Fig. 5
Treatment of low-dose mitoxantrone in combination with TGFβ and PD-1 blockade delays the growth of subcutaneously transplanted 9464D tumors and reshapes the intratumoral infiltrate. A Schematic representation of the drug treatment and timing of tumor immune infiltrate analysis. B Tumor growth of 9464D injected subcutaneously in C57BL/6 mice and treated as indicated. Significance at day 41 (Mann Whitney test). C Weight of explanted tumors at day 41 after cell inoculation. D Flow-cytometry analysis of the immune compartment in 1 day-MTX-treated 9464D tumors. Levels of significance for comparison between samples were determined by two-tailed Student’s t test. E Chemokine expression in 1 day-MTX-treated 9464D lysates by protein array. Relative chemokine expression based on densitometric analysis is shown on the right. F-H Flow-cytometry analysis of the immune content in 9464D tumors treated for 12 days. I Flow-cytometry analysis of the activation status of tumor-infiltrating NK cells. L Multiple immunofluorescence staining of drug-treated 9464D tumor specimens for NK1.1 (green) and granzyme B (red), shown at original magnification × 40 (zoom), scale bar 30 μm. Images with nuclei (Hoechst) are shown in the bottom panel. Granzyme B-positive NK cells are indicated by yellow arrows. Quantitative analysis of the indicated immune cells from n = 6 biologically independent 9464D specimens is shown on the right. Levels of significance for comparison between samples in F-L were determined by ANOVA. CTR, vehicle control; MTX, mitoxantrone; MTP, mitoxantrone, anti-TGFβ and anti-PD-1; GZMB, granzyme B. Statistically significant, P values are shown