Extended Data Fig. 2: Pancreatic stellate cell ACLY contributes to acetate secretion and tumor burden.
a, Relative mRNA levels of ACLY in human pancreatic stellate (HPS) cells expressing scrambled control (shScr) and shACLY plasmids (n = 3 in each group from independent biological replicates). b, Experiment scheme and immunoblots showing the expression of ACLY protein in PDAC cells upon treatment with CM-derived from HPS and CAF-0911 cells. Representative image of two independent experiments. c, Experiment scheme and immunoblots showing the expression of ACLY protein in S2-013, HPAF-II, CFPAC-1, and T3M4 cells upon co-culture with HPS and CAF-0911 cells for 24 hrs. Representative image of two independent experiments. d, Representative images of tumors excised from athymic-nude mice implanted with S2-013 cells alone (n = 9) or co-implanted with control (shScr; n = 7) and ACLY knockdown (shACLY; target a, n = 9; target b, n = 9) HPS cells. e,f, Representative IHC images for Ki-67 staining, nuclear staining (DAPI), cytokeratin 19 (epithelial/tumor cells), and merge in tumor sections from athymic-nude mice implanted with S2-013 cells alone or co-implanted with shScr or shACLY HPS cells (along with the quantitation of percent Ki-67 and CK19 dual positive cells (f). Scale bar = 100 µm. Ki-67 and CK19 positive cells were counted manually in three different fields from 3 tumor sections of each group (n = 9 in each group). g, Tumor volumes, upon necropsy of athymic-nude mice implanted with CFPAC-1 cells alone (n = 7) or co-implanted with shScr (n = 8) and shACLY (shACLY-a, n = 7; shACLY-b, n = 6) HPS cells. h,i, Representative IHC images for Ki-67 staining (h) in tumor sections from athymic-nude mice implanted with CFPAC-1 cells alone or co-implanted with shScr or shACLY HPS cells along with the quantitation of percent positive cells (i). Scale bar = 100 µm. Ki-67 positive cells were counted manually in three different fields from 3 tumor sections of each group (n = 9 in each group). j, Representative immunofluorescent images showing co-expression of αSMA and ACLY proteins in tumor sections from athymic-nude mice implanted with CFPAC-1 cells alone or co-implanted with shScr or shACLY HPS cells. Scale bar = 250 µm. k, Levels of acetate in the interstitial fluid of tumor tissues from athymic-nude mice implanted with S2-013 cells alone (-) or co-implanted with shScr or shACLY HPS cells. l,m, Tumor weights (l) and tumor volumes (m), upon necropsy from athymic-nude mice implanted with S2-013 cells alone or co-implanted with shScr or shACLY HPS cells. For k,l,m, n = 9 (S2-013 alone), 9 (co-implanted with shScr HPS), 8 (co-implanted with shACLY HPS) mice. One-way ANOVA with Bonferroni’s post-hoc test; mean ± s.e.m. (a,f,g,i,k,l,m).
