Fig. 2

Dynamic changes of the tumor microenvironment and tumor-intrinsic features in tumor tissues obtained pre-treatment of atezolizumab plus bevacizumab combination therapy and post-progression. A Top row: Hematoxylin and eosin staining; Second row: CD8 immunohistochemistry (IHC) revealed that the percentage of CD8⁺ T cells in the central tumor area was also decreased in the post-progression tumor tissue. Third row: PD-L1 IHC showing decreased PD-L1 expression in tumor-infiltrating immune cells (ICs) after disease progression but no change in expression in tumor cells (TCs). Bottom row: Multiplex immunofluorescence assay and the digital pathology algorithm showed an immune-excluded pattern in the post-progression tumor tissue. CD8⁺ T cells were surrounded outside the tumor area, which was defined by HepPar-1 and arginase-1 (Hep/Arg) positivity. A digital pathology algorithm showed the expression of CD8 decreased in tumor area but increased in peritumor stroma. The activated fibroblast marker (FAP) was increased while the blood vasculature marker, CD31, was decreased in the disease progression sample. MHC class I expression in TCs was increased in the tissue collected at the time of disease progression compared with that collected at baseline. B RNA sequencing revealed increased expression of hepatoblast/progenitor signature (i.e., BUB1, DLGAP5, DUSP9, E2F5, IGSF1, NLE1, and RPL10A) and imprinted genes (i.e., DLK1, PEG3, and ZIM2), decreased expression of cytochrome p450 genes (i.e., ADH1C, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, and CYP3A4), and decreased expression of PD-L1 mRNA (CD274) and an T effector signature (GZMB, CXCL9, and PRF1) in the post-progression tissue