Article Figures & Data
Figures
- Figure 1.
Gene expression profiles in MDA-MB-231 cells co-cultured with murine adipose tissue. A, Heat maps of the 50 top up- and downregulated genes in MDA-MB-231 cells co-cultured with adipose tissue of HFD induced obese mice vs. control (basal medium). Gene expression data of MDA-MB-231 cells co-cultured with adipose tissue of mice fed a NC diet were included accordingly. B, Overrepresented cellular pathways (KEGG database) of ≥1.5-fold up- and downregulated genes in MDA-MB-231 cells co-cultured with adipose tissue of HFD mice. C, mRNA expression of selected candidate genes in MDA-MB-231 cells in basal medium and co-cultured with adipose tissue of NC or HFD mice (n = 3/group; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant).
- Figure 2.
Human ACM induces the expression of PPAR target genes and Angptl4 secretion in TNBC cells via PPARα, which is dependent on patient's BMI. A and B, MDA-MB-231 and HCC38 cells were incubated with basal medium or ACM of patients with BMI ≥25<30 (ACM<30) or BMI≥40 (BMI>40). In C and D, the PPARα inhibitor GW6471 (10 μmol/L) was added to cells. Gene expression was quantified by qPCR (A and C). Protein concentration of Angptl4 in cell culture supernatants was assessed by ELISA (B and D). Data are presented as mean + SD of triplicates of one representative experiment (*, P < 0.05; **, P < 0.01; ***, P < 0.001; nd, not detectable).
- Figure 3.
Exogenous FFAs activate PPARα signaling in TNBC cells. A, FFA and mono-, di-, and triacylglycerol were quantified in ACM<30 (n = 4) and ACM>40 (n = 8) by FIA-FTMS. B, Total fatty acid analysis by GC-MS; displayed are concentrations of oleic, palmitic, and linoleic acid in ACM<30 (n = 5) and ACM>40 (n = 8). C, Correlations of total FFA, oleic, palmitic, or linoleic acid in ACMs with ANGPTL4 gene expression in MDA-MB-231 cells treated with the corresponding ACMs. D–F, ANGPTL4 gene expression in MDA-MB-231 and HCC38 cells cultured with BSA-conjugated oleic acid (BSA-OA) or linoleic acid (BSA-LA) and treated with or without PPARα inhibitor GW6471 (n = 3). G, Angptl4 protein concentrations in cell culture supernatants after incubation with BSA-OA and with or without GW6471 (n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001; nd, not detectable).
- Figure 4.
Incubation with ACM alters cellular lipid metabolism in MDA-MB-231 cells. A, Gene expression of SCD1 and FASN in MDA-MB-231 cells cultured with basal medium, ACM<30 or ACM>40. B, Immunoblots of Srebp1 precursor (pSrebp) and nuclear Srebp1 (nSrebp) with cell lysates of MDA-MB-231 cells. C, Fraction of de novo synthesized FA 16:0 in MDA-MB-231 cells cultured with basal medium, ACM<30 or ACM>40 (n = 3). D, OCR and ECAR of MDA-MB-231 cells was assessed after ACM>40 treatment (24 hours) under basal or stressed conditions (1 μmol/L oligomycin, 1 μmol/L FCCP) by a Seahorse cell energy phenotype assay. Data are shown as mean ± SEM of n = 5 replicates. E, Gene expression of CPT1A, SLC25A20, and ACAA2 in MDA-MB-231 cells after treatment with basal medium, ACM<30 or ACM>40. F, To evaluate FAO, OCR was measured in the presence of palmitate and limitation of other substrates (0.5 mmol/L glucose, no glutamine). Displayed are basal, coupled, and maximal respiration calculated as described in Materials and Methods (n = 10–11). G, Inhibition in OCR induced by treatment with etomoxir (Etx, 4 μmol/L), calculated by subtracting basal OCR with etomoxir from basal OCR without etomoxir. H, Measurement of FAO activity (oxidation of octanoyl-CoA) in cell lysates of MDA-MB-231 cells incubated with basal medium or ACM>40 (n = 10; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant).
- Figure 5.
