Fig. 4

Low-dose mitoxantrone recalls activated CD8⁺ T cells and NK cells in MDOTS when combined with TGFβ and PD-1 blockade. A Experimental scheme. Explanted tumors are reduced to small pieces, cultured to form MDOTS and then co-cultured with syngeneic splenocytes from tumor-bearing mice in ULA plates or microfluidic devices. Representative images of MDOTS cultured with or without splenocytes are shown. Original magnification, 20x. Scale bar, 30 μm. B Representative IHC staining of hematoxylin and eosin (HE) and synaptophysin (syn) in 975A2 MDOTS and the tumors from which they were derived. Brown, synaptophysin positive cells. Nuclei were counterstained with hematoxylin (blue). Original magnification, 20x. Scale bar, 30 μm. C, D Flow-cytometry analysis of IFNγ and granzyme B expression of CD8⁺ T cells (C) and NK cells (D) from splenocytes co-cultured 24 hours with drug-treated and untreated 975A2 MDOTS. E Representative images of the migration of red-labeled splenocytes in microfluidic devices to drug-treated and untreated 975A2 MDOTS after 24 hours of co-culture. The number of splenocytes migrating versus drug-treated and untreated 975A2 MDOTS was assessed by ImageJ software. Data are shown as fold change ± SD. Levels of significance for comparison between samples were determined by ANOVA (C-E). CTR, vehicle control; MTX, mitoxantrone; aTGFβ, anti-TGFβ; aPD-1, anti-PD-1; MT, mitoxantrone and anti-TGFβ; MP, mitoxantrone and anti-PD-1; MTP, mitoxantrone, anti-TGFβ and anti-PD-1; GZMB, granzyme B. Statistically significant P values are shown