Integrating single-cell and bulk RNA sequencing reveals CK19 + cancer stem cells and their specific SPP1 + tumor-associated macrophage niche in HBV-related hepatocellular carcinoma

Abstract

Purpose: Cytokeratin 19-positive cancer stem cells (CK19 + CSCs) and their tumor-associated macrophages (TAMs) have not been fully explored yet in the hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC).

Experimental design: Single-cell RNA sequencing was performed on the viable cells obtained from 11 treatment-naïve HBV-associated HCC patients, including 8 CK19 + patients, to elucidate their transcriptomic landscape, CK19 + CSC heterogeneity, and immune microenvironment. Two in-house primary HCC cohorts (96 cases-related HBV and 89 cases with recurrence), TCGA external cohort, and in vitro and in vivo experiments were used to validate the results.

Results: A total of 64,581 single cells derived from the human HCC and adjacent normal tissues were sequenced, and 11 cell types were identified. The result showed that CK19 + CSCs were phenotypically and transcriptionally heterogeneous, co-expressed multiple hepatics CSC markers, and were positively correlated with worse prognosis. Moreover, the SPP1 + TAMs (TAM_SPP1) with strong M2-like features and worse prognosis were specifically enriched in the CK19 + HCC and promoted tumor invasion and metastasis by activating angiogenesis. Importantly, matrix metalloproteinase 9 (MMP9) derived from TAM_SPP1, as the hub gene of CK19 + HCC, was activated by the VEGFA signal.

Conclusions: This study revealed the heterogeneity and stemness characteristics of CK19 + CSCs and specific immunosuppressive TAM_SPP1 in CK19 + HCC. The VEGFA signal can activate TAM_SPP1-derived MMP9 to promote the invasion and metastasis of CK19 + HCC tumors. This might provide novel insights into the clinical treatment of HCC patients.

Keywords: Cancer stem cell; Hepatitis B virus; Hepatocellular carcinoma; Heterogeneity; Keratin 19; Matrix metalloproteinase 9; SPP1; Single-cell transcriptome sequencing; Tumor-associated macrophage; VEGFA signal.