Fig 2: Autophagy stimulation enriches CSCs with primitive mesenchymal cells.Normalized quantification by flow cytometry of the CD45-/Flk1-/Sca1+ population (A), the Sca1+/CD90+/DDR2- mesenchymal primitive subpopulation (B), the whole CD45-/Flk1-/DDR2+ fibroblast-like fraction (C), and of the Sca1-/CD90-/DDR2+ subpopulation (D) in CSCs after 32 hours of nutrient deprivation stress, with or without pretreatment with 50 mM trehalose (TRE) for 24 hours (n=8). *P<0.05, **P<0.01.

Fig 3: CSC characterization.A Representative microscopy image of a fixed GIEMSA-stained CSC colony. Scale bar=500 µm. B Quantification of clonogenic efficiency (n=6). C Quantification of positive cells (n=5), and representative immunofluorescence panels (D) for selected cardiac markers (GATA4, Nkx2.5, TnI, SMA). Arrows indicate positive nuclei. Scale bars=20 µm. E Quantification of single marker positivity by flow cytometry analysis of the whole CSC population (n=5). F Conceptual scheme of the association among the Sca1, CD90, and DDR2 markers with corresponding phenotypes. G Quantification by flow cytometry of the percentage of CD90 or DDR2 cells among the CD45-/Flk1-/Sca1+ population (n=3).

Fig 4: Combination treatment with transcutaneous arginase inhibition and systemic PD-1 inhibition is associated with increased recruitment of intratumoral T cells and dendritic cells than either treatment, alone. (A) Percentage of intratumoral CD45+ leukocytes that are CD3+ CD4+, indicating tumor-infiltrating CD4+ T cells; combination nor-NOHA + nivolumab treatments had significantly higher proportions than either treatment alone. (B) Percentage of intratumoral CD45+ leukocytes that are CD3+ CD8+, indicating tumor-infiltrating CD8+ T cells; combination nor-NOHA + nivolumab treatments had significantly higher proportions than either treatment alone. (C) Percentage of intratumoral CD45 + leukocytes that are CD11c+ MHCII+, indicating tumor-infiltrating dendritic cells; combination nor-NOHA + nivolumab treatments had significantly higher proportions than either treatment alone. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 using the unpaired t-test adjusted for multiple comparisons. Two separate experiments with n > 5 per group.

Fig 5: The α-PD-L1 treatment reduces the PD-L1+ cells in TME.B6 mice were intraperitoneally inoculated with 5 × 105 MC38-luc cancer cells and treated with VV and/or α-PD-L1 as described. Tumour-bearing mice were killed at day 5 post first treatment and primary tumours were collected and analysed to determine the PD-L1+ CD45− cells (a,b), PD-L1+ DC (defined as CD45+ CD11b+CD11c+Ly6G−PD-L1+) (c), PD-L1+G-MDSC (defined as CD45+CD11c−CD11b+Ly6G+Ly6clowPD-L1+) (d), PD-L1+ M-MDSC (defined as CD45+CD11c−CD11b+Ly6G−Ly6chiPD-L1+) (e), PD-L1+ TAM1 (defined as CD45+CD11c−CD11b+Ly6G−F4/80+CD206−PD-L1+) (f), PD-L1+ TAM2 (defined as CD45+CD11c−CD11b+Ly6G−F4/80+CD206+PD-L1+) (g). Of note, the anti-PD-L1 antibody clone 10F.9G2 was used for therapy while clone MHI5 was used for subsequent analysis. Data were analysed using Student's t-test (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).