ACM promotes tumor cell proliferation and motility. A, Cell proliferation of MDA-MB-231 and HCC38 cells was assessed by BrdUrd assay. Cells were treated with basal medium or ACM>40 and BrdUrd incorporation was analyzed after 24 hours (n = 6). B, Migration capabilities of cells were analyzed by scratch assay. Wound closure was determined after treatment with basal medium or ACM>40 after 24 hours (n = 4). C, To determine invasion capability, MDA-MB-231 and HCC38 cells were seeded in the top chamber of a Matrigel coated transwell system and cultured with basal medium or ACM>40 (n = 4). After 24 hours, invaded cells were counted and invasion was calculated as the mean of invaded cells/field (**, P < 0.01; ***, P < 0.001).
- Figure 6.
ACM promotes cell motility mediated by Angptl4 and phosphorylation of FAK. A, MDA-MB-231 cells were transfected with siRNAs targeting ANGPTL4 (siANGPTL4_1 or siANGPTL4_2) or scrambled siRNA (siNTC). ANGPTL4 expression was determined after 24, 48, and 72 hours (n = 3). B, ANGPTL4 expression in MDA-MB-231 cells with stable ANGPTL4 depletion, generated by lentiviral-mediated transduction of shRNAs. Control cells were transduced with scrambled shRNA (shNTC; n = 3). C and D, Angptl4 protein levels in cell culture supernatants was determined in ANGPTL4-depleted MDA-MB-231 cells, incubated with basal medium or ACM>40 (n = 3). E and F, ANGPTL4-depleted MDA-MB-231 cells were seeded in the top chamber of a Matrigel coated transwell system and treated with basal medium or ACM>40 (n = 4). After 24 hours, invaded cells were visualized using DAPI and counted. G, Relative wound closure was assessed following treatment with ACM>40 (siANGPTL4_1: n = 10; shANGPTL4_1: n = 8). H, Immunoblots for FAK, phosphorylated FAK (Tyr576/577 and Tyr397), and Gapdh in cell lysates of MDA-MB-231 cells treated with basal medium or ACM>40. I, Matrigel invasion and (J) wound scratch assays of MDA-MB231 cells treated with basal medium or ACM>40 and with or without 10 μmol/L FAK inhibitor PF-573228 (n = 4). K, Cell lysates of ANGPTL4-depleted MDA-MB-231 cells were prepared after incubation with basal medium or ACM>40 and subjected to immunoblotting (*, P < 0.05; **, P < 0.01; ***, P < 0.001; nd, not detectable).
- Figure 7.
Schematic representation of cellular, molecular, and metabolic changes in TNBC cells in response to ACM of obese individuals.
Additional Files
Supplementary Data
- Supplementary Data, Tables 1-7, Supplementary Figures 1-11 - Table S1: Primersequences used for human and murine cell lines. Table S2: Upstream regulators of genes with increased expression in MDA-MB-231 cells co-cultured with murine adipose tissue (HFD). Table S3: Upstream regulators of genes with decreased expression in MDA-MB-231 cells co-cultured with murine adipose tissue (HFD). Table S4: Characterization of the free fatty acid profile of ACMs<30 and ACMs>40. Table S5: Enriched GO terms (Biological Process) in genes with {greater than or equal to}1.5-fold downregulation in MDAMB-231 cells co-cultured with adipose tissue of HFD mice vs control. Table S6: Enriched GO terms (Biological Process) in genes with {greater than or equal to}1.5-fold upregulation in MDA-MB231 cells co-cultured with adipose tissue of HFD mice vs control. Fig. S1: Pathway Analysis and gene expression data of co-cultured MDA-MB-231 cells. Fig. S2: ACM increases PPAR target gene expression in murine E0771 cells. Fig. S3: ANGPTL4 mRNA is increased in human triple negative breast cancer and associated with reduced survival. Fig. S4: PPAR target gene expression is dependent on PPARα. Fig. S5: Free fatty acids activate PPAR signaling in TNBC cells. Fig. S6: Incubation of TNBC cells with ACM or BSA-OA decreases de novo FA synthesis. Fig. S7: Cultivation of TNBC cells with ACM or BSA-OA induces the expression of β-oxidation genes. Fig. S8: Treatment with oleic acid increases intracellular lipid droplet formation in TNBC cells. Fig S9: ACM promotes proliferation and migration of E0771 cells. Fig. S10: ANGPTL4 knockdown in MDA-MB-231 cells does not affect cell proliferation. Fig. S11: MMP2 is upregulated in MDA-MB-231 cells upon ACM cultivation.
Volume 18, Issue 12
