Figure 2.
Low-dose SN-38 significantly increases the animal survival rate, regulates PD-L1 and FOXO3 expression in mouse ID-8 tumor tissues, and engages mouse CD49b/NK1.1/NKp46-positive NK cells infiltrating the ID-8 TME in a syngeneic mouse ID8 tumor model. Mouse ID-8 cells (2×105 cells/injection) were injected into the peritoneal cavity of female C57BL6 mice (n=9/group). (A) The scheme of the therapeutic model is shown. Twenty days after the inoculation of ID-8 cells, the mice were given an intraperitoneal (i.p.) injection (0.1 mL) of SN-38 (20 µg/kg body weight (BW)/mouse) or metformin (20 mg/kg BW/mouse) or an equal amount of DMSO (control) three times per week at even intervals for several weeks (up to 18 weeks). (B) Animal survival rates of the different groups were shown as Kaplan-Meier survival curves, which were calculated by the Kaplan-Meier method. The median survival days were shown. The significance was compared using the log-rank test. P values between the SN-38 group versus the DMSO group and between the metformin group versus the DMSO group were 0.0093 and 0.0328, respectively. (C) Three slides of tumor samples from mice treated with DMSO or SN-38 were incubated with anti-PD-L1 or anti-FOXO3 Abs and followed by Alexa Fluor 488-conjugated or 594-conjugated secondary Abs and IF analysis as described previously. Four archetypal IF images were displayed; all IF images and DAPI (the nuclear staining) images were shown in online supplemental figure S1). (D and E) The relative expression of PD-L1 and FOXO3 between tumors treated with DMSO and SN-38 in vivo are shown in the histograms as indicated, respectively. Scale bar: 20 µm. (F and G) Three slides of samples from ID8 tumors treated with DMSO or SN-38 were incubated with antimouse CD49b Ab (F) or NK1.1 Ab (G) and followed by fluorescent secondary Abs and IF analysis. The IF images and DAPI images were shown in online supplemental figure S2. (H and I) Total lysates of tumor samples were analyzed by immunoblotting with the indicated anti-NKp46 and anti-NKp44 Abs. (J) Similarly, tumor samples were incubated with antimouse IFN-γ Ab and secondary Ab. GAPDH represents loading controls. Abs, antibodies; DMSO, dimethylsulfoxide; IF, immunofluorescence; IFN-γ, interferon-γ; NK, natural killer; PD-L1, programmed death-ligand 1; TME, tumor microenvironment.