Journal: Nature Communications

Article Title: Acid-exposed and hypoxic cancer cells do not overlap but are interdependent for unsaturated fatty acid resources

doi: 10.1038/s41467-024-54435-3

Figure Lengend Snippet: a Representative pictures and quantification of Carbonic Anhydrase 9 (CA9, red) and pimonidazole (green) immunostaining in FaDu and HCT116 spheroid sections ( N = 3, n = 2); DAPI (blue) nuclear staining was used to normalize measurements. The white delimitation represents the rim of the spheroid. b Representative pictures and quantification of CA9 (red) and Hypoxia-Responsive Element-dependent GFP reporter (HRE, green) wholemount fluorescence in FaDu and HCT116 spheroids ( N = 5 and 7, respectively). c 3-dimensional modeling of CA9 (red), HRE-GFP (green), and DAPI (blue) staining in Fadu spheroids; this experiment was repeated twice with similar results. d Representative CA9 (purple) and pimonidazole (yellow) immunostaining in FaDu and HCT116 tumor sections; this experiment was repeated twice with similar results. e Representative flow cytometry analysis of CA9 staining in FaDu and HCT116 cancer cells maintained under normoxia at physiological pH 7.4 or at acidic pH 6.5; this experiment was repeated twice with similar results. The scale bar represents 100 µm for 3D tumor spheroids ( a – c ) and 200 µm for mouse tumor sections ( d ). Data are plotted as the means ± SD (** P = 0.0017, **** P < 0.0001); N indicates the number of independent experiments and n indicates the number of biological replicates (when >1). Significance was determined by two-sided Student’s t -test ( a , b ). Source data are provided as a Source Data file.

Article Snippet: The staining was performed with PE-coupled anti-CA9 antibody (130-123-340, Miltenyi, 1:200) for 20 min at 4 °C.

Techniques: Immunostaining, Staining, Fluorescence, Flow Cytometry

Journal: Nature Communications

Article Title: Acid-exposed and hypoxic cancer cells do not overlap but are interdependent for unsaturated fatty acid resources

doi: 10.1038/s41467-024-54435-3

Figure Lengend Snippet: a , b Schematic protocol of cell sorting from 3D FaDu spheroids based on CA9 immunostaining and HRE-GFP expression, created in BioRender. Feron, O. (2024) BioRender.com/i40c288 ( a ) and representative FACS plots showing gating strategy for each of the four quadrants ( b ). c Principal component analysis (PCA) discriminating the four quadrants based on RNA-seq analysis performed on 3 independent sorting experiments. d Volcano plot of differentially expressed genes between CA9+/HRE− and CA9+/HRE+ FaDu cell populations. e . KEGG pathway enrichment analysis of the differentially expressed genes between CA9+/HRE− and CA9+/HRE+ FaDu cell populations. f , g Changes in the mRNA expression of the indicated desaturases in the four distinct FaDu spheroid compartments ( f ) ( N = 3, n = 2), and in FaDu cancer cells maintained at physiological pH 7.4 or at acidic pH 6.5 ( g ) ( N = 4); results are expressed as fold-change vs . mRNA levels in CA9-/HRE- double negative cell populations and in cancer cells at pH 7.4, respectively. Data are plotted as the means ± SD ( P -values as indicated or *** P < 0.001, **** P < 0.0001); N indicates the number of independent experiments and n indicates the number of biological replicates (when >1). Significance was determined by a two-sided Student’s t -test with FDR adjustment ( d ), one-way ANOVA with Tukey’s multiple comparison test ( f ), or two-sided Student’s t -test ( g ). Source data are provided as a Source Data file.

Article Snippet: The staining was performed with PE-coupled anti-CA9 antibody (130-123-340, Miltenyi, 1:200) for 20 min at 4 °C.

Techniques: FACS, Immunostaining, Expressing, RNA Sequencing, Comparison

Journal: Nature Communications

Article Title: Acid-exposed and hypoxic cancer cells do not overlap but are interdependent for unsaturated fatty acid resources

