(A) Western blot analysis of PC expression levels in single-cell cloned sgPC knockout PSCs compared to sgControl clones using β-actin as a control. (B) Fractional labeling of aspartate M+1 following 24 hr of 1-13C-pyruvate tracing in sgPC PSC cell lines compared to sgControl cell lines. Mean +/- SD is shown. (C) Aspartate M+1 isotopomer labeling from (P) was normalized to pyruvate M+1 labeling as a surrogate for pyruvate carboxylation activity following 24 hr of 1-13C-pyruvate tracing. Mean +/- SD is shown. (D) Fluorescent images of murine PDAC organoids cultured in DMEM-pyruvate with 10% dialyzed FBS alone (top), with murine sgControl PSCs (middle), or with murine sgPC PSCs (bottom). (E) Quantification of tdTomato fluorescence from images in (R). The differences in tdTomato fluorescence between organoids alone and organoids with sgControl PSCs (0.0023), organoids alone and organoids with sgPC PSCs (p=0.0122), and organoids with sgControl PSCs and organoids with sgPC PSCs (p=0.0041) are significant based on unpaired, two-tailed student’s t-tests. Mean +/- SD is shown. (F) Growth of sgControl AL1376 murine PDAC cell lines alone (blue), with sgControl PSCs (green), or sgPC PSCs (black) after subcutaneous transplantation into syngeneic Bl6 mice. There is no significant difference in final tumor volume between sgControl AL1376 cells and sgControl AL1376 cells co-injected with sgPC PSCs (p=0.1313), but differences are significant at intermediate time points. The difference in final tumor volume is significant between sgControl AL1376 cells and sgControl AL1376 cells with sgControl PSCs (p<0.0001), based on unpaired, two-tailed student’s t-tests. Mean +/- SEM is shown. n = 6 for each group.