Fig. 6: TGFβ2-driven LD formation under acidic conditions fosters in vivo cancer cell invasiveness.

a Representative immunofluorescent pictures of Alexa Fluor 568-conjugated pHLIP and BODIPY 493/503 staining in 10-day-old HT-29 spheroids. Scale bar: 100 µm. b Time-course accumulation of pHLIP during HT-29 spheroid growth. c–e Distribution (from the periphery to the core of the spheroids) of pHLIP (c), BODIPY 493/503 (d), and Ki67 (e) staining. f, g Representative immunofluorescent pictures (f) and quantification (g) of BODIPY 493/503 staining in 10-day-old HT-29 spheroids treated or not with 20 µM TGF-βRI inhibitor SB431542. h Time course of the growth inhibitory effects of 20 µM SB431542 on 3D tumor spheroid growth. I, j Representative immunohistochemical pictures (i) and quantification (j) of GFP- or mCherry-positive lung metastases in mice 5 weeks after i.v. injection of GFP-expressing native and mCherry-expressing acidosis-adapted HT-29 cancer cells (n = 5–8 mice per group). Scale bar: 1 mm. k, l Representative H&E staining (k) and quantification of metastasis area (l) in lungs from mice 5 weeks after i.v. injection of acidosis-adapted HT-29 cells and i.p. treatment with 40 mg/kg etomoxir (n = 6–7 mice per group). Scale bar: 1 mm. m, n Kaplan–Meier curves depicting mouse survival after i.v. injection of native and acidosis-adapted FaDu (m) and HCT-116 (n) cancer cells. o, p Representative H&E staining (o) and quantification of metastasis area (p) in lungs from mice 5 weeks after i.v. injection of lipid droplet-deprived (forskolin-treated; or not) acidosis-adapted HCT-116 cells (n = 5 mice per group). Scale bar: 250 µm. Data are represented as mean ± SEM of three independent experiments (with ≥ 6 technical replicates). Significance was determined by Student’s t-test (b, g, j, l, p). *p < 0.05; **p < 0.01; ***p < 0.001. Source data are provided as a Source Data file.