (GCTCGGCCTCTACGGTCTCAT). These shRNAs were synthesized by\nTsingke Biotechnology Co., Ltd. (Beijing, China) and subcloned into the\npLKO.1-PURO vector.The resulting shRNA-expressing plasmids were co-transfected\nwith packaging plasmids into HEK293T cells to produce lentiviral particles. The\n8\ncollected viral supernatants were used to transduce human hepatocellular carcinoma\ncell lines HepG2 and HCCLM3. After infection, cells were cultured in medium\ncontaining 2 μg/mL puromycin to select for stably transduced clones. The selected\nstablecell lines were used for subsequent experiments, and knockdown efficiency was\nassessed byWestern blotanalysis.”\nChanges intheSupporting Information on page42:\nSupplementary Fig. 25 Knockdown of ATP6V0C inhibits the malignant\nphenotypes of HepG2 cells. a Western blot analysis of ATP6V0C expression in\nHepG2 cells transfected with sh1-ATP6V0C or sh2-ATP6V0C. b Wound healing\nassay showing representative bright-field images of HepG2 cells transfected with\nsh1-ATP6V0C or sh2-ATP6V0C at 0 h and 24 h. c Representative bright field images\nof HepG2 cell Clone formation. d Representative microscopic images of invaded\nHepG2 cells transfected with sh1-ATP6V0C or sh2-ATP6V0C in the Transwell\ninvasion assay (Scale bar = 200 μm). e Quantitative analysis of the wound healing\nrate. The error bars represent the mean ± SD(n =3 independent experiments; ****p<\n9\n0.0001). f Quantitative analysis of Clone formation rate. The error bars represent the\nmean ± SD (n = 3 independent experiments; ***p < 0.001, ****p < 0.0001). g\nQuantitative analysis of the invasion rate. The error bars represent the mean ± SD(n =\n3independent experiments; ****p<0.0001).\nChanges intheSupporting Information on page43:\nSupplementary Fig. 26 Knockdown of ATP6V0C inhibits the malignant\nphenotypes of HCCLM3 cells. a Western blot analysis of ATP6V0C expression in\nHCCLM3 cells transfected with sh1-ATP6V0C or sh2-ATP6V0C. b Wound healing\nassay showing representative bright-field images of HCCLM3 cells transfected with\nsh1-ATP6V0C or sh2-ATP6V0C at 0 h and 24 h. c Representative bright field images\nof HCCLM3 cell Clone formation. d Representative microscopic images of invaded\nHCCLM3 cells transfected with sh1-ATP6V0C or sh2-ATP6V0C in the Transwell\ninvasion assay (Scale bar = 200 μm). e Quantitative analysis of the wound healing\nrate. The error bars represent the mean ± SD(n =3 independent experiments; ****p<\n10\n0.0001). f Quantitative analysis of Clone formation rate. The error bars represent the\nmean ± SD (n = 3 independent experiments; ****p < 0.0001). g Quantitative analysis\nof the invasion rate. The error bars represent the mean ± SD (n = 3 independent\nexperiments;****p<0.0001).\nChanges intheSupporting Information on page44:\nSupplementary Fig. 27 Confocal Laser Scanning Microscopy (CLSM) images of\nHepG2 and HCCLM3 cells stained with BCECF-AM after different treatments (Scale\nbar = 50 μm). b-c Quantification offluorescence intensity from (a) (n = 3 independent\nexperiments).\nThe results of this series of functional rescue experiments demonstrate that\nknockdown ofATP6V0C effectively inhibits themigration, proliferation, invasion and\nacidification capabilities of HCC cells. We believe that these additional data\nstrengthen the scientific rigor of our study and fully address your concerns. Once\nagain, thankyou for helping us enhance thequalityof thiswork.\nComment3:TherapeuticEfficacy and Mechanisms\nPTT-ESOM Synergy: The combinatorial effect is promising, but the mechanism\nremains speculative. Does ESOM directly sensitize cells to PTT by decreasing\nintracellularpHlevels?\nRequest: Provide intracellular pH measurements post-PTT+ESOM to clarify the\nmechanism.\npromising synergistic effects of our PTT-ESOM combination strategy. We appreciate\ntheopportunity tofurther clarify theunderlying mechanism.\nWe agree that inhibition of proton extrusion by ESOM is a key contributor to the\nobserved synergy. ESOM does not directly acidify the cytoplasm; instead, it inhibits\nthe proton pump activity ofV-ATPase, thereby blocking H+ efflux from the cytosol1,2.\nThis disruption of intracellular pH homeostasis results in the accumulation of acidic\nmetabolites and a subsequent decrease in intracellular pH(pHi)3.\nIn our study,we adopted a sequential therapeutic strategy to utilize this mechanistic\ninterplay. The PPS was first used for PTT targeting the pre-existing acidic tumor\nmicroenvironment (pHe ≈ 6.5), effectively eliminating the majority of tumor cells.\nESOM was then administered to inhibit proton extrusion in the residual tumor cells,\nfurther disturbing pH regulation and inducing acid-mediated apoptosis and necrosis\n(Fig. 1c). This sequential regimen, PTT followed by ESOM, maximizes therapeutic\nefficacy: if ESOM were administered prior to PTT, the transient reduction in\nextracellular acidity caused by blocked proton efflux would attenuate the\npH-responsive photothermal activation of PPS, thereby diminishing treatment\nefficiency.\nFig. 1 Schematic illustration of the design and application of pH-responsive\nratiometric photoacoustic sensor (PPS). c Schematic illustration of PPS-mediated\npH-responsivephotothermal synergistic PPIs-based acid suppression tumor therapy.\nthat ESOM alone decreased intracellular pH due to impaired H+ extrusion (Fig. 7c),\nthereby reducing the proliferation and invasion of hepatocellular carcinoma cells.\nHowever, ESOM monotherapy only partially suppressed tumor growth in vivo\n(Supplementary Fig. 28). In contrast, the sequential combination treatment\n(L+PPS+ESOM; Group 6) produced significantly enhanced tumor regression\ncompared with PTTalone(L+PPS; Group 5) (Fig. 8).\nTogether, these findings confirm that ESOM enhances PTT efficacy by disrupting\nintracellular pH homeostasis through inhibition of proton extrusion, supporting the\nmechanistic rationale of our sequential PTT-ESOM treatment strategy. To further\nclarify ourresearch strategy,we havemade thefollowing revisions to themanuscript:\nChanges inthemanuscripton page 28:(Discussion section)\n“...... Sequential PTT-ESOM treatment further enhances therapeutic efficacy by\nintegrating photothermal ablation with proton pump inhibition. PPS-mediated PTT\neliminates tumor cells within the acidic microenvironment, while subsequent ESOM\nadministration blocks V-ATPase-driven proton extrusion, intensifying intracellular\nacid stress and reinforcing tumor eradication through pH-targeted synergy.\nNevertheless, this dual functionality positions PPS as a comprehensive clinical tool\nfor cancer management and paves the way for developing probes tailored to unique\nTME characteristics across various cancer types ......”\nReferences\n1. Perut, F. et al. V-ATPase as an effective therapeutic target for sarcomas. Exp. Cell\nRes. 320,21-32(2014).\n2. Marino, M. L. et al. Proton pump inhibition induces autophagy as a survival\nmechanism following oxidative stress in human melanoma cells. Cell Death Dis. 1,\ne87 (2010).\n3. Chen, F., Kang, R., Liu, J. & Tang, D. The V-ATPases in cancer and cell death.\nCancer Gene Ther. 29,1529-1541 (2022).\nreassuring, but long-term toxicity(e.g., 28days)is notassessed.\nRequest: Include chronic toxicity studies, especially given PPS’s potential clinical\ntranslation.\nResponse: We thank you for raising this important point regarding long-term toxicity\nassessment of the PPS, which is indeed crucial for evaluating its clinical translation\npotential. In response, we performed chronic toxicity studies over a 28-day period\nfollowing intravenous administration ofPPSin mice.