Chemical, Molecular, and Single-nucleus Analysis Reveal Chondroitin Sulfate Proteoglycan Aberrancy in Fibrolamellar Carcinoma.

ABSTRACT

Fibrolamellar carcinoma (FLC) is an aggressive liver cancer with no effective therapeutic options. The extracellular environment of FLC tumors is poorly characterized and may contribute to cancer growth and/or metastasis. To bridge this knowledge gap, we assessed pathways relevant to proteoglycans, a major component of the extracellular matrix. We first analyzed gene expression data from FLC and nonmalignant liver tissue (n = 27) to identify changes in glycosaminoglycan (GAG) biosynthesis pathways and found that genes associated with production of chondroitin sulfate, but not other GAGs, are significantly increased by 8-fold. We then implemented a novel LC/MS-MS based method to quantify the abundance of different types of GAGs in patient tumors (n = 16) and found that chondroitin sulfate is significantly more abundant in FLC tumors by 6-fold. Upon further analysis of GAG-associated proteins, we found that versican (VCAN) expression is significantly upregulated at the mRNA and protein levels, the latter of which was validated by IHC. Finally, we performed single-cell assay for transposase-accessible chromatin sequencing on FLC tumors (n = 3), which revealed for the first time the different cell types in FLC tumors and also showed that VCAN is likely produced not only from FLC tumor epithelial cells but also activated stellate cells. Our results reveal a pathologic aberrancy in chondroitin (but not heparan) sulfate proteoglycans in FLC and highlight a potential role for activated stellate cells. Significance: This study leverages a multi-disciplinary approach, including state-of-the-art chemical analyses and cutting-edge single-cell genomic technologies, to identify for the first time a marked chondroitin sulfate aberrancy in FLC that could open novel therapeutic avenues in the future.