Abstract A024: Multiple resistance mechanisms identified via Single-cell RNAseq in patient derived xenograft model of rectal cancer during treatment
Authors: Chen Yu , Irwin Regina K. , Williams Gregory W. , Smithson Mary , Chong Zechen , Hardiman Karin M. 
Published: May 2022

Rectal cancer (RC) accounts for one third of all colorectal cancer. Locally advanced RC is treated with neoadjuvant chemoradiotherapy (nCRT) and subsequent surgery. Most patients are at least partially resistant to nCRT and resistance is correlated with both local recurrence and distant metastasis. Our previous work assessed the role of genetic subclones in response to nCRT via sequencing of pre-and post-treatment primary human rectal cancers and found that RC genetic sub-clones exist in individual patients and are differentially responsive to treatment. Here, we utilized single-cell RNA sequencing (scRNA-Seq) to study cellular clusters over time following chemoradiotherapy in a patient derived xenograft (PDX) model of RC. We generated a patient-derived xenograft model in athymic nude mice from a primary tumor collected from a pre-treatment RC patient. Multiple mice were implanted with tumor and treated for a total of 2 weeks daily with oral capecitabine and 2 Gy radiation. Tumor was collected from untreated animals, animals that had been treated for 2 days and 1 week and also from animals that had been treated for 2 weeks and then given a 1 week break to allow for tumor re-growth (3 weeks from the start of treatment). Single-cell analysis revealed five tumor cellular clusters with distinct gene expression profiles. The cellular fraction of clusters 1 and 3 both decrease at 2 days after initiation of treatment but then increase again at subsequent time points. The cellular fraction for cluster 2 remains high until 1 week of treatment when it falls but by 3 weeks, it has rebounded again. Cluster 4 initially represents a very low fraction but increases with treatment and then subsequently decreases again. Cluster 5 remains at a low cellular fraction throughout treatment and is unchanged. Several of the marker genes for cluster 1 are those with prior evidence that they confer radiation resistance due to their role in DNA repair such as FANCD2, FANCI, and H2Ax and pathway analysis identified enrichment of cell cycle, DNA replication, and DNA repair. Despite a pattern similar to cluster 1 with initial decrease after treatment and then recovery, Cluster 3 marker genes were related to stress response including response to metal ions and hypoxia. Pathway analysis of Cluster 2, which was responsive to treatment at 1 week but then rebounded after treatment was stopped, revealed an increase in epithelial proliferation and Wnt signaling which are both common characteristics in colorectal cancer. These results reveal that individual rectal cancers grown as PDX models are heterogeneous and that their resistant mechanisms are complex with multiple different mechanisms being identified in the same tumor. How to approached multiple mechanisms at once with targeted therapy is a critical area for future study.

Citation Format: Yu Chen, Regina K. Irwin, Gregory W. Williams, Mary Smithson, Zechen Chong, Karin M. Hardiman. Multiple resistance mechanisms identified via Single-cell RNAseq in patient derived xenograft model of rectal cancer during treatment [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A024.