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: 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