Journal: Journal for Immunotherapy of Cancer

Article Title: Carbonic anhydrase 9 as a circulating biomarker and therapeutic target in patients with hepatocellular carcinoma treated with atezolizumab plus bevacizumab

doi: 10.1136/jitc-2025-013384

Figure Lengend Snippet: High-throughput proteomics identifies circulating proteins correlated with the response to Atez and Bev treatment in unresectable HCC. 78 patients with unresectable HCC treated with Atez/Bev were included in the discovery cohort. ( A–B ) Volcano plots showing plasma levels of 92 immune-oncology-related proteins measured by Olink in patients who achieved DC versus those with PD, evaluated by ( A ) RECIST and ( B ) mRECIST. The x-axis indicates log₂ fold-change of protein levels in PD versus DC, and the y-axis indicates –log₁₀ (p) values for the comparison between the two groups. ( C ) ORR and DCR of patients with high and low plasma CA9 levels on Atez/Bev therapy evaluated by mRECIST. ( D–E ) Kaplan-Meier PFS curves for patients with high and low plasma CA9 levels on Atez/Bev therapy evaluated by RECIST ( D ) and mRECIST ( E ). ( F ) Kaplan-Meier OS curves for patients with high and low plasma CA9 levels on Atez/Bev therapy. Group comparisons were performed using appropriate statistical tests (eg, χ 2 test or log-rank test), as detailed in the Methods; p<0.05 was considered statistically significant. Atez, atezolizumab; Bev, bevacizumab; CA9, carbonic anhydrase 9; CSF, colony-stimulating factor; DC, disease control; DCR, disease control rate; FC, fold change; HCC, hepatocellular carcinoma; IL, interleukin; mDCR, disease control rate by mRECIST; mPFS, modified PFS; mRECIST, modified RECIST; MCP-3, monocyte chemoattractant protein 3; MMP12, matrix metalloproteinase 12; mORR, objective response rate by mRECIST; OS, overall survival; ORR, overall response rate; PD, progressive disease; PFS, progression-free survival; RECIST, Response Evaluation Criteria in Solid Tumors; VEGF, vascular endothelial growth factor.

Article Snippet: CA9 inhibitor S4 (MedChemExpress, Cat# HY-110243; 5 mg/kg, i.p.).

Techniques: High Throughput Screening Assay, Clinical Proteomics, Comparison, Control, Modification

Journal: Journal for Immunotherapy of Cancer

Article Title: Carbonic anhydrase 9 as a circulating biomarker and therapeutic target in patients with hepatocellular carcinoma treated with atezolizumab plus bevacizumab

doi: 10.1136/jitc-2025-013384

Figure Lengend Snippet: Patients with unresectable HCC with high plasma CA9 levels are associated with poor disease control and shorter survival on Atez/Bev therapy. 89 patients with unresectable HCC treated with Atez/Bev therapy were enrolled in the validation cohort. ( A–B ) Plasma CA9 levels in patients with HCC who achieved disease control with Atez/Bev treatment and those with progressive disease, evaluated by RECIST (A) and mRECIST (B). Each dot represents one patient. ( C ) ROC curve of plasma CA9 levels for predicting initial PD; the area under the ROC curve is shown. ( D ) Proportions of patients with DC or PD stratified by high versus low plasma CA9 levels, according to RECIST (left) and mRECIST (right), based on the CA9 cut-off defined in ( C ). ( E, F ) Kaplan-Meier curves for PFS stratified by high versus low plasma CA9 levels, assessed by ( E ) RECIST and ( F ) mRECIST. ( G ) Kaplan-Meier curves for OS stratified by plasma CA9 level in the same cohort. Group comparisons were performed using appropriate statistical tests (eg, χ 2 test, or log-rank test), as detailed in the Methods; p<0.05 was considered statistically significant. Atez, atezolizumab; Bev, bevacizumab; CA9, carbonic anhydrase 9; CR, complete response; DC, disease control; DCR, disease control rate; HCC, hepatocellular carcinoma; mDCR, disease control rate by mRECIST; mPFS, modified PFS; mRECIST, modified RECIST; OS, overall survival; PD, progressive disease; PR, partial response; PFS, progression-free survival; RECIST, Response Evaluation Criteria in Solid Tumors; ROC, receiver operating characteristic; SD, stable disease.

Article Snippet: CA9 inhibitor S4 (MedChemExpress, Cat# HY-110243; 5 mg/kg, i.p.).