doi: 10.1038/s41467-024-54435-3

Figure Lengend Snippet: a , b Representative contrast phase pictures ( a ) and quantification ( b ) of the effects of CRISPR-Cas9-based SCD1 gene invalidation on FaDu spheroid growth at day 7 post-formation (vs. control sgRNA); N = 3, n = 2. c – e Quantification of SCD1 (violet) ( c ) ( N = 5) and CA9 (red) immunostaining ( d ) ( N = 5), and representative pictures ( e ) of spheroids made of CRISPR-Cas9-based SCD1-silenced FaDu cells at day 10 post-formation. f , g Spheroid growth ( f ) ( N = 3, n = 3–4) and cytotoxicity (Incucyte Cytotox Green reagent) follow up ( g ) ( N = 3, n = 4) after exposure to SCD1 inhibitor (or vehicle) for 72 h in FaDu spheroids. h , i Representative pictures ( h ) and quantification of CA9 (red) ( i ) in sections of FaDu spheroids collected after 72 h exposure to SCD1 inhibitor ( N = 3, n = 2). j Flow cytometry analysis of CA9 labeling from FaDu cells isolated from spheroids after 72 h exposure to SCD1 inhibitor (or vehicle) ( N = 3). k Quantification of pimonidazole (green) in sections of FaDu spheroids collected after 72 h exposure to SCD1 inhibitor ( N = 3, n = 2). l , m Mitochondrial oxygen consumption rate (OCR) of l FaDu ( N = 3, n = 7) and HCT116 ( N = 3, n = 7–8) spheroids after 72 h SCD1 inhibition and ( m ) FaDu cancer cells transduced with the indicated SCD1 sgRNA ( N = 3, n = 3) or control sgRNA ( N = 3, n = 3–4). n Non-mitochondrial OCR of FaDu cells transduced with an SCD1-expressing vector or control plasmid and exposed to palmitate ( N = 3, n = 3) or vehicle (FA-free BSA) ( N = 3, n = 4). o – r Representative pictures ( o ) and quantification of SCD1 (violet) ( p ), CA9 (red) ( q ), and pimonidazole (green) ( r ) staining in FaDu spheroids exposed to PPAR-γ inhibitor (or vehicle) for 72 h ( N = 3); the effects of a PPAR-α inhibitor are also shown in graphs ( p – r ). All treatments ( f – l ) with SCD1 inhibitor (A939572, 32 µM) were initiated at day 7 after spheroid formation (i.e., timing 0 on graphs). All immunostaining quantifications ( c , d , i , k , p – r ) were normalized to the DAPI nuclear staining area. Data are plotted as the means ± SD ( P -values as indicated or *** P < 0.001, **** P < 0.0001); N indicates the number of independent experiments and n indicates the number of biological replicates (when >1). Significance was determined by one-way ANOVA with Tukey’s multiple comparison tests ( b – d ), Dunnet’s multiple comparison tests ( p – r ), Sidaks’s multiple comparison tests ( f , g ), two-way ANOVA with Tukey’s multiple comparison tests ( l – n ) or two-sided Student’s t -tests ( i – k ). Source data are provided as a Source Data file.

Article Snippet: The staining was performed with PE-coupled anti-CA9 antibody (130-123-340, Miltenyi, 1:200) for 20 min at 4 °C.

Techniques: CRISPR, Control, Immunostaining, Flow Cytometry, Labeling, Isolation, Inhibition, Transduction, Expressing, Plasmid Preparation, Staining, Comparison

Journal: Nature Communications

Article Title: Acid-exposed and hypoxic cancer cells do not overlap but are interdependent for unsaturated fatty acid resources

doi: 10.1038/s41467-024-54435-3

Figure Lengend Snippet: a , b Representative pictures ( a ) and quantification ( b ) of colorectal cancer patient-derived organoids after treatment with SCD1 inhibitor (A939572, 20 µM) in the presence of OA (100 µM) and/or DHA (50 µM) for 96 h ( N = 2, n = 2). c , d Effect of SCD1 inhibitor (12 µM) on the viability (96 h) ( c ) and lipid peroxidation BODIPY-C11 staining (72 h) ( d ) of 6.5/Fadu ( N = 3, n = 2) and 6.5/HCT116 ( N = 3, n = 4) cancer cells in the presence of OA (100 µM) and/or DHA (50 µM) and/or α-Tocopherol (10 µM) or vehicle(s). e Representative pictures of CA9 immunofluorescence signal (red) in equatorial sections of FaDu and HCT116 spheroids exposed for 72 h to SCD1 inhibitor (A939572, 32 µM) in the presence of OA (100 µM) or not; this experiment was repeated twice with similar results. f Representative flow chart depicting the effects of SCD1 inhibitor on the uptake of TopFluor oleate by 6.5/Fadu and 6.5/HCT116 cancer cells maintained under normoxia or hypoxia (1% O 2 ) ( N = 4, n = 4). g Schematic representation of the symbiotic relationship between hypoxic cancer cells (unable to synthesize MUFA and thus dependent on exogenous MUFA) and acidic, non-hypoxic cancer cells that may use both MUFA sources. Inhibition of SCD1 however forces the latter cell compartment to capture exogenous MUFA thereby depriving hypoxic cells from a vital source of MUFA. h Viability of FaDu and HCT116 cancer cells maintained under hypoxia (1% O 2 ) and exposed for 48 h to the conditioned medium (CM) from normoxic 6.5/FaDu and 6.5/HCT116 exposed or not to SCDi (24 h, 15 and 25 µM A939572, respectively) ( N = 3, n = 7). In some experiments, CM was supplemented by oleate (50 µM) ( N = 3, n = 4). Control conditions consist of CM + fresh addition of SCDi ( N = 3, n = 7), and non-conditioned medium (NCM) ( N = 3, n = 3). i , j MUFA amounts ( i ) and SFA/MUFA ratio ( j ) determined in the conditioned medium (CM) of normoxic 6.5/FaDu and 6.5/HCT116 cancer cells exposed or not to SCDi as above ( N = 2). Data are plotted as the means ± SD (ns: non-significant, P -values as indicated or *** P < 0.001, **** P < 0.0001); N indicates the number of independent experiments and n indicates the number of biological replicates (when >1). Significance was determined by two-way ANOVA with Tukey’s multiple comparison test ( b – h ). Source data are provided as a Source Data file.

Article Snippet: The staining was performed with PE-coupled anti-CA9 antibody (130-123-340, Miltenyi, 1:200) for 20 min at 4 °C.

Techniques: Derivative Assay, Staining, Immunofluorescence, Inhibition, Control, Comparison

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