Single-cell RNA sequencing reveals B cell-related immunosuppressive landscape and a potential suppressor in hepatocellular carcinoma
Background Sophisticated tumor microenvironment is responsible for malignant progression and poor prognosis of hepatocellular carcinoma (HCC) patients. Discovering new therapeutic targets was desired for preferable treatments of HCC patients. Methods To uncover the HCC microenvironment, single-cell transcriptomes of HCC tissues and corresponding non-cancerous tissues were analyzed. Differentially expressed genes (DEGs) and enriched pathways were analyzed in B cells. Moreover, heterogeneity between malignant and normal hepatocytes was further investigated, which revealed potential biomarkers in HCC progression. The candidate biomarkers were further explored in datasets from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Of which, serum amyloid A2 (SAA2) was found out and further validated in HCC tissues by immunohistochemistry (IHC) and western blot. The biological roles of SAA2 were further investigations in HCC cells. Results The results suggested that B cells in HCC tissues was significantly decreased compared to non-cancerous tissues, which might result in the immunosuppressive status of HCC microenvironment. Following differentially expressed genes (DEGs) and functional enriched analysis indicated B cells might participate in immunosuppression of HCC by regulating lipid metabolism. Further analysis on hepatocytes found out highly expressed genes in normal hepatocytes derived from non-cancerous liver tissues, which were validated in datasets from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Interestingly, we found serum amyloid A2 (SAA2) was highly expressed in normal liver tissues compared to HCC tissues. The results were validated in clinical HCC specimens by immunohistochemistry (IHC) and western blot assays. Moreover, investigations in HCC cells revealed SAA2 acted as a tumor suppressor in HCC progression. Conclusions Taken together, our study might be of great significance for revealing B cell-related immunosuppressive landscape in HCC, as well as discovering SAA2 as a novel suppressor in HCC, which made great sense for better understanding of HCC landscape and offered a promising therapeutic target for HCC patients. Keywords: B cell-related immunosuppressive landscape, hepatocellular carcinoma, serum amyloid A2, single-cell RNA sequencing, suppressor, tumor microenvironment Overall design: Although the HCC microenvironment has been explored extensively, the tumor ecosystem at single-cell level remains to be further resolved. To investigate the HCC microenvironment at cellular level, we designed the following study. The complete study process was shown in Figure 1A. In order to build a comprehensive single-cell reference for HCC, we recruited 5 newly treated HCC patients for scRNA-seq. Detailed clinical and pathological information including gender, age, tumor size, alpha-fetoprotein (AFP) levels, Child-Pugh stage, and Barcelona Clinic Liver Cancer (BCLC) stage were shown in Supplementary Table S1. These patients were diagnosed as HCC pathologically and their surgical HCC specimens as well as paired corresponding non-cancerous tissues were collected. Sequencing libraries of pooled single cells were obtained and subjected to a sequencing run. The Chromium platform (10×Genomics) was used to capture single cells in prepared single-cell suspensions. The single cells were collected in form of droplets and marked with specific barcodes and then subjected to a subsequent series of manipulation procedures. The captured mRNAs were reverse-transcribed into cDNA and amplified for single-cell library preparation . We applied QC with key metrics including number of genes, total counts, and percentage of mitochondrial counts . After applying the QC criteria, a total of 73707 single cells were retained and included in the downstream analysis, of which 36323 single cells were from HCC tissues and the rest were from corresponding non-cancerous tissues (Supplementary Table S2). These identified cells produced an average of 2783 detected genes. Then, the cell compositions in HCC ecosystem were resolved and cell clusters closely related to the immunosuppressive landscape of HCC especially B cells, were analyzed for DEGs and corresponding signal pathways.