Abstract PR004: In vitro and in vivo CRISPR screens in colorectal cancer organoids identify suppressors of metastasis
Authors: Cramer Zvi , Wang Xin , Leu Nicolae Adrian , Monaghan Keara , Rhoades Joshua H. , Tian Yuhua , Rico Joshua , Mendez Diego , Petroni Ricardo , Kim Melissa S. , Li Ning , Lengner Christopher J. , Blanco M. Andres 
Published: December 2022

Abstract
Colorectal cancer (CRC) is the 2nd-leading cause of cancer-related deaths worldwide and metastatic disease remains a major unmet clinical need. Nevertheless, the molecular underpinnings of metastatic spread and colonization of distant sites remain elusive. To systematically identify functional genes contributing to metastasis in a physiologically relevant model of CRC, we developed a novel platform enabling robust, pooled CRISPR/Cas9 screening in primary colonic organoids engineered with common CRC driver mutations in Apc, Kras, and Tp53. We performed parallel in vitro invasion and in vivo metastasis gain-of-function CRISPR/Cas9 knockout screens with high-content gRNA libraries to uncover novel suppressors of metastasis. We confirmed resulting hits as bona-fide metastatic suppressors using both pooled validation screens and single-gene approaches. Genes scoring in both in vitro and in vivo screens included previously identified regulators of migration and invasion, such as Ctnna1, and genes scoring only in the in vivo screen included novel negative regulators of metastasis such as Bcl2l13. These hits in the murine system were supported by human TCGA data, indicating that low expression of these genes is associated with worse 5-year survival in colon and rectal adenocarcinoma patients (COAD and READ). Utilizing transwell and live-imaging assays, we found that sgRNAs targeting Ctnna1 markedly enhanced invasion and migration of CRC organoids while sgRNAs targeting Bcl2l13 had no effect, an observation consistent with our in vitro screen. Therefore, while loss of Ctnna1 enhances metastasis by augmenting invasion and migration, loss of Bcl2l13 acts through alternative mechanisms. Single cell RNA sequencing of primary and metastatic lesions derived from Bcl2l13 knockout and control CRC tumors identified novel putative pathways promoting metastatic spread via migration-independent mechanisms, including upregulation of hypoxia signalling and recruitment of pro-tumorigenic macrophages. Thus, our CRISPR/Cas9 screening pipeline is a viable approach for the identification of novel regulators of CRC metastasis in vitro and in vivo, uncovering both motility and migration-dependent and independent paths to metastatic success. These findings will help untangle the complex web of molecular events underlying CRC metastasis and provide clinically-relevant insights into