Abstract 892: Single-cell RNA sequencing of patient-derived organoid reveals treatment-induced tumor resistance through cancer stem cells
Authors: Fujino Shiki , Ito Aya , Yasui Masayoshi , Matsuda Chu , Ohue Masayuki , Horie Masafumi , Yachida Shinichi , Doki Yuichiro , Eguchi Hidetoshi , Miyoshi Norikatsu 
Published: June 2022

Abstract
It is thought that a few treatment-resistant cells form heterogenous recurrences and metastases, but what cells make complex tumor lineage is unexplored in colorectal cancer (CRC). To elucidate the cells that escape from treatment and form recurrences and metastases, the gene expression landscape, and drug susceptibility were explored using patient-derived organoids (PDOs). Drug sensitivities of 16 PDOs including eight PDOs derived from patients with distant metastases were evaluated. t-SNE analysis clearly showed four clusters, including two clusters based on response to therapy (good and poor). RNA sequence analysis showed that poor-response PDOs were enriched in gene sets related to distant metastasis, recurrence, and treatment resistance compared with good-response PDOs. Expression of nine genes (CBS, E2F8, GCNT1, NETO2, PLPP5, POU5F1, XRCC2, RECQL4, and ZNF681) were characteristic in the poor-response PDOs. Among them, we focused on POU5F1 gene that characterized liver metastasis in CRC from our previous report. Further analysis revealed that POU5F1-positive cells survive after anticancer drug treatment, and they were produced from POU5F1-expressing cells, not POU5F1-negative cells. Notably, isolated single POU5F1-expressing cell produced a heterogeneous population of cells expressing various differentiation markers both in vitro and in vivo. Single POU5F1-expressing cell also reconstructed ductal structure with mucus-producing ability. Cells produced from POU5F1-expressing cells were thought to construct tumor diversity remaining various differentiation stage. Single-cell RNA sequencing was performed to reveal the heterogeneous population with treatment resistance. The seven characteristic clusters were identified and the number of cells in clusters 2, 4, and 6 increased at the time of tumor re-growth after anticancer drug treatment. Cluster 2 was characterized by cell-cycle score. When cluster 4 was set as the root of the proliferative trajectory, the trajectory of cluster 4→3→6 emerged with characteristic gene expression profiles. The Wnt signaling pathway was enriched in cluster 4, suggesting stem cell characteristics. Moreover, Glycolysis, HIF-1 signaling, and ferroptosis pathways, which are also reported as properties of persister cells were enriched in cluster 6. Herein, we report the features derived from single-cell RNA seq analysis and further examinations in vitro, in vivo, and in clinical specimens.
Citation Format: Shiki Fujino, Aya Ito, Masayoshi Yasui, Chu Matsuda, Masayuki Ohue, Masafumi Horie, Shinichi Yachida, Yuichiro Doki, Hidetoshi Eguchi, Norikatsu Miyoshi. Single-cell RNA sequencing of patient-derived organoid reveals treatment-induced tumor resistance through cancer stem cells [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 892.