Identification of Cancer-Associated Fibroblast Subtypes That Promote HCC Progression and Their Differentiation Programs
Kodai MIYAZAKI *, Akira OOKA, Ryota SHIZU, Kouichi YOSHINARI
Laboratory of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka
[Purpose] Recent progress in molecular-targeted drugs and immunotherapy has helped in the treatment of various cancers, but hepatocellular carcinoma (HCC) often recurs and resists treatment. Growing evidence indicates that the tumor microenvironment (TME) is a key factor in these problems. Cancer-associated fibroblasts (CAFs), a major stromal cell type in the TME, were once viewed as a homogeneous tumor-promoting population, but recent studies have revealed heterogeneous subtypes, including tumor-suppressive ones. Hepatic stellate cells (HSCs) are the principal precursors of CAFs in the liver, so understanding how HSCs differentiate into each CAF subtype may open novel therapeutic options for HCC. Here, we aim to (i) identify tumor-promoting and tumor-suppressive CAF subtypes in HCC and (ii) clarify the molecular mechanisms that drive their differentiation from HSCs.
[Methods] Single-cell RNA-seq data from multiple HCC patients were integrated using Seurat (v5.3.0). We identified a fibroblast cluster defined by high expression of COL1A1, ACTA2, FAP, and PDGFRB and subclustered it into CAF subtypes. The signatures obtained from these subtypes were used to deconvolute stage-stratified bulk RNA-seq data with CIBERSORTx (v1.0). Any CAF subtype whose estimated proportion increased with tumor stage was classified as tumor-promoting, while any subtype whose proportion decreased was classified as tumor-suppressive. Differentiation trajectories were inferred with Monocle3 (v1.4.26) pseudotime analysis, starting from a quiescent HSC-like cluster. Any gene whose expression changed along these trajectories was subjected to Bayesian network analysis to find hub regulators.
[Results and Discussion] Sixteen distinct CAF subtypes were identified, revealing a new level of heterogeneity that includes clusters associated with metabolic and protein folding processes. Stage-stratified analysis classified seven subtypes as tumor-promoting and four as tumor-suppressive. Pseudotime analysis indicated multiple pathways from quiescent HSC-like cells to each subtype. Bayesian networks identified hub genes, such as SERPINA1, whose expression changed dramatically at branching points, suggesting that they influence the fate of CAFs. Some subtypes were enriched in metabolic or protein folding pathways, highlighting new layers of CAF diversity. Given that these CAFs are suggested to differentiate from HSCs, further functional validation using human HSC cell lines, such as LX-2, is warranted to confirm the roles of these identified genes.
[Conclusion] By integrating single-cell and bulk RNA-seq analysis, we delineated CAF differentiation pathways in HCC and identified potential hub regulators for both tumor-promoting and tumor-suppressive CAF subtypes. These candidates may serve as therapeutic targets to improve responses across cancer treatments, including immunotherapy.