\nComprehensive hematological, histological, and physiological evaluations were\nconducted. No significant abnormalities were observed in blood parameters, body\nweight, or major organ histopathology compared to control groups. These findings\nconfirm the favorable biocompatibility and biosafety of PPS during prolonged\nsystemic exposure, further supportingits potential for futureclinical translation.\nThe added chronic toxicity data have been incorporated into the revised\nSupplementary Information:\nChanges inthemanuscripton page 26:(Resultssection)\n“...... To assess the potential for the clinical translation of PPS, its biosafety was\nevaluated both in vitro and in vivo. As shown in Supplementary Fig. 37, PPS at\nconcentrations up to 500 μg/mL had no significant cytotoxicity in normal liver cells\n(THLE-3) or the liver cancer cell lines HepG2 and HCCLM3. Hemocompatibility\ntesting demonstrated minimal hemolysis following a 4-hour incubation with red blood\ncells (Supplementary Fig. 38). To further evaluate long-term safety, chronic toxicity\nstudies were performed over a 28-day period following intravenous administration of\nPPS. Comprehensive hematological, histological, and physiological analyses showed\nno significant abnormalities in blood parameters, body weight, or major organ\nhistopathology compared to control mice (Supplementary Fig. 39-42). Furthermore,\nhistological examinations of major organs revealed no significant pathological\nchanges compared with those in saline-treated controls (Supplementary Fig. 43).\nunder prolonged systemic exposure, supporting its potential for future clinical\ntranslation ......”\nChanges intheSupporting Information on page13:(Methodssection)\n“...... In vivo biosafety evaluation: Healthy BALB/c nude mice were randomly\ndivided into three groups: PBS control, PPS (0.5 mg/kg), and PPS (1 mg/kg). Body\nweight was recorded every four days, as a general indicator of systemic toxicity and\noverallhealth status.\nFor hematological and histopathological analyses, healthy BALB/c nude mice were\nintravenously injected with either PBS or PPS (1 mg/kg). At 7, 14, and 28 days\npost-injection, the mice were euthanized, and blood samples as well as major\norgans—including the heart, liver, kidneys, lungs, and spleen—were collected.\nComplete blood count (CBC) analyses were performed using an automated\nhematology analyzer (BC-2800vet, Mindray). The collected organs were sectioned\nand stained with hematoxylin and eosin (H&E), followed by imaging and\nhistopathological evaluation using adigital microscope......”\nChanges intheSupporting Information on page57:\ninjection. a White blood cell (WBC) count. b Neutrophil (NEUT) count. c Red blood\ncell (RBC)count. d Mean corpuscular hemoglobin (MCH) concentration. e Redblood\ncell distributionwidth (RDW). fMean corpuscular volume(MCV).\nChanges intheSupporting Information on page58:\nSupplementary Fig. 40 Liver function of mice on days 7, 14, and 28 following PPS\ninjection. a γ-glutamy transpeptidase (γ-GT). b Serum albumin (ALB). c Total bile\nacid (TBA). d Direct bilirubin (DBIL). e Total bilirubin (TBIL). f Alanine\namiotransferase (ALT).\nChanges intheSupporting Information on page59:\ninjection. a Blood urea nitrogen (BUN). b Serum creatinine (CREA). c Uric acid\n(UA).\nChanges intheSupporting Information on page60:\nSupplementary Fig. 42 Body weights as functions of time in mice treated with PPS\n(0.5mg/kgor1mg/kg,100μL)or PBS through intravenous injection.\nChanges intheSupporting Information on page61:\nincluding the heart, liver, spleen, kidneys, and lungs, collected from the mice on days\n7,14,and 28following PPSinjection (Scale bar=100 μm).\nComment5:Technical Considerations\nPA Imaging Depth and Resolution: The study claims deep-tissue imaging (15 mm,\nSupplementary Fig. 9), but tumor models (subcutaneous, orthotopic) may not fully\nrecapitulatedeep HCClesions.\nRequest: Compare PPS performance with current standards of clinical imaging (e.g.,\nMRIorPET)in deeper tumors.\nResponse: We appreciate this valuable suggestion. To address this, we have now\nestablished a rat orthotopic HCC model to assess the performance of PPS-based PA\nimaging in deep-seated liver tumors. Comparative imaging with MRI and PET was\nperformed.\nAs shown in the Supplementary Fig. 23, PPS enabled precise visualization of\nintraparenchymal liver tumors consistent with MRI and PET results. In vitro phantom\nstudies also confirmed the capability of PPS to image at depths up to 15 mm\n(Supplementary Fig. 9). Furthermore, by comparing PPS-based photoacoustic\nimaging with PET imaging, we found that PPS-assisted photoacoustic imaging\nexhibited comparable tumor detection performance to PET in identifying small tumor\nlesions (Supplementary Fig. 19), indicating that this PPS-based imaging strategy\npossesses reliable sensitivityandaccuracy fortumordetection.\nWhile we acknowledge that the penetration depth of PA imaging (typically within\n2-3 cm) is lower than MRI or PET, PPS-based PA imaging offers superior spatial\nresolution (~200 μm) and real-time imaging capability, making it suitable for\ndetecting superficial or moderately deep lesions.\nComparative imaging data have been incorporated into the revised Supplementary\nInformation,and thelimitations are discussed in theDiscussion section.\n“......To further evaluate the performance of PPS in deep-seated liver tumors, we\nestablished a rat orthotopic HCC model and conducted comparative imaging with\nMRI and PET. PPS-enabled PA imaging precisely visualized intraparenchymal liver\ntumors, showing tumor localization consistent with MRI and PET results\n(Supplementary Fig. 23). These findings highlight the potential of PPS as a sensitive\nand specific imaging modality for detecting and characterizing acidic TMEs in\northotopic and metastatic liver tumors, including deep-seated liver tumors comparable\ntoclinical imaging standards such as MRIand PET......”\nChanges inSupplemental Information on Page40:\nSupplementary Fig. 23 Multimodal imaging in the rat orthotopic hepatocellular\ncarcinoma model. a MRI images (transverse, coronal, sagittal, and 3D views)\nshowing the localization of deep-seated liver tumors (outlined in magenta). b PET\nimages (coronal and sagittal views) of orthotopic hepatocellular carcinoma in rats. c\nSchematicillustration of photoacoustic(PA)imaging of orthotopic liver tumors in rats.\nd Representative ultrasound (US) and PA images at 780 nm and 1064 nm, showing\nPPS-enhanced signal contrast at thetumorsite(outlined in magenta).\n“..... In addition, we validated the progressive and dynamic acidification of the TME\nacross various stages of HCC progression, providing in vivo evidence that proton\npump-driven acidification represents a continuous process in tumor development.\nThese findings highlight the potential of proton pump-driven acid imaging strategies\nfor both HCC diagnosis and therapy monitoring. Notably, these results were further\ncorroborated in resected clinical HCC tissues. While PPS-based PAimaging is limited\nin\npenetration depth compared with MRI or PET, it provides superior spatial\nresolution (~200 μm) and real-time monitoring of dynamic pH changes. These\nfeatures enable mechanistic insights into early TME acidification and temporal proton\ndynamics, complementing conventional clinical imaging ......”\nComment6:\nMinorConcerns\nFigure Clarity: Fig. 8 (RNA-seq heatmap) lacks gene labels—highlight key DEGs\n(e.g., metabolic regulators).