(A–B) CRISPR/Cas9 was used to disrupt PC and/or ME1 as indicated in AL1376 murine PDAC cells. (A) Fractional labeling of M+1 aspartate following culture of the indicated cells for 24 hr in media containing 1-13C-pyruvate. M+1 aspartate labeling was significantly decreased in double knockout cells compared to control cells (p=0.0012, 0.0001, and 0.0131) or to double knockout cells with exogenous ME1 expression (ME rescue) (p=0.0006) based on unpaired, two-tailed student’s t-tests. Mean +/- SD is shown. (B) Aspartate M+1 isotopomer labeling was normalized to pyruvate M+1 labeling as a surrogate for pyruvate carboxylation activity following 24 hr of 1-13C-pyruvate tracing. Pyruvate carboxylation activity was significantly decreased in double knockout cells compared to control cell lines (p=0.0004, 0.0002, and 0.0100) or to double knockout cells with exogenous ME1 expression (ME rescue) (p<0.0001) based on unpaired, two-tailed student’s t-tests. Mean +/- SD is shown. (C) Sections from tumors arising in LSL-KrasG12D/+; Trp53fl/fl; Pdx1-Cre (KP-/-C) mice were stained with an antibody against ME1. Scale bar represents 100 μm. (D) Expression of ME1 was measured by qPCR in the indicated cells sorted from tumors arising in KP-/-C mice. The expression of ME1 was not significantly different in sorted cancer cells compared to the whole tumor (p=0.1114), but was significantly higher in sorted cancer cells compared to fibroblasts (p=0.0009) based on unpaired, two-tailed student’s t-tests. Mean +/- SEM is shown. 36B4 was used as a housekeeping gene control. (E) Representative image from a human pancreatic tumor tissue microarray stained with an antibody against ME1. Scale bar represents 100 μm. (F) Proliferation rate of sgControl and sgME1 AL1376 murine PDAC cells in standard 2D culture. (G) Quantification of tdTomato fluorescence from images of sgControl or sgME1 PDAC cancer cell organoids isolated from KP-/-CT tumors cultured in DMEM-pyruvate with 10% dialyzed FBS alone or with murine PSCs. sgControl organoids with PSCs trended towards higher tdTomato fluorescence compared to sgME1 organoids with PSCs (p=0.0579) but was not significant based on an unpaired, two-tailed student’s t-test. Mean +/- SD is shown. The sgControl data are also shown in Figure 6B–C. (H) Fluorescent images of sgControl or sgME1 PDAC cancer cell organoids cultured DMEM-pyruvate with 10% dialyzed FBS alone (top) or with murine PSCs (bottom). The sgControl images are also shown in Figure 6B–C. (I) Growth of sgControl (black) and sgME1 (blue) AL1376 murine PDAC cells as tumors following subcutaneous transplantation into syngeneic B6 mice. The final tumor volume is significantly greater in sgControl AL1376 cells compared to sgME1 cells based on unpaired, two-tailed student’s t-tests (p<0.0001 to 0.0017). Mean +/- SEM is shown. n = 6 for each group.