Extended Data Fig. 10: Combined blockade of Tgfβr1 and Pdl1 eliminates doxorubicin-resistant mouse HCC.

a. Schematic showing the establishment of mouse model of control (n=5 mice) or doxorubicin-resistant syngeneic orthotopic allografts (n=11 mice) which were then treated with control antibody (n=6 mice) or combination therapy with TgfbrI and anti-Pdl1 inhibitor (n=10 mice). b.Representative gross images, H&E and IHC images of control or doxorubicin-resistant orthotopic HCC-bearing mice treated with control antibody (n=6 mice) or combination therapy with TgfbrI and anti-Pdl1 inhibitor (n=10 mice). IHC for CD8 T cells shows in the bottom panel. c. Quantification of tumor burden and CD8T cells in the liver of control or doxorubicin-resistant orthotopic HCC-bearing mice treated with control antibody (n=6 mice) or combination therapy with TgfbrI and anti-Pdl1 inhibitor (n=10 mice). Bar plots compare the mean between the groups with two-sided unpaired t-tests. Data are presented as mean values +/− SEM. d. Flow cytometry-based quantification of tumor-infiltrating leukocytes, T cells, M2-like (PDL1+ or CD206+) and M1-like macrophage (CD86+ or MHCII+) subsets in doxorubicin-resistant orthotopic HCC-bearing mice treated with control antibody (n=4 mice) or combination therapy with TgfbrI and anti-Pdl1 inhibitor (n=4 mice). Bar plots compare the mean between the groups two-sided with unpaired t-tests. Data are presented as mean values +/− SEM. e. Representative flow cytometry images and quantification of M1-like and M2-like macrophages within the tumor in the doxorubicin-resistant orthotopic HCC-bearing mice treated with control antibody or combination therapy with TgfbrI and anti-Pdl1 inhibitor. f. Hep 53.4 HCC cell lines are treated with control or doxorubicin for 96 hours and viable cells are selected for orthotopic implantation into mouse liver, confirmed by MRI. g. Flow cytometry analysis shows no difference in leukocyte or T cell or macrophage infiltration in WT mice bearing control HCC treated with either control antibody (n=2 mice) of combined inhibition of Tgfbr1 and anti-Pdl1 antibody (n=3 mice). Data are presented as mean values +/− SEM. h. Schematic summarizing the main findings- i. The spatial organization of post-TACE residual HCC is unveiled through integrated analysis of single-cell spatial profiling employing CODEX and GeoMx spatial transcriptomics of human HCC. ii. Mouse model of minimal residual disease (MRD) reveals that TGFβ1 derived from PDL1+ macrophages enable the persistence of residual stem-like tumor cells and induces exhaustion in CD8T cells. iii. In two mouse models of MRD, we target the TGFβ pathway and PDL1, eliminating residual tumor cells and preventing HCC recurrence. Abbreviations: HCC- hepatocellular carcinoma, Tgfb -transforming growth factor beta, H&E-hematoxylin and eosin, Ctrl-control, Res-residual, MRD- Minimal residual disease, DoxR- doxorubicin-resistant.