\nResponse: We sincerely thank you for this valuable suggestion regarding the clarity\nof Fig. 8 (the RNA-seq heatmap). To maintain figure readability, the complete\nheatmap with all labeled genes has been provided in the Supplementary Information,\nand the figure legend has been updated to reflect this. The revised RNA-seq heatmap\nis presented as follows:\nChanges inthemanuscripton page 26:(Resultssection)\nFig 8 i Heatmap showing hierarchical clustering of samples based on gene expression\nlevels. The complete heatmap with all labeled genes is shown in Supplementary Fig.\n44.\nSupplementary Fig. 44 Heatmap showing hierarchical clustering of samples on the\nbasis ofgene expression level.\nand therapy. The findings are well-supported but would benefit from additional\nmechanisticand translational validation.\nAcceptance Decision: MinorRevision (address majorconcerns before publication).\nResponse: Wesincerely thank you for the encouraging evaluation and for recognizing\nour work as “a compelling advance in TME imaging and therapy” with\n“well-supported” findings. We are grateful for the recommendation of “Minor\nRevision” and have carefully addressed all major and minorconcerns raised.\nFinally, we would like to thank you for the constructive suggestions on how to\nfurther strengthen this manuscript. We believe that the additional experiments and\nrevisions have significantly strengthened the overall scientific rigor and presentation\nofourwork.\nReviewer#2(ECR):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nResponse: We would like to express our sincere gratitude to the co-reviewer who\nparticipated in the evaluation of our manuscript through the co-review scheme. We\ngreatly appreciate the time, careful consideration, and valuable feedback provided\nduring the peer review process. The constructive comments jointly provided by you\nand the primary reviewer were significant in improving the quality of our work. We\nhave carefully addressed all the comments point by point and conducted additional\nexperiments as suggested to further strengthen our study. Thank you for your\ncontribution.\nThe study by Zeng et al. reports the development of a real-time imaging protocol to\nmonitor acidity in time in the context of hepatocellular carcinoma (HCC). In\nparticular,the authors identifiedATP6V0C as an important factor for acidification and\nlevels of ATP6V0C correlated with acidification and poor prognosis. Therefore they\ndesigned a sensor for tumor detection and targeted HCC therapy. The manuscript is\nwell written, the results convincing and the conclusions are supported by the\nnumerous data shown in themanuscript figures andin thesupplemental material.\nResponse: We sincerely thank you for the positive evaluation of our work and for the\nconstructive suggestions provided. In response to your insightful comments, we have\nmadetwo key revisions to improvethemanuscript:\n1. We have expanded the Discussion section to incorporate a more critical analysis of\ntheimplicationsand limitationsof ourfindings.\n2. We have revised the caption of Fig. 1 to improve the clarity and logical flow of the\ngraphical abstract, ensuring that the conceptual framework is more accessible and\naccuratelyconveys thestudy’s core message.\nWe believe these revisions have significantly strengthened the academic rigor and\nreadabilityof themanuscript. Detailed point-by-point responses are provided below.\nComment1:\nMajor point: Discussion is quite short and it seems more a summary of the data\npresented in the results section. Indeed, data are not discussed critically comparing\nthem with literature data (in the discussion there is not even a single citation).\nMoreover, even suggestions about the use of these sensors together with other\ntherapeutical means remain general and superficial withoutdetails.\nI believe that the authors should discuss their data throughly, comparing their\nsensors with others and underlining the advantages of their sensors together with their\nlimitations. In addition, they should discuss throughy their applications in\ncombination withdefined therapeutical means.\nthe importance of comparing PPS with existing literature data and discussing its\npotential in combination with other therapeutic modalities. We have thoroughly\nrevised the Discussion section in accordance with this guidance. The specific\nenhancements are as follows:\n1. We have added a critical comparison between PPS and existing pH-sensing\ntechnologies. Specifically, we now discuss the limitations of small-molecule\nfluorescent probes and single-wavelength photoacoustic probes, thereby clearly\nunderscoring the distinct advantage of our ratiometric PPS for quantitative deep-tissue\nimaging.\n2. We have deepened the discussion of our biological findings by actively engaging\nwith the existing literature. Our observation onearlyTME acidification is nowframed\nwithin the context of the “acidic niche” concept, and we clarify how our findings on\nits role as an early biomarker extend beyond its well-established function in\npromotinginvasion.\n3. We have substantially elaborated on the potential for combination therapies and\nacknowledged the limitations of our sensor. The previously general suggestion has\nbeen replaced with a specific, actionable example of how PPS could guide\nacid-suppression therapy to enhance the efficacy of chemotherapy/immunotherapy.\nFurthermore, we explicitly state the limitation of PPS-mediated photothermal therapy\ndueto light penetration depth, providing abalanced perspective.\nChanges inthemanuscripton page 27:(Discussion section)\n“...... In this study, we have developed a non-invasive, real-time imaging strategy to\nmonitor spatiotemporal changes in TME acidity during tumor initiation, progression,\nand metastasis. By integrating bioinformatics analyses with validation using clinical\nsamples, we identified ATP6V0C as a key driver of TME acidification during tumor\ndevelopment. Elevated ATP6V0C was strongly correlated with increased HCC acidity\nand poor patient prognosis. Based on these findings, we designed a pH-responsive\nratiometric photoacoustic sensor with excellent biocompatibility. PPS undergoes a\n24\npH-induced transition from EB to ES within the physiological pH range (pH 8-6),\nenabling sensitive H+ activation and amplified PA signals at 780 nm. Compared with\nexisting pH sensors, such as small-molecule fluorescent probes that exhibit limited\ntissuepenetration and single-wavelength PA probes that are susceptible to nonspecific\nsignal fluctuations62,63, PPS provides intrinsic ratiometric calibration, significantly\nimproving quantitative accuracy in deep-tissue imaging. This feature establishes PPS\nas apromising and reliable tool for precisetumordetection and targeted HCC therapy.\nLeveraging PPS's acid-responsive properties, we identified a strong association\nbetween early-stage TME acidification in HCC and multiple molecular events,\nincluding hypoxia and angiogenesis, which collectively drive tumor proliferation and\nmetastasis. Our observation that acidification precedes macroscopic tumor growth\naligns with the emerging concept of \"acidic niche\" formation as a facilitator of tumor\nprogression64. This finding extends beyond the well-established role of acidosis in\ndriving invasion by highlighting its potential as an early diagnostic biomarker6, 65. In\naddition, we validated the progressive and dynamic acidification of the TME across\nvarious stages of HCC progression, providing in vivo evidence that proton\npump-driven acidification represents a continuous process in tumor development.