murine car9 open reading frame  (OriGene)

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: Stable Car9-overexpressing and vector control lines were generated using lentiviral particles encoding the murine Car9 open reading frame (ORIGENE, Rockville, Maryland, USA).

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: Stable Car9-overexpressing and vector control lines were generated using lentiviral particles encoding the murine Car9 open reading frame (ORIGENE, Rockville, Maryland, USA).

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