Single-cell RNA sequencing unravels the immunosuppressive landscape and tumor heterogeneity of HBV-associated hepatocellular carcinoma

Abstract

Introduction: The fundamental understanding of the immunocellular and molecular landscapes of hepatocellular carcinoma (HCC) remains elusive. Methods: We performed single-cell RNA sequencing (scRNA-seq) on HBV-associated HCCs. The cellular composition, subclonal diversity, high-resolution multifaceted landscapes of individual HCC cells and the immunosuppressive landscape were analyzed. Functional analyses were performed to validate the findings. Results: From the scRNA-seq, we detected a statistically significant association between CD163 (M2 macrophage marker) and LAIR1 expression, which was further confirmed in both in-house and TCGA RNA-seq datasets. We observed frequent overlap of LAIR1 and CD163 immunofluorescence staining and detected a significant association between LAIR1 and CD163 expression in HCC. Besides, we established stable LAIR1 knockdown (shLAIR1) in macrophages using THP-1 cells. Co-culturing the shLAIR1 macrophages with CD8 T cells upregulated T cell activation, as exemplified by the increased proportion of CD44+CD62L- effector T cells. Of significance, using scRNA-seq, we identified a prominent co-inhibitory immune checkpoint signal via TIGIT-NECTIN2 axis in complementary T cells and antigen-presenting cells. Moreover, in both our in-house and TCGA RNA-seq cohorts as well as using IHC in our clinical specimens, we observed NECTIN2 was significantly upregulated in HCCs. We also detected a significant association between TIGIT and NECTIN2 expression in HCC but not in non-HCC, HBV-associated cirrhotic livers. This suggests that the TIGIT-NECTIN2 axis may likely be a tumor evasion strategy, instead of viral evasion one. Upon ligation to NECTIN2, TIGIT acts an inhibitory receptor on T and NK cells. To investigate whether NECTIN2 expressed on HCC cells would lead to T cell exhaustion, we established Nectin2 knockout (KO) stable cells in mouse HCC cell line Hepa1-6. Upon co-culturing mouse splenic T cells with Hepa1-6 parental cells, anti-Nectin2 neutralizing antibody significantly restored both CD4+ and CD8+ T cell proliferation. Consistently, KO of Nectin2 in HCC cell lines also restored both CD4 and CD8 T cell proliferation. To extend our observation in vivo, we performed hydrodynamic tail-vein injection to generate wildtype (WT) and Nectin2 KO mouse HCC. The Nectin2 KO HCC tumors were significantly smaller as compared to WT controls, accompanied with increased infiltration of T effector, CD4+, and CD8+ cells in HCC. Similar findings were also observed in our Nectin2 stable knockdown orthotopic implantation model. In addition, the cell state transition of immune cells towards a more immunosuppressive and exhaustive status exemplified the overall cancer-promoting immunocellular landscape. Using global transcriptomic profiling, liver cancer stem cell markers, inferred copy number variation, and expression profile of receptor tyrosine kinase families, our findings collectively suggested the degree of inter-tumoral heterogeneity was more prominent than intra-tumoral one. Conclusion: Taking advantage of the multi-dimensional capacity of scRNA-seq, we have revealed the novel cellular and immunosuppressive landscapes and demonstrated the important roles of LAIR1-expressing TAMs and TIGIT-NECTIN2 axis in shaping a cancer-promoting tumor microenvironment in HCC. Our overview of the immunosuppressive landscape and intercellular interactions provide useful mechanistic information for the design of efficacious immune-oncology treatments in HCC. Funding support: Hong Kong Research Grants Council Theme-based Research Scheme (T12-704116-R).