\nThese findings highlight the potential of proton pump-driven acid imaging strategies\nfor both HCC diagnosis and therapy monitoring. Notably, these results were further\ncorroborated inresected clinical HCC tissues. WhilePPS-based PAimaging islimited\nin penetration depth compared with MRI or PET, it provides superior spatial\nresolution (~200 μm) and real-time monitoring of dynamic pH changes. These\nfeatures enable mechanistic insights into early TME acidification and temporal proton\ndynamics, complementing conventional clinical imaging.\nBeyond its diagnostic capabilities, PPS demonstrated the ability to provide\nreal-time feedback during PPIs-based acid-suppressive treatments, representing a\npromising approach for assessing therapeutic responses in HCC patients. Our findings\nsuggest that proton pump-driven acid imaging strategies can dynamically monitor and\nprovide feedback on TME acidification, enhancing the precision of cancer therapy.\nThisis particularly relevant for combination therapies. For instance, theacidic TME is\n25\nknown to dampen theefficacy of chemotherapy and immunotherapy66.The PPS-based\nimaging detection can be employed to dynamically assess TME acidification\nthroughout different treatment courses, thereby enabling the dynamic adjustment of\ndosing regimens to maintain a therapy-friendly TME. Moreover, PPS exhibited\npH-dependent photothermal properties, expanding its application to theranostics by\ncombining diagnostic imaging with potential therapeutic interventions. While the\nphotothermal conversion efficiency of PPS is competitive with other organic\ntheranostic agents like polydopamine nanoparticles67, its primary limitation for\nstandalone therapy remains the limited penetration depth of light, which may restrict\nits efficacy in deep-seated tumors. Sequential PTT-ESOM treatment further enhances\ntherapeuticefficacy byintegrating photothermal ablation with proton pump inhibition.\nPPS-mediated PTT eliminates tumor cells within the acidic microenvironment, while\nsubsequent ESOM administration blocks V-ATPase-driven proton extrusion, intensifying\nintracellular acid stress and reinforcing tumor eradication through pH-targeted synergy.\nNevertheless, this dual functionality positions PPS as a comprehensive clinical tool\nfor cancer management and paves the way for developing probes tailored to unique\nTMEcharacteristics across various cancer types.\nOverall, this work establishes a novel framework for precise cancer diagnosis and\ntreatment using proton pump-driven acid imaging. Our preliminary results suggest\nthat this approach demonstrates superior performance in evaluating TME acidity and\nits associated pathological processes. However, the generalizability of PPS across\ncancer types may be influenced by the heterogeneous expression ofATP6V0C. Future\nresearch should focus on integrating PPS with other TME metabolic feature-based\nmultifunctional molecular imaging strategies to further optimize its diagnostic and\ntherapeutic utility. Additionally, exploring its compatibility with other therapeutic\nmodalities, specifically testing the proposed combination with\nchemotherapy/immunotherapy and its application in personalized medicine could\nexpand its clinical impact, providing new avenues for managing HCC and other\nmalignancies ......”\nand balance of the Discussion. We deeply appreciate your comments, which have\nbeen instrumental in strengthening the conceptual and translational impact of our\nmanuscript.\nReferences\n62. Wang, X. et al. Fluorescent Probes for Disease Diagnosis. Chem. Rev. 124,\n7106-7164 (2024).\n63. Fu, Q., Zhu, R., Song, J., Yang, H. & Chen, X. Photoacoustic Imaging: Contrast\nAgents andTheir BiomedicalApplications.Adv.Mater.31,e1805875(2019).\n64. Chen, J. et al. Lactate and lactylation in cancer. Signal Transduction and Targeted\nTherapy.10,38(2025).\n65. Chen, F., Kang, R., Liu, J. & Tang, D. The V-ATPases in cancer and cell death.\nCancer GeneTher.29,1529-1541 (2022).\n66. Wang, B. Y. et al. Intermittent high dose proton pump inhibitor enhances the\nantitumor effects of chemotherapy in metastatic breast cancer. J Exp Clin Cancer\nRes. 34,85(2015).\n67. Cai, Y. et al. Phototherapy in cancer treatment: strategies and challenges. Signal\nTransductTargetTher.10,115 (2025).\nComment2:Minorpoints\nFigure 1 is not cited in the text. I believe that it is a sort of graphical abstract of the\nentire manuscript but it is named figure 1, and should be cited in the text. Moreover,\nthe figure legend of this figure should be more informative giving details on the\nproposed ways to use the sensor developed, in particular because it is the first figure\nthat oneencounters even before reading results.\nResponse: Thank you for this valuable feedback. We have revised the manuscript\naccordingly based onyour suggestions.The specific modifications are as follows:\n(1)Wehave nowcited Fig.1in themain textto ensureits proper contextualization.\n(2) We have expanded the figure legend of Fig. 1 to provide more detailed\ninformation on the potential applications and operational context of the PPS, thereby\nclarifying its practical usescenarios.Therevisions are as follows:\n“...... In this study, we have developed a high-specific and -sensitive PA molecular\nimaging method to detect TME acidity during tumor initiation, progression, and\nmetastasis (Fig.1a,b)......”\n“...... Furthermore, we evaluated the therapeutic potential of combining PPS-based\nH+-activated photothermal therapy (PTT) with proton pump inhibitors (PPIs), which\ndemonstrated its efficacy ininhibiting tumorgrowth (Fig.1c)......”\nChanges intheManuscriptpage5:\nFig. 1 Schematic illustration of the design and application of pH-responsive\nratiometric photoacoustic sensor (PPS). a Schematic illustration of multiscale\nproton pump-driven acid photoacoustic imaging mediated by PPS. PPS-based\npH-responsive photoacoustic ratio imaging allows dynamic monitoring of tumor\nmicroenvironment acidity from early lesions to metastatic tumors, validated in clinical\nhepatocellular carcinoma samples. b Schematic illustration of metabolic changes in\nthe tumor microenvironment during tumor initiation and progression. During tumor\ninitiation, cells proliferate with altered oxygen and glucose levels. Tumor growth\nenhances glycolysis and hypoxia, increasing H⁺ concentration. Later stages involve\nMMPs-mediated ECM degradation, EMT, and angiogenesis, promoting invasion and\nmetastasis, while extracellular pH (pHe) remains ~6.5. c Schematic illustration of\nPPS-mediated pH-responsive photothermal synergistic PPIs-based acid suppression\ntumortherapy.\nWe are deeply grateful to you for the expert feedback. The expanded Discussion\nand improved Fig. 1 now provide a more rigorous, contextualized, and reader-friendly\npresentation. These revisions have considerably strengthened both the scientific and\nconceptual dimensionsof themanuscript.\nThe article presents a highly innovative approach to detecting and monitoring the\nacidity of the tumor microenvironment (TME) during tumor progression and\nmetastasis using a pH-responsive ratiometric photoacoustic sensor (PPS). The study\ncombines cutting-edge photoacoustic imaging technology with the molecular\ntargeting of ATP6V0C, a subunit of V-ATPase, which plays a crucial role in TME\nacidification. The results show that the sensor can effectively visualize spatiotemporal\nchanges in tumor acidity, providing real-time monitoring of TME dynamics in vivo.\nAdditionally, the article explores the potential therapeutic implications of combining\nthePPSwith protonpump inhibitors, offering anovel approach to cancer therapy.