Reciprocal interactions between malignant cells and macrophages enhance cancer stemness and M2 polarization in HBV-associated hepatocellular carcinoma

Abstract

<p><strong>Background:</strong> The tumor microenvironment of cancers has emerged as a crucial component in regulating cancer stemness and plays a pivotal role in cell-cell communication. However, the specific mechanisms underlying these phenomena remain poorly understood. <strong>Methods:</strong> We performed the single-cell RNA sequencing (scRNA-seq) on nine HBV-associated hepatocellular carcinoma (HCC) patients. The heterogeneity of the malignant cells in pathway functions, transcription factors (TFs) regulation, overall survival, stemness, as well as ligand-receptor-based intercellular communication with macrophages were characterized. The aggressive and stemness feature for the target tumor subclone was validated by the conduction of <em>in vitro</em> assays including sphere formation, proliferation, Annexin V apoptosis, flow cytometry, siRNA library screening assays, and multiple <em>in vivo</em> preclinical mouse models including mouse hepatoma cell and human HCC cell xenograft models with subcutaneous or orthotopic injection. <strong>Results:</strong> Our analysis yielded a comprehensive atlas of 31,664 cells, revealing a diverse array of malignant cell subpopulations. Notably, we identified a stemness-related subclone of HCC cells with concurrent upregulation of CD24, CD47, and ICAM1 expression that correlated with poorer overall survival. Functional characterization both <em>in vitro</em> and <em>in vivo</em> validated S100A11 as one of the top downstream mediators for tumor initiation and stemness maintenance of this subclone. Further investigation of cell-cell communication within the tumor microenvironment revealed a propensity for bi-directional crosstalk between this stemness-related subclone and tumor-associated macrophages (TAMs). Co-culture study showed that this interaction resulted in the maintenance of the expression of cancer stem cell markers and driving M2-like TAM polarization towards a pro-tumorigenic niche. We also consolidated an inverse relationship between the proportions of TAMs and tumor-infiltrating T cells. <strong>Conclusions:</strong> Our study highlighted the critical role of stemness-related cancer cell populations in driving an immunosuppressive tumor microenvironment and identified the S100A11 gene as a key mediator for stemness maintenance in HCC. Moreover, our study provides support that the maintenance of cancer stemness is more attributed to M2 polarization than the recruitment of the TAMs.</p>