ScRNA-seq to examine heterogeneity of transcriptional regulation of CAFs between left-sided and right-sided CRC.

Abstract

e15548 Background: Cancer-associated fibroblasts (CAFs) play prominent and diverse tumor-promoting function roles in tumor microenvironment (TME) and influence cancer hallmarks, but without systematic investigation between left-sided and right-sided CRC (colorectal cancer). Methods: We collected a public scRNA-seq dataset (Pelka et al., 2021) with 21 samples from left-sided CRC and 79 from right-sided CRC. Only CAFs were extracted for downstream analysis. Seurat (Stuart et al., 2019), Monocle2 (den Berge et al., 2020) and pySCENIC (Aibar et al., 2017) were used to perform dimensionality reductions, differential analysis, pseudo-time trajectory analysis, and the transcriptional regulation analysis, respectively. Results: Firstly, we performed differential analysis on 2,049 cells from 62 of 100 samples that contained CAFs. The results showed that highly expressed genes including IGFBP1, IGFBP2, LCN2, ALDH1A1 and CASP1 in left-sided CRC were mainly enriched in immune response pathway, while up-regulated genes in right-sided CRC were neutrophil-migration-chemotaxis-related such as PPBP1, PF4V1, CCL7, CXCL3 and CXCL8. Then, we classified 2,049 CAFs into four subclusters based on the markers of CCL8+ CAF, CXCL14+ CAF, GREM1+ CAF and MMP3+ CAF. The proportion of CXCL14+ CAF was higher in left-sided CRC while MMP3+ CAF was preferentially enriched in right-sided CRC, which was also independently validated by 33 TCGA cohorts. Further investigation on those subclusters showed that CXCL14+ CAF was myCAF, while the rest (CCL8+ CAF, MMP3+ CAF and GREM1+ CAF) were iCAF. After investigating the heterogeneity of CAFs, we focused on the transcriptional regulatory networks (TRNs). The results showed that the dominant regulons in left-sided CRC ( HOXD9, HSF2, MYBL1, HES6, XRCC4, and ZNF585B) and right-sided CRC ( CREB5, ZNF274, IRF7, TFEC, NR2F1, and PITX3) were distinct, which highlighted the heterogeneity and plasticity of CAF-based TRNs between left-sided and right-sided CRC. Conclusions: Collectively, although further experimental verification is required to establish the functional roles of each CAF cluster and the diverse origins of CAFs, this study may facilitate better understanding molecular mechanism of CAF-targeted therapy strategies in CRC.