Techniques: Clinical Proteomics, Control, Biomarker Discovery, Modification

Journal: Journal for Immunotherapy of Cancer

Article Title: Carbonic anhydrase 9 as a circulating biomarker and therapeutic target in patients with hepatocellular carcinoma treated with atezolizumab plus bevacizumab

doi: 10.1136/jitc-2025-013384

Figure Lengend Snippet: Tumorous CA9 may be the source of circulating CA9 and confers resistance to the combination immunotherapy of anti-PD-L1 and anti-VEGF antibodies in HCC. ( A–C ) Single-cell RNA sequencing data from the HCCDB V.2.0 (Lifeome) portal showing ( A ) UMAP of major cell types and feature plots of ( B ) CA9 and ( C ) AFP expression. ( D ) Western blot analysis of Car9 and Actb in Hep55.1c cells with NC or Car9 OE. ( E ) mRNA levels of Car9 in syngeneic subcutaneous tumors derived from Hep55.1c cells with either negative control or Car9 overexpression (NC: n=28; OE: n=25). ( F ) Growth curves of syngeneic subcutaneous tumors derived from Hep55.1c cells with either negative control (left) or Car9 overexpression (right) (n=8 per group). ( G ) Tumor volume 14 days after inoculation in NC and OE tumors (NC: n=28; OE: n=25). ( H ) Growth curves of syngeneic subcutaneous tumors derived from NC or Car9-overexpression Hep55.1c cells treated with vehicle or anti-PD-L1/VEGF antibodies (treatment) (n=8 per group). ( I ) Relative treatment response calculated for each mouse as the tumor volume in the anti-PD-L1/VEGF-treated group divided by the mean tumor volume of the corresponding vehicle-treated group within the same genotype (NC or Car9 OE) at day 14. Each dot represents an individual tumor, allowing comparison of relative treatment effects between NC-derived and Car9-OE-derived tumors. Data are shown as mean±SEM. Group comparisons were performed using two-tailed unpaired Student’s t-tests or two-way repeated-measures ANOVA, as appropriate; p<0.05 was considered statistically significant. AFP, alpha-fetoprotein; ANOVA, analysis of variance; CA9, carbonic anhydrase 9; HCC, hepatocellular carcinoma; mRNA, messenger RNA; NC, negative control; NK, natural killer; OE, overexpression; PD-L1, programmed death-ligand 1; UMAP, Uniform Manifold Approximation and Projection; VEGF, vascular endothelial growth factor.

Article Snippet: CA9 inhibitor S4 (MedChemExpress, Cat# HY-110243; 5 mg/kg, i.p.).

Techniques: Single Cell, RNA Sequencing, Expressing, Western Blot, Derivative Assay, Negative Control, Over Expression, Comparison, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Carbonic anhydrase 9 as a circulating biomarker and therapeutic target in patients with hepatocellular carcinoma treated with atezolizumab plus bevacizumab

doi: 10.1136/jitc-2025-013384

Figure Lengend Snippet: Tumorous CA9 flourishes the immunosuppressive and angiogenic tumor microenvironment in HCC. ( A–C ) Relative mRNA expression of the indicated genes in syngeneic subcutaneous tumors derived from Hep55.1c cells with NC or Car9 OE, measured by quantitative RT-PCR (NC: n=28; OE: n=25). Each dot represents one tumor; bars indicate mean±SEM. ( D ) UMAP of single-cell RNA sequencing data from NC and CA9 OE tumors, colored by annotated cell type (eg, malignant cells, T cells, TAMs, endothelial cells). ( E ) Frequency of CTLs per tumor sample, calculated as CTLs divided by total cells profiled by scRNA-seq (NC: n=3; CA9 OE: n=3). ( F, G ) Differential gene expression and representative cytotoxicity-related genes in CD8 + T cells from CA9 OE versus NC tumors. ( H, I ) Differential gene expression and representative TAM-related genes in TAMs from CA9 OE versus NC tumors. ( J–L ) Gene Set Enrichment Analysis (Gene Ontology) showed the top 20 pathways in TAMs ( J ), dendritic cells ( K ), and endothelial cells ( L ). ( M ) Tumor growth curves of syngeneic subcutaneous tumors derived from Car9-overexpressing Hep55.1c cells treated with vehicle, CA9 inhibitor alone, anti-PD-L1 plus anti-VEGF antibodies (Combi), or Combi plus CA9 inhibitor (Combi+CA9 inhibitor) (n=8–12 per group). Data are shown as mean±SEM. Group comparisons were performed using two-tailed unpaired Student’s t-tests or Wilcoxon rank-sum tests, as appropriate; p<0.05 was considered statistically significant. CAF, cancer-associated fibroblast; CA9, carbonic anhydrase 9; CTLs, cytotoxic T lymphocytes; HCC, hepatocellular carcinoma; mRNA, messenger RNA; NC, negative control; NK, natural killer; OE, overexpression; PD-L1, programmed death-ligand 1; RT-PCR, reverse transcription PCR; scRNA-seq, single-cell RNA sequencing; TAM, tumor-associated macrophage; TAN, tumor-associated neutrophils; TPM, transcripts per million; UMAP, Uniform Manifold Approximation and Projection; VEGF, vascular endothelial growth factor; WT, wild type.

Article Snippet: CA9 inhibitor S4 (MedChemExpress, Cat# HY-110243; 5 mg/kg, i.p.).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Single Cell, RNA Sequencing, Gene Expression, Two Tailed Test, Negative Control, Over Expression, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription