Figure 6.
Orthotopic mouse oral squamous cell carcinoma tumors exhibit inhibited mitophagy when orally infected with P gingivalis
(A) Orthotopic oral squamous cell carcinoma tumors were generated in female C57BL/6 mice (6–8 weeks old) by injecting MOC2 cells intramuscularly into the mylohyoid. Tumors developed over at least 2.5 weeks before uninfected (NP) mice were treated with 10 mg/kg LCL768 intraperitoneally every other day for 2.5 weeks. Otherwise, the resident mouse oral microbiota was treated with antibiotic for 3 days in drinking water prior to starting concurrent P. gingivalis infection (109 CFU, oral gavage) and LCL768/vehicle intraperitoneal treatment every other day. P. gingivalis infection via oral gavage was performed four times total.
(B) Fluorescent in situ hybridization (FISH) was conducted on mouse OSCC tumor sections infected/uninfected with P. gingivalis and treated with LCL768 or vehicle control to confirm the presence of the bacterium in tumor samples. Insets (right panels) show higher magnifications marked with white squares in the middle panels.
(C) Quantification of P. gingivalis signal foci mean fluorescence intensity in (B) was completed with ImageJ Fiji and normalized to uninfected, untreated control. Values indicate mean ± SD of n = 3 independent experiments. Ns, not significant, ∗∗∗∗p < 0.0001.
(D–G) Immunofluorescence was completed on sections of female C57BL/6 mouse orthotopic oral squamous carcinoma tumor infected or uninfected with P. gingivalis (109 CFU) via oral feeding and treated with LCL768 or vehicle control intraperitoneally (10 mg/kg). Quantification of confocal images of LC3 and Tom20 (D), ceramide and LC3 (E), Tom20 and LAMP1 (F), ANXA2 and ceramide (G) association are shown. Pearson’s correlation coefficient values indicate mean ± SD of n = 3 independent experiments, normalized to uninfected, untreated control. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
(H) Mouse OSCC tumors were stained with Ki67 antibody, indicating proliferating tumor cells.
(I) Quantification of Ki67+ nuclei staining in mouse orthotopic tumor sections in (H).
(J) Basal percentage of tumor infiltrating lymphocytes in mouse orthotopic tumors.