Figure 7. Lung cancer cells require Pcx for tumor formation in vivo.
(A) sgRNAs targeting Pcx or control were introduced into lung cancer cells derived from KP lung tumor. Proliferation of control and Pcx disrupted cells in culture is shown (n=3), as is a Western blot analysis showing Pcx expression from isogenic clones.
(B) The same cells described in (A) were cultured in the presence of [U-13C]glucose. Percent labeling of aspartate isotopomers is shown for Control and Pcx-disrupted cell lines. The percent M3 isotopomer of aspartate that can be generated from glucose via flux through Pcx is also presented in green, and percent labeling of other isotopomers of aspartate displayed, n = 3.
(C) The same cells described in (A) were introduced as allografts into the flanks of nu/nu mice. Immunohistochemistry assessing Pcx expression in lung tumors derived from control cells or material present at the injection site from Pcx-disrupted cells is shown.
(D) The same cells described in (A) were introduced as allografts into the flanks of mice. The percent labeling of aspartate (Asp), citrate (Cit), glutamate (Glu), and glutamine (Glu) in lung tumors derived from control cells (black) or material present at the injection site from Pcx-disrupted cells (green) was determined following a 6-hour [U-13C]glucose infusion. The M0 and M3 isotopomers are shown for each metabolite. All values in mean ± SEM. *Difference is statistically significant by two-tailed T-test, p < 0.05.
(E) The same cells described in (A) were introduced as allografts into the flanks of nu/nu mice. Immunohistochemistry assessing Pcx expression in lung tumors derived from control cells or material present at the injection site from Pcx-disrupted cells is shown. Scale bar: 200μM.
(F) Representative H&E staining of the same cells described in (A) 4 weeks after orthotopic transplantation into the lungs of mice. Scale bar: 200μM.
(G) Representative hematoxylin and eosin staining of KP mice infected with pSECC containing a control sgRNA (Control) and sgPcx (n = 4 mice analyzed, no tumors were observed in the sgPcx mice). Scale bar indicates 2mm.