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. 2021 Dec 8;9(12):e002772. doi: 10.1136/jitc-2021-002772

Figure 4.

Figure 4

SN-38 sensitizes unresponsive tumors to respond to anti-PD-1 mAb therapy, regulates PD-L1 and FOXO3 expression in tumors, and engages mouse NK1.1/CD49b-positive NK cells and/or some CD3/CD8-positive T cells to infiltrate the TME in a syngeneic mouse tumor model. (A) Mouse ID8 cells were injected into female C57BL6 mice subcutaneously (n=4/group). When palpable solid ID8 tumors were detected, mice were given an intraperitoneal injection (0.1 mL) of an anti-PD-1 Ab (8 mg/kg BW/mouse) or SN-38 (100 µg/kg BW/mouse) or a combination of both drugs (PD-1 Ab + SN38) or equal amounts of controls, mouse isotype IgG or DMSO, once per week at even intervals for 5 weeks. (B) The ID8 tumor tissues were excised from the treated mice for IF analysis. Three slides of samples from ID8 tumors treated with the indicated regimens or controls were incubated with antimouse PD-L1 or antimouse FOXO3 Abs and followed by Alexa Fluor 488-conjugated or 594-conjugated secondary Abs and IF analysis as described previously. Archetypal IF images were illustrated; all IF and DAPI images were shown in online supplemental figure S3. Scale bar: 50 µm. (c) The relative expression of PD-L1 and FOXO3 between tumors treated with the indicated regimens or controls in vivo are shown in the histograms, respectively. (D) Three slides of samples from ID8 tumors treated with the indicated regimens or controls were incubated with antimouse NK1.1 or antimouse CD3 Abs and followed by secondary Abs and IF analysis. Paradigmatic IF images were shown; all IF and DAPI images were displayed in online supplemental figure S4. Scale bar: 20 µm. (E) The relative expression of mouse NK1.1 and CD3 among tumors treated with the indicated regimens or controls in vivo are shown in the histograms as indicated, respectively. (F–I) Similarly, three slides of samples from these tumors were incubated with antimouse Abs against CD49b or CD3 or CD8, and IF analysis was performed as described previously. Scale bar: 20 µm. The relative expression of mouse CD49b and CD3 (G) or CD8 (I) among tumors treated with the indicated regimens in vivo are shown in the histograms as indicated, respectively. Paradigmatic IF images were shown; all IF and DAPI images were displayed in online supplemental figure S5 and S6. Scale bar: 20 µm. Abs, antibodies; BW, body weight; DMSO, dimethylsulfoxide; IF, immunofluorescence; mAb, monoclonal antibody; PD-L1, programmed death-ligand 1; TME, tumor microenvironment.