\nResponse: Weare deeply grateful to you for the comprehensive evaluation and highly\npositive feedback on our work. We sincerely appreciate the recognition of the\ninnovation underlying the PPS developed in our study, as well as its potential for\nreal-time monitoring of acidity in the TME. We are particularly encouraged by the\nreviewer’s comment emphasizing the biological relevance of targetingATP6V0C and\nthe translational prospects of combining PPS with proton pump inhibitors. The\nassessment further highlights the significance of our work and its potential\ncontribution toadvancing molecular imaging and cancer therapy.\nWe have carefully considered each of the reviewer’s constructive comments and\naddressed them in detail below.\nComment1:\n(i) Clarification on the Sensitivity Range of PPS:The manuscript indicates that PPS is\nsensitive to pH changes within the physiological range (pH 8-6). Could you provide\nmore detailed information about the sensor's performance outside this range,\nparticularly inmore extreme acidic conditionsthat mayoccur inadvanced tumors?\nResponse: We appreciate your insightful comment regarding the sensitivity range of\nPPS. To directly address this concern, we performed additional imaging experiments\nusing an advanced orthotopic HCC model with intrahepatic metastases to evaluate the\nimaging performance ofPPSunderhighly acidic conditions inlate-stage tumors.\nacross a broad pH range of 3–7,with increased absorbance at 780nm as pH decreased\n(Fig. 2m and Supplementary Fig. 12). Notably, PPS maintained high sensitivity even\nunder strongly acidic conditions (pH 3–4). Furthermore, in vitro studies revealed a\nclear ratiometric photoacoustic (PA) response, where the PA /PA ratio increased\n780 1064\nmonotonically as pH decreased from 7 to 4 (Supplementary Fig. 12). Collectively,\nthese results confirm that PPS remains highly responsive well beyond the acidity\nlevels typically observed in tumormicroenvironments (TME).\nDuring tumor progression, metabolic reprogramming gradually acidifies the\nmicroenvironment, promoting an acidic TME that facilitates proliferation and\nmetastasis1. In our in vivo imaging validation (Fig. 4d), we employed the pancreatic\ncancer liver metastasis model that mimics the late stages of tumor development.\nPPS-mediated PA imaging showed significantly enhanced signal intensity compared\nto normal liver tissue. Unlike the localized signal observed in primary orthotopic\nHCC lesions, the metastatic model displayed a diffuse and widespread PA signal\nenhancement across the hepatic parenchyma, further underscoring the high specificity\nand sensitivityof PPSfor acidic lesions.\nPrevious studies have reported that the extracellular pH of advanced HCC typically\napproaches ~6.52, 3. In our supplementary imaging experiments, PPS exhibited\nmarkedly enhanced PA signals within these highly acidic tumor regions, with strong\nconcordance between PA signal intensity and ex vivo histopathological findings.\nThese results further validate the reliability of PPS under extremely acidic TME\nconditions.\nTaken together, the broad pH responsiveness of PPS (pH 3-7) and its robust\nimaging performance in advanced HCC models demonstrate its suitability for\nmonitoring intratumoral acidity throughout the entire course of tumor progression.\nThe additional imaging data have been incorporated into the revised Supplementary\nInformation.\n“Quantification of the PA signal ratios further confirmed a significant increase in\ntumor-bearing livers compared with that observed in normal controls (Fig. 4f, g).\nAdditionally, in an advanced orthotopic HCC model with intrahepatic metastases,\nPPS-mediated PA imaging enabled clear visualization of intraparenchymal tumor\nlesions, showing diffuse and markedly enhanced PAsignals (Supplementary Fig. 22).\nThe PA imaging results were corroborated by H&E staining (Fig. 4h), which\nconfirmed thepresenceof tumors andan associated acidicTME.”\nChanges intheSupplemental Information onPage39:\nSupplementary Fig. 22 Validation of the sensitivity of PPS PA imaging in\nadvanced-stage hepatocellular carcinoma models. a PAimaging of the advanced-stage\nhepatocellularcarcinomamodels at 9hours postinjection of PPS.b-cPhotographs and\nH&E stainingof tumortissues (indicated byarrows).\nReferences\n1.Liu, Z. et al. Multi-stage mechanisms of tumormetastasis and therapeutic strategies.\nSignalTransductionandTargetedTherapy.9,270(2024).\n2. Swietach, P., Boedtkjer, E. & Pedersen, S. F. How protons pave the way to\naggressivecancers. Nat. Rev.Cancer.23,825-841 (2023).\n3. Feng, Q. et al. Severely polarized extracellular acidity around tumour cells. Nat\nBiomed Eng. 8,787-799 (2024).\n31\n(ii) Specificity and Sensitivity of PPS. The study shows that PPS exhibits significant\nphotoacoustic signal enhancement under acidic conditions. However, in complex in\nvivo environments, how do the authors rule out interference from other factors (e.g.,\ninflammation or local ischemia) that may also alter pH? Are there experimental data\nsupportingPPS's specificity in non-tumoracidic conditions?\nResponse: We sincerely thank you for this important question regarding PPS\nspecificity in complex biological settings. We totally agree that distinguishing\ntumor-specific acidity from non-tumor pathological acidification (e.g., inflammation\nor local ischemia) is indeed essential for evaluating translational applicability. To\naddress this, we established two well-characterized non-tumor models that can induce\nlocal acidification andinflammation:\n1. Thioacetamide (TAA)-induced acute liver failure model, which causes pronounced\nhepaticinflammation and metabolicacidosis.\n2. Hepatic ischemia-reperfusion injury (HIRI) model, characterized by transient\ntissuehypoxia and ischemic acidification.\nPhotoacoustic imaging revealed that, unlike tumor models, PPS exhibited\nnegligible signal enhancement in both non-tumor acidic models (Supplementary Fig.\n21). This indicates that PPS activation requires a sustained and localized acidic\nmicroenvironment, as occurs uniquelyin tumortissues.\nMoreover, PPS demonstrated strong anti-interference stability in vitro. Neither\nreactive oxygen species (ROS), proteases, nor other microenvironmental components\naffected its pH-responsiveoptical orPAbehavior (Supplementary Fig. 11).\nCollectively, these findings confirm that PPS selectively responds to persistent\ntumor-specific acidity rather than transient acidosis associated with inflammation or\nischemia.\nChanges intheManuscripton page13:(Results section)\n“...... Real-time imaging of H⁺ levels in liver tumor lesions is crucial for\nunderstanding pathological processes within the acidic TME. Before in vivo tumor\nimaging, we evaluated whether other pathological factors could interfere with pH\n32\nmonitoring. Using two non-tumor disease models that induce local inflammation or\ntissue acidosis (acute liver failure and hepatic ischemia-reperfusion injury), PA\nimaging showed that PPS exhibited minimal or negligible signal enhancement\ncompared with tumor tissues (Supplementary Fig. 21), indicating excellent pH\nspecificity and insensitivity toother metaboliccomponents intheTME ......”\nChanges intheSupplemental Information onPage38:\nSupplementary Fig. 21 In vivo specificity validation of PPS. a-c Serum levels of\nALT,AST, and TBil. d Photoacoustic images of a mouse model of acute liver failure.\ne Photographs of the modeling surgery for hepatic ischemia-reperfusion injury (the\nyellow arrow indicates the occluded portal vein branch). f Photoacoustic images of a\nmousemodel ofhepatic ischemia-reperfusion injury.