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Figure 4. Adipocyte-derived secretome enhances fatty acid metabolism and mitochondria biogenesis in STn-positive gastric cancer cells. (A) Schematic illustration of the workflow used, in which 3T3-L1 adipocytes were treated with Mock or MST6-I secretome/CM to generate transdifferentiated white adipocytes (TWAT)(Mock) or TWAT(MST6-I). The secretome/CM from these adipocytes, enriched in free fatty acids, was used to treat the gastric cancer cells. Created in BioRender. Duarte, H. (2026) https://BioRender.com/iz3w8pf; (B) Seahorse analysis of glycolytic activity in Mock and MST6-I cells treated with secretome/CM from white or transdifferentiated/beige adipocytes [TWAT(MST6-I)]. When indicated, etomoxir was added. Quantification of basal (left panel) and compensatory glycolysis (right panel) is shown as mean ± SEM. One-way ANOVA was used for statistical analysis; unless otherwise indicated, differences were not significant (ns); (C) Quantification of fatty acid uptake by Mock and MST6-I gastric cancer cells relative to untreated controls. Results are shown as mean ± SEM, with statistical significance determined by One-way ANOVA; (D) Fluorescence microscopy images and quantification of fluorescence intensity in Mock (upper panel) and MST6-I (bottom panel) cells, untreated or incubated with secretome/CM from white or transdifferentiated/beige adipocytes pre-labeled with BODIPY FL C16 (green). Where indicated, etomoxir was added. Nuclei were counterstained with DAPI (blue). Images were acquired at 630× magnification. Scale bar: 10 µm. Results are shown as mean ± SEM, with statistical significance determined by an unpaired t-test; (E) Representative electron microscopy images of Mock and MST6-I cells after treatment with white or transdifferentiated/beige [TWAT(MST6-I)] secretome/CM (left panel) and quantification of mitochondrial number per cell (right panel). Arrows indicate mitochondria (yellow), the Golgi apparatus (purple), lipid droplets (pink), and endosomes (orange). Images were acquired at 20,000× magnification. Scale bar: 500 nm. Results are shown as mean ± SEM, with statistical significance determined by One-way ANOVA. Biological replicates are represented as individual data points. ns: P-value > 0.05; *: P-value ≤ 0.05; **: P-value ≤ 0.01; ***: P-value ≤ 0.001; ****: P-value ≤ 0.0001. CM: Conditioned medium; STn: sialyl-Tn; SEM: standard error of the mean; ANOVA: analysis of variance; DAPI: 4',6-diamidino-2-phenylindole.