Multiplex immunohistochemistry to explore the tumor immune microenvironment in HCC patients with different GPC3...

GPC3 has been recognized as a promising target for immunotherapy in hepatocellular carcinoma (HCC). However, the GPC3-targeted immunotherapies have shown limited therapeutic efficacy. The use of anti-PD-1/PD-L1 monoclonal antibodies in HCC treatment is considerably constrained. Furthermore, there is still a notable lack of understanding concerning the immune landscape in HCC, especially regarding varied GPC3 expression levels. Therefore, thorough exploration of the intricate tumor immune microenvironment at different GPC3 expression levels is essential for guiding and improving HCC treatment strategies.

Sixty patients with HCC were enrolled in this study, receiving a first-line treatment that combined anti-angiogenesis targeted drugs and immunotherapy. Immunohistochemistry was used to assess the levels of GPC3 expression. Multiple immunohistochemical markers, such as CD8, PD-1, LAG3, TIGIT, TIM-3, CD103, Claudin18.2, PD-L1, CD4, Foxp3, CD68, CD163, GPC3, CD11C, CD14, CD66b, and HLA-DR, were used to characterize the immune microenvironment and spatial distribution of immune cells in HCC tumors with different levels of GPC3 expression. Cell expression levels and spatial distribution were determined by fluorescence staining and subsequent analysis of fluorescence intensity using the Panoramic Pathology Workstation (Pano ATLAS). This approach facilitated a detailed examination of cell characteristics and spatial information within the samples.

Based on the result of GPC3 immunohistochemical analysis, patients with strong positive GPC3 expression were classified as high GPC3 expression, while the others were classified as low GPC3 expression. Patients in the low GPC3 expression group had longer overall survival (OS) than in the high group (P = 0.003, HR = 2.9240). Further exploration of the immune microenvironment based on different GPC3 expression levels revealed that in high GPC3 expression group, the proportions of CD8⁺ T cells(P = 0.0435), TIM-3⁺ T cells(P = 0.0447), CD103⁺CD8⁺ tissue-resident T cells(P = 0.0410), CD11C⁺CD14^-DC cells(P = 0.0497), CD11C⁺HLA-DR^-DC cells(P = 0.0309), CD11C⁺CD14^-HLA-DR^-DC cells(P = 0.0233), and CD11C⁺CD14^-CD66b^-DC cells(P = 0.0474) were all higher compared to low expression group. At spatial distances of 10 μm, 20 μm, and 30 μm, the levels of CD8⁺ T cells were higher in the high expression group compared to the low expression group (high vs. low: P = 0.0281, P = 0.0236, P = 0.0220).

Multiple immunohistochemistry is a powerful technique for exploring the intricate immune microenvironment of hepatocellular carcinoma, enabling the precise identification of diverse cell subsets and their spatial distribution within the tumor microenvironment. This methodology provides valuable insights into the complex interactions and spatial organization of immune cells in the context of hepatocellular carcinoma progression. Low GPC3 expression in HCC patients indicates potential benefits from combined targeted and immunotherapy. Different levels of GPC3 expression levels can predict the effectiveness of targeted combination immunotherapy in HCC patients. Additionally, different GPC3 expression patterns in HCC patients correspond to unique tumor immune microenvironments, which have implications for guiding HCC treatment approaches.