\nSupplementary Fig. 11. Anti-interference properties of PPS. a Photographs of PPS\nincubated with various substances at pH 6.5, 7.0 and 7.4. b PAimages of PPS at 780\nnm incubated with different substances at pH 6.5, 7.0 and 7.4. c Absorbance\nmeasurements of PPS at 780 nm incubated with different substances. d Quantitative\nanalysis ofphotoacoustic intensityin(b).\nComment 2: PPS Application in Different Cancer Types: While the study focuses on\nhepatocellular carcinoma (HCC), it would be valuable to discuss the potential\napplicability of PPS in other types of cancers with different TME characteristics. Are\nthere any limitations in the current approach for other cancer types, such as those with\nloweracidity ordifferent metabolicprofiles?\nResponse: We thank you for this valuable suggestion. To further explore the\napplicability of PPS across various cancer types, we performed additional\nexperiments and data analyses focusing on the relationship between the expression\nlevel ofATP6V0C, a key regulator of proton transport and TME acidification, and the\nefficacy ofPPS.\npublicly available transcriptomic and single-cell sequencing datasets (GEO database\nof GSE203024, GSE290925, GSE29282, and GSE277905). The results revealed that\nATP6V0C expression was markedly elevated in hepatocellular carcinoma (HCC),\nsignificantly higher than in breast cancer (BC) and colon adenocarcinoma (COAD)\n(Supplementary Fig. 29). This finding supports that HCC, characterized by its\nintrinsically high acidification potential, serves as an ideal tumor model for\ninvestigatingPPS-mediated acidity detection.\nWefurther validated the in vivo imaging performance of PPS in three tumor models\nwith distinct ATP6V0C expression profiles, including subcutaneous xenografts of\nHCC, breast cancer, and colon cancer. After intravenous administration of PPS,\nphotoacoustic (PA) imaging was performed for comparative analysis. As shown in\nSupplementary Fig. 30, HCC exhibited the strongest PAsignal, followed by moderate\nsignal intensity in COAD, while BC displayed the weakest response. This trend\nclosely correlated with the increasing order of ATP6V0C expression levels (HCC >\nCOAD > BC), indicating that PPS signal intensity is positively associated with the\ntumor’s intrinsicacidification capability.\nCollectively,these results suggest that the performance of PPS is directly correlated\nwith the acidity of the TME. Cancers with lower ATP6V0C expression and reduced\nproton pump activity exhibit a less acidic baseline, thereby diminishing PPS\nactivation efficiency. Consequently, different tumor types may display varying levels\nof responsiveness to PPS-based PA imaging. The additional imaging data have been\nincorporated into therevised Supplementary Information.\nChanges intheManuscripton page20:(Results section)\n“...... To further assess PPS applicability across tumor types, we analyzed ATP6V0C\nexpression pan-cancer and found it highest in hepatocellular carcinoma (HCC), higher\nthan in colon adenocarcinoma (COAD) and breast cancer (BC) (Supplementary Fig.\n29). Consistently, PPS-mediated PA imaging in HCC, COAD, and BC xenografts\nshowed the strongest signal in HCC, moderate in COAD, and weakest in BC\n35\n(Supplementary Fig. 30), indicating that PPS signal intensity correlates with\nATP6V0C-driven tumoracidification ......”\nChanges intheSupplemental Information onPage46:\nSupplementary Fig. 29 Expression of ATP6V0C in different cancer types. a\nexpression of ATP6V0C in cancer types from the GSE203024 (BLCA: Bladder\nCOAD: Colon Adenocarcinoma; EC: Endometrial Carcinoma; HCC: Hepatocellular\ncarcinoma; NPC: Nasopharyngeal carcinoma; OV: Ovarian Cancer; PRAD: Prostate\nAdenocarcinoma; STAD: Stomach Adenocarcinoma). b Single-cell analysis of\nATP6V0C expression in HCC, BC and COAD from the GSE290925, GSE29282, and\nGSE277905. c-e UMAP projection of tumor cells from the corresponding cell type\nannotation panels for HCC, BC,and COAD cells.\nChanges intheSupplemental Information onpage48:\nSupplementary Fig. 30 PPS-assisted PA imaging in different cancer types. a\nRatiometric PA imaging of HCC, BC, and COAD. b Quantification of the\nPA /PA signal ratios from (a). c pH values for tumor from HCC, BC, and COAD,\n780 1064\nas calculated from (a).\nComment3: In Vivo Imaging Depth:The study demonstrates the ability to detect PPS\nsignals at depths of up to 15 mm in tissues. However, this depth is quite limited in\nclinical settings, particularly for larger tumors. how does imaging quality and\nquantitative accuracy perform in even deeper tissues (e.g., visceral tumors)?Are there\nsupportingdata?\nResponse: We sincerely appreciate your insightful comments concerning the\ntranslational potential of our imaging approach, particularly regarding its applicability\n37\nto deep-seated visceral tumors. We fully agree that achieving reliable imaging\nperformance indeep tissuesis crucial for potentialclinical translation.\nTo address this important point, we conducted additional in vivo experiments in a\nrat orthotopic hepatocellular carcinoma model to evaluate the performance of PPS in\ndeep-tissue environments. Moreover, we complemented the photoacoustic (PA)\nimaging results withMRIand PETimaging for cross-validation.\nResults demonstrated that PPS exhibited specific pH-responsive activation within\ndeep hepatic tumor regions, generating distinct PA signals that clearly differentiated\ntumor tissue from surrounding normal liver parenchyma. The localization and signal\npatterns observed were highly consistent with those obtained from MRI and PET\nimaging modalities, thereby reinforcing the reliability and quantitative accuracy of the\nPAimaging datain deep-tissuesettings.\nWhile we acknowledge that PAimaging still faces inherent challenges in achieving\nlarge imaging depths in clinical contexts, it retains unique advantages in terms of\nspatial resolution and molecular specificity compared to conventional modalities.\nFurthermore, we are currently exploring advanced strategies such as PAendoscopy to\nenhanceimaging penetration and enable real-timemonitoringof visceral tumors.\nThese additional imaging results and discussions have been incorporated into the\nrevised manuscript. We have also expanded the Discussion section to clarify the\ncurrent advantages, limitations, and future directions of deep-tissue PA imaging for\npotential clinical applications.\nChanges inthemanuscripton page 14:(Resultssection)\n“...... To further evaluate the performance of PPS in deep-seated liver tumors, we\nestablished a rat orthotopic HCC model and conducted comparative imaging with\nMRI and PET. PPS-enabled PA imaging precisely visualized intraparenchymal liver\ntumors, showing tumor localization consistent with MRI and PET results\n(Supplementary Fig. 23). These findings highlight the potential of PPS as a sensitive\nand specific imaging modality for detecting and characterizing acidic TMEs in\northotopic and metastatic liver tumors, including in deep-seated liver tumors\ncomparableto clinical imaging standards such as MRIand PET......”\nChanges intheSupplemental Information onPage40:\nSupplementary Fig. 23 Multimodal Imaging in rat Orthotopic Model of\nHepatocellularCarcinoma.aMRIimages (transverse, coronal, sagittal, and 3Dviews)\nshowing the localization of deep-seated liver tumors (outlined in magenta). b PET\nimages (coronal and sagittal views) of orthotopic hepatocellular carcinoma in rats. c\nSchematicillustration of photoacoustic(PA)imaging of orthotopic liver tumors in rats.