Figure 8. NLRX1 in cancer cells inhibits STING signaling in vivo and excludes functional effectors from TME.

(A) Control and shNLRX1 MOC2-E6/E7 cells were stimulated by 1.0 μg/mL poly(dA:dT) for 16 hours, and qPCR was performed to quantitate the mRNA levels of indicated IFN-I signature genes. Experiments were performed 3 times. Comparisons between 2 groups were made using a 2-tailed unpaired t test (**P < 0.01, ****P < 0.0001). (B) Control and NLRX1-deficient MOC2-E6/E7 cells were transfected with 1.0 μg/mL expression plasmid encoding murine STING and incubated for 16 hours. Cell lysates were immunoblotted against the indicated markers. Immunoblotting results represent 2 independent repeats. (C) The proliferation of EV control and shNLRX1 MOC2-E6/E7 cells was measured by an alamarBlue assay. Each group included 5 replicate wells. Experiments were performed twice. (D) One million EV control or NLRX1-deficient MOC2-E6/E7 cells were implanted subcutaneously in the right flank of C57BL/6 hosts. Tumor measurements were performed every 2–3 days. Tumor burden was compared using the generalized estimating equations model (n = 8 in each group; *P < 0.05). In vivo experiments were performed 3 times with n = 19 total in each group. A representative set is shown. (E) Total RNA was isolated from 1 representative set of tumors and subjected to qPCR. (F) After harvesting of tumors, TILs of 1 representative set were isolated and analyzed by flow cytometry (n = 8 in control group, n = 4 in shNLRX1 group due to tumor rejection). (G) Lymphocytes were isolated from draining lymph nodes of 1 representative set and assessed by flow cytometry (n = 5 in each group). Comparisons between 2 groups from E–G were made using a 2-tailed unpaired t test (*P < 0.05, **P < 0.01). Quantifications indicate the mean ± SEM. Results represent 3 independent experiments.