Abstract 4079: Single-cell RNA-seq revealed important pathways in response to neoadjuvant therapy resistance in rectal cancer

Abstract

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-related death in Europe and United States and is often diagnosed at advanced stage. Rectal cancer (RC) accounts for one third of all CRCs. The treatment options of RC include surgery, chemotherapy, and radiation. Locally advanced RC is treated with neoadjuvant chemoradiotherapy (nCRT) and subsequent surgery. Less than 20% of RC patients have a complete response to nCRT. Our previous work demonstrated that RC subclones respond differently to nCRT. However, the resistance mechanisms remain elusive. To explore the mechanisms underlying therapy resistance in RC, we performed single-cell RNA sequencing (scRNA-Seq) to identify and characterize the therapy-resistant RC cells. We generated patient-derived xenograft model in athymic nude mice with primary tumor cells collected from pre-treatment advanced RC patients. The compete treatment for xenograft animals includes 2 weeks of chemoradiotherapy and 1 week off. Tumor tissues were collected from mice for scRNA-Seq at a series of time points: no treatment, 2 days, 1 week, and 3 weeks after treatment. Single-cell analysis revealed five tumor cellular clusters with distinct gene expression profiles. The marker genes of cluster 1 were related to cell cycle, DNA replication, and p53 signaling pathway based on KEGG pathway enrichment analysis, which suggests that this cluster of cells are actively proliferating. Marker genes of cluster 3 were related to mineral absorption, PPAR signaling pathway, and HIF-1 signaling pathway. We compared the composition of the five cell clusters at each time point and found that while the cellular fractions of cluster 1 and cluster 3 initially dropped (2 days), they later increased (1 week and 3 weeks), suggesting that these two clusters were therapy-resistant and continued proliferating after the nCRT. Consistent with our previous DNA sequencing results, different cell populations also demonstrated tumor heterogeneity based on scRNA-seq clustering analysis of the RC xenograft model. We further compared the gene expression levels before and after treatment and identified 147 differently expressed genes (DEGs). Pathway enrichment analysis of these DEGs highlighted that signaling pathways of IL-17, TNF, MAPK, and ErbB were up-regulated posttreatment, suggesting that they may be responsible for the therapy resistance of the tumor cell clusters. These results shed light on the importance of these signaling pathways on the mechanisms of rectal cancer therapy resistance. Further work is needed to validate this discovery. Citation Format: Yu Chen, Regina K. Irwin, Gregory W. Williams, Mary Smithson, Karin M. Hardiman, Zechen Chong. Single-cell RNA-seq revealed important pathways in response to neoadjuvant therapy resistance in rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4079.