Journal: Cell

Article Title: HIF Regulates Multiple Translated Endogenous Retroviruses: Implications for Cancer Immunotherapy

doi: 10.1016/j.cell.2025.01.046

Figure Lengend Snippet: Key resource table

Article Snippet: Carbonic Anhydrase 9 antibody, antihuman, PE, REAfinity , Miltenyi Biotec , Cat#130–123-299, RRID: AB_2857582.

Techniques: Virus, Recombinant, SYBR Green Assay, cDNA Synthesis, Purification, Sample Prep, Sequencing, Amplification, Software, Binding Assay, Transferring, Reverse Transcription, Low Protein Binding, Antibody Purification, Adjuvant, Enzyme-linked Immunospot

Journal: Acta Pharmaceutica Sinica. B

Article Title: An immunostimulant nanomedicine enhances radioimmunotherapy by remodeling the tumor immunosuppressive landscape after radiotherapy

doi: 10.1016/j.apsb.2025.11.012

Figure Lengend Snippet: Immunosuppression after radiotherapy promotes breast cancer progression. Tumor growth curves (A) and weights (B) of mice before and after RT ( n = 6). (C) Survival curve of mice after different treatments ( n = 6). Tumor growth curves (D) and weights (E) of mice treated with RT and RT plus α -PD-L1 ( n = 6). (F) Survival curve of mice after different treatments ( n = 6). (G) Cluster analysis of differential expression genes between untreated and RT-treated tumors 48 h post-RT ( n = 3). (H) Gene Ontology (GO) analysis of tumor tissues before and after RT (select the top 10 for each item). (I) Heat map of differentially expressed genes related to apoptosis and immune suppression ( n = 3). (J) Immunofluorescence staining images of tumor tissue in saline and RT groups (scale bar = 20 μm). (K) Quantitative analysis of tumor-infiltrating CD45 + cells ( n = 6). Representative flow cytometry images (M) and quantitative analysis (L) of tumor-infiltrating MDSCs ( n = 6). (N) The TUNEL staining of tumor section (scale: 25 μm). (O) Adenosine content detection in tumor tissue ( n = 6). Data are presented as mean ± SD. ∗∗∗∗ P < 0.0001 determined by Student’s t-test.

Article Snippet: InVivo MAb anti-mouse PD-L1 was purchased from Bioxcell (USA).

Techniques: Quantitative Proteomics, Immunofluorescence, Staining, Saline, Flow Cytometry, TUNEL Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: An immunostimulant nanomedicine enhances radioimmunotherapy by remodeling the tumor immunosuppressive landscape after radiotherapy

doi: 10.1016/j.apsb.2025.11.012

Figure Lengend Snippet: FD@ATRA-enhanced radiotherapy combined with α -PD-L1 to enhance the efficacy of large-volume tumors. (A) Schematic diagram of the therapeutic process for evaluating the anti-tumor effects in a bilateral 4T1 tumor-bearing mouse model of large volume. (B) Distant tumor growth curves of mice after different treatments ( n = 5). (C) In vitro images of the distant tumors ( n = 5). (D) In vitro distant tumors mass ( n = 5). (E) Immunohistochemical staining of CD3 in tumor tissue slices after different treatments (scale bars = 100 μm). The images below were the corresponding enlarged parts (scale bars = 25 μm). (F) Immunohistochemical staining of CD8 in tumor tissue slices after various treatments (scale bars = 100 μm). The images below were the corresponding enlarged parts (scale bars = 25 μm). Data are presented as mean ± SD. ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 determined by Student’s t -test.

Article Snippet: InVivo MAb anti-mouse PD-L1 was purchased from Bioxcell (USA).

Techniques: In Vitro, Immunohistochemical staining, Staining

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

Journal: International Journal of Molecular Sciences

Article Title: PIMT Binding to C-Terminal Ala459 of CAIX Is Involved in Inside-Out Signaling Necessary for Its Catalytic Activity

doi: 10.3390/ijms21228545

Figure Lengend Snippet: C -terminal amino acid Ala459 residue cooperates in the regulation of CAIX catalytic function: ( a ) Immunoblot analysis of cell lysates from C33a cells transfected with mock control, CAIX wild type (wt) or mutant (A459G) cultured 48 h in hypoxia. CAIX was detected using mouse monoclonal antibody M75 diluted 1:10 and β-actin was detected using mouse monoclonal antibody (CS3700, Cell Signaling Technology, Danvers, Massachusetts) diluted 1:5000. HRP-conjugated anti-mouse antibody (Dako Agilent, Santa Clara, California) diluted 1:5000 was used as a secondary antibody. ( b ) Fluorescence staining of CAIX in non-fixed and non-permeabilized C33a-CAIX wild type as well as A459G transfectants measured by flow cytometry. CAIX was detected using PG-domain specific mouse monoclonal antibody M75 diluted to 1 µg/mL and AlexaFluor 488-conjugated anti-mouse secondary antibody (Invitrogen, Carlsbad, California) diluted 1:1000. Results clearly demonstrate plasma membrane localization of CAIX and showed that 42.7% of C33a-CAIX-wt and 50.9% of C33a-CAIX-A459G transfectants expressed CAIX protein. C33a cells transfected with mock control plasmid were used as a negative control. The data are presented as the mean, n = 2. ( c ) Effect of Ala459 mutation on CAIX-mediated extracellular acidification. The graph shows the differences between pHe values (ΔpH) of culture media from CAIX wt or A459G-transfected and mock-transfected cells cultured 48 h in hypoxia. A459G mutant reduced acidification of extracellular pHe when compared to control wild type C33a-CAIX transfectants. The data are presented as the mean ± s.d., n = 5. Statistical significance was analyzed using the Student’s t -test and expressed as a p -value (* p < 0.05). ( d ) Effect of Ala459 mutation on migration capacity of C33a cells. The graph depicts the results of the wound healing assay given as a % of the area covered by cells migrating to close the wound at 30 h after the scratch, measured at various positions along the wounds. Area covered by C33a cells expressing CAIX-wt was set as 100%. C33a cells expressing CAIX with mutated Ala459 exhibited slower migration. The data are presented as the mean ± s.d., n = 10. Statistical significance was analyzed using the Student’s t -test and expressed as a p -value (* p < 0.05). ( e ) Accumulation of the fluorescent sulfonamide (FITC-CA-i) occurred in hypoxic MDCK cells expressing the CAIX-wt, whereas it was diminished in hypoxic MDCK cell transfected with the CAIX-A459G mutant. Images were taken using objective 10×. ( f ) In situ detection of the interaction between CAIX and AE2 using a proximity ligation assay (PLA). Analysis was performed in C33a cells transiently transfected with CAIX-wt and A459G mutant and cultured in hypoxia for an additional 48 h. Red PLA signal indicates the existence of interaction or close proximity localization also between CAIX with mutated Ala459 and AE2. C33a-mock transfectants served as a negative assay control due to a lack of one target protein. CAIX protein was post-labelled in green, nuclei are blue. Images were taken using objective 40× and zoom 3.

Article Snippet: After 48 h of hypoxic incubation, transiently transfected cells were scraped into culture medium, centrifuged at low speed, washed twice with Versene solution and incubated with the CAIX-specific mouse monoclonal M75 antibody (1 µg/mL, BMC SAS, Bratislava, Slovakia) for 30 min at 4 °C.

Techniques: Western Blot, Transfection, Mutagenesis, Cell Culture, Fluorescence, Staining, Flow Cytometry, Plasmid Preparation, Negative Control, Migration, Wound Healing Assay, Expressing, In Situ, Proximity Ligation Assay