\nd Representative ultrasound (US) and PA images at 780 nm and 1064 nm, showing\nPPS-enhanced signal contrast at thetumorsite(outlined in magenta).\n“..... In addition, we validated the progressive and dynamic acidification of the TME\nacross various stages of HCC progression, providing in vivo evidence that proton\npump-driven acidification represents a continuous process in tumor development.\nThese findings highlight the potential of proton pump-driven acid imaging strategies\nfor both HCC diagnosis and therapy monitoring. Notably, these results were further\ncorroborated in resected clinical HCC tissues. While PPS-based PAimaging is limited\nin penetration depth compared with MRI or PET, it provides superior spatial\nresolution (~200 μm) and real-time monitoring of dynamic pH changes. These\nfeatures enable mechanistic insights into early TME acidification and temporal proton\ndynamics, complementing conventional clinical imaging ......”\nComment 4: Mechanism of Synergistic Photothermal Therapy. The study suggests\nthat combining PPS with the proton pump inhibitor (PPI) esomeprazole (ESOM)\nsynergistically suppresses tumor growth, but the underlying molecular mechanism is\nnot thoroughly explored. Are there data showing whether ESOM directly enhances\nPPS's photothermal effect byinhibitingATP6V0C,or dothetwo act independently?\nResponse: We sincerely thank you for raising this insightful point, which allows us to\nfurther clarify the mechanistic basis underlying the synergistic interaction between\nPPS and the proton pump inhibitor esomeprazole (ESOM). The combined application\nof PPS-mediated photothermal therapy (PTT) and ESOM effectively suppresses\ntumor growth. However, this synergistic antitumor effect does not primarily result\nfrom ESOM directly enhancing the photothermal conversion efficiency of PPS.\nInstead, the two components act through distinct yet complementary mechanisms that\ncooperatively disrupt tumorpHhomeostasis and inducecell death.\nTo elucidate the antitumor efficacy and mechanism of ESOM monotherapy, we\nconducted systematic in vitro and in vivo investigations. In vitro studies revealed that\nESOM significantly reduced intracellular pH (pHi) by inhibiting proton extrusion,\nleading to intracellular acidification (Fig. 7c). Moreover, ESOM effectively\nsuppressed theproliferation, invasion, and migration ofhepatocellular carcinoma cells.\nIn vivo, while ESOM monotherapy exhibited measurable tumor growth inhibition, it\nwas insufficient to achieve complete tumor eradication (Supplementary Fig. 28).\nNotably, as shown in Figure 8, the sequential treatment strategy, PTT followed by\nESOM administration (Group 6: L+PPS+ESOM), produced a markedly stronger\ntumor-suppressive effect than PTT alone (Group 5: L+PPS), confirming a synergistic\n40\ntherapeutic enhancement. Mechanistically, this synergy arises from the following\nsequential and cooperativeprocesses:\n1. PPS-mediated PTT targets the acidic TME: PPS is specifically activated within the\npre-existing acidic tumor microenvironment (pHe ~6.5). Upon laser irradiation, it\ngenerates localized hyperthermia that ablates the majority of tumor cells within these\nacidicregions—serving as the primary tumor-eradication step.\n2. ESOM disrupts pH homeostasis in residual cells: Following PTT, ESOM inhibits\nvacuolar-type H⁺-ATPase (including ATP6V0C) in surviving tumor cells, thereby\nblocking proton extrusion. This disrupts intracellular pH homeostasis, accumulates\nacidicmetabolites, and lowers pHi.\n3. Synergistic outcome: Residual tumor cells that survive initial PTT become\nparticularly vulnerable to ESOM-induced intracellular acidification. This sequential\napproach cripples their ability to restore pH balance, promoting acid-triggered\napoptosisand necrosis, thereby preventing recurrence andmetastasis.\nIn summary, the PPS-ESOM combined therapy operates through a two-tier\nmechanism: PPS-mediated acid-activated photothermal ablation eliminates tumor\ncells in acidic niches, while ESOM inhibits proton pumps to further destabilize pH\nregulation in residual cells. Together, these actions synergistically enhance apoptosis,\nsuppress proliferation and invasion, and effectively remodel the acidic “soil” that\nsupports tumorsurvival.\nWe apologize for not having clearly articulated this mechanistic explanation in the\noriginal manuscript. The revised version has now been updated to clarify this synergy\nindetail.\nChanges inthemanuscripton page 28:(Discussion section)\n“...... Sequential PTT-ESOM treatment further enhances therapeutic efficacy by\nintegrating photothermal ablation with proton pump inhibition. PPS-mediated PTT\neliminates tumor cells within the acidic microenvironment, while subsequent ESOM\nadministration blocks V-ATPase-driven proton extrusion, intensifying intracellular\nacid stress and reinforcing tumor eradication through pH-targeted synergy.\nfor cancer management and paves the way for developing probes tailored to unique\nTME characteristics across various cancer types ......”\nComment 5: Reproducibility and Generalizability.The study employs multiple tumor\nmodels (e.g., subcutaneous, orthotopic, and metastatic), but are there performance\ndifferences in PPS across these models? Do the authors plan to validate PPS's\ngeneralizability in other cancer types? Additionally, some experiments have small\nsamplesizes(e.g., n=3)—could this affect statistical power?\nResponse: We sincerely thank you for this important comment. In the revised\nmanuscript, we have incorporated additional imaging validation experiments across\ndifferent tumor types and increased the number of independent biological replicates\nfor therapeutic studies to n=5. All relevant data and sample size information have\nbeen updated accordingly.Ourdetailed responses are provided below.\n1. Reproducibility and generalizability across models: In this study, we systematically\nevaluated the pH-responsive imaging performance of PPS across multiple tumor\nmodels representing different stages of tumor development, including early\nmicro-lesions (subcutaneous model), orthotopic liver tumors, and metastatic lesions.\nThese models correspond to the progressive stages of tumor evolution—from\ninitiation to local invasion and distant metastasis. The results consistently\ndemonstrated that PPS-enabled photoacoustic imaging effectively detects and\ndifferentiates acidic tumor microenvironments at each stage, confirming its robust\nreproducibility and generalizability across diverse biological and pathological\ncontexts.\n2. Validation in other cancer types: We appreciate the reviewer’s suggestion. Guided\nby our bioinformatic analysis (Supplementary Fig. 30), we expanded our validation to\ninclude mouse models of breast (4T1) and colon (CT26) cancer, guided by our\nbioinformatic analysis. Our results indicate that PPS efficacy across tumor types is\nclosely associated with TME acidity. Tumors with low ATP6V0C expression exhibit\n42\nreduced proton pump activity and weaker baseline acidity, leading to lower PPS\nactivation efficiency. These findings suggest that PPS responsiveness varies among\ntumor types in a manner proportional to their acidification potential, thereby defining\nitsbiological specificity and translational scope.\n3. Sample size and statistical power: We understand the reviewer’s concern about\nsample size and reproducibility. In revised experiments, the number of biological\nreplicates (n=5) (Fig. 7l-n) was increased for key in vivo tumor acid-suppression\nimaging validations to ensure sufficient statistical robustness. It is important to note\nthat the reported sample size refers to independent biological replicates-tumors grown\nin different animals-rather than technical repeats. To adhere to ethical animal use and\n3R principles (Replacement, Reduction, and Refinement), our study design balanced\nstatistical power with responsible animal welfare considerations. Furthermore,\nappropriate statistical methods suitable for small-sample biological data were\nemployed to maintain analytical validity and reproducibility.\nChanges inthemanuscripton page 15:(Resultssection)\nFig. 4 pH-responsive ratiometric PA imaging of tumors in the liver. .....d PA\nimages at 780 nm and 1064 nm in the region of the liver after PPS (1 mg/mL, 200 μL\n43\ni.v.) injection (Scale bars = 5 mm). e Quantification of PA intensity from (d). The\n780\nerror bars represent means ± SDs (n=5 independent experiments). f Ratiometric\nanalysis of the PA signal intensity in tumors at 9 hours postinjection of PPS, as\ncalculated from the data in (d) (n=5 independent experiments). g Ratiometric PA\nimaging of normal liver the HCC orthotopic and metastasis models at 9 hours\npostinjectionof PPS (Scale bars = 5mm).\nChanges inthemanuscripton page 22:(Resultssection)\nFig. 7 Ratiometric PA imaging for the real-time monitoring of pH changes\ninduced by anti-acid treatment and proton pump inhibition. ......k Schematic\nillustration of the treatment procedure. Tumor-bearing mice were imaged as indicated.\nl Ratiometric PA imaging of subcutaneous HepG2 xenografts treated with ESOM or\nPBS at different time points. PPS was intravenously injected before each scan (Scale\nbar = 2.5 mm). m Quantification of the PA /PA signal ratios from (l). The error\n780 1064\nbars represent means±SDs (n = 5 independent experiments). n pH values of tumors\nwith and without anti-acid treatment. The error bars represent means±SDs (n = 5\nindependent experiments).\nWe once again express our sincere appreciation for your constructive feedback,\nwhich has significantly improved the transparency,reproducibility,and scientific rigor\nofourrevised manuscript.\nReviewer #1(Remarks to theAuthor):\nGeneralAssessment\nThis study presents a novel pH-responsive ratiometric photoacoustic sensor (PPS) for\nreal-time monitoring of tumour microenvironment (TME) acidification in\nhepatocellular carcinoma (HCC). The authors integrate bioinformatics, molecular\nimaging, and therapeutic intervention to demonstrate the role of ATP6V0C in TME\nacidification and its implications for tumour progression and metastasis. The work is\nwell-structured, methodologically rigorous, and clinically relevant.\nOverall Recommendation\nThisstudy presents acompelling advance inTME imaging andtherapy.\nAcceptanceDecision: Accept. Concerns were addressed.\nImpact: High potential toinfluence HCCdiagnosis and targeted therapy.\nResponse: We sincerely appreciate the reviewer’s highly positive assessment and are\ngrateful for the recognition of our work’s methodological rigor,clinical relevance, and\npotential impact, as well as for theacceptance recommendation.\nReviewer #2(Remarks to theAuthor):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nResponse: We are grateful to the reviewer for insightful comments and suggestions\nthat helped improve ourstudy.\nReviewer #3(Remarks to theAuthor):\nTheauthors responded satisfactorily to mycriticisms\nResponse: We sincerely appreciate the reviewer’s positive evaluation and are glad\nthat ourrevisions have satisfactorily addressed thereviewer’s concerns.\nReviewer #4(Remarks to theAuthor):\nThe authors have made a thorough effort to address thereviewers’previous comments.\nThe revised manuscript shows clear improvement in terms of data analysis and\ninterpretation. The additional experiments and clarifications have strengthened the\nvalidityof theconclusions.\nWhile some minor aspects could still benefit from further polishing, the current\nversion of the manuscript adequately supports the main claims. The data are\nconsistent and reasonable, and theconclusions are well justified.\nIn light of these improvements, I believe the manuscript is now suitable for\npublication inNatureCommunications,pending final editorial consideration.\nResponse: We sincerely thank the reviewer for the positive and encouraging\nassessment, and we are grateful that the revised manuscript is considered suitable for\npublication pending final editorial review."}, {"round": 2, "reviewer_comments": {"reviewer_1": "General Assessment\nThis study presents a novel pH-responsive ratiometric photoacoustic sensor (PPS) for real-time monitoring of tumour\nmicroenvironment (TME) acidification in hepatocellular carcinoma (HCC). The authors integrate bioinformatics, molecular\nimaging, and therapeutic intervention to demonstrate the role of ATP6V0C in TME acidification and its implications for\ntumour progression and metastasis. The work is well-structured, methodologically rigorous, and clinically relevant. However,\ncertain aspects require clarification and additional validation to strengthen the claims.\nStrengths\n1. Innovative Imaging Tool:\nThe development of PPS for pH-sensitive photoacoustic imaging is a significant technical advance, enabling non-invasive,\nhigh-resolution monitoring of TME acidification.\nThe dual-wavelength ratio-metric approach enhances specificity and minimizes background interference with potential to\ninfluence HCC diagnosis and targeted therapy.\n2. Mechanistic Insights:\nStrong bioinformatics and clinical validation linking ATP6V0C overexpression to HCC progression and poor prognosis.\nMolecular docking and functional assays convincingly demonstrate the interaction between esomeprazole (ESOM) and\nATP6V0C, supporting the therapeutic rationale.\n3. Multimodal Validation:\nThe study employs multiple models (subcutaneous, orthotopic, metastatic) and human specimens to validate PPS utility.\nCorrelative analyses (hypoxia, angiogenesis) strengthen the biological relevance of TME acidification.\n4. Therapeutic Implications:\nThe combination of PPS-mediated photothermal therapy (PTT) and ESOM provides a synergistic anti-tumour effect,\nsupported by RNA-seq and single-cell transcriptomics.\nPrevious Major Concerns and Subsequent Revision\n1. Specificity and Sensitivity of PPS\nInterference from Other Metabolites:\nWhile Supplementary Fig. 10 shows minimal interference from ions (K⁺, Na⁺, Mg²⁺), tumour TME contains lactate, ROS, and\nproteases. PPS response might not\nRequest: Test PPS response in the presence of lactate (due to Warburg effect) or H₂O₂ (ROS from metabolic stress and\nimmune activity) to rule out other possibilities of false positives or signal modulation.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_3": "The authors responded satisfactorily to my criticisms", "reviewer_4": "The authors have made a thorough effort to address the reviewers’ previous comments. The revised manuscript shows clear\nimprovement in terms of data analysis and interpretation. The additional experiments and clarifications have strengthened the\nvalidity of the conclusions.\nWhile some minor aspects could still benefit from further polishing, the current version of the manuscript adequately supports\nthe main claims. The data are consistent and reasonable, and the conclusions are well justified.\nIn light of these improvements, I believe the manuscript is now suitable for publication in Nature Communications, pending\nfinal editorial consideration."}, "author_response": ""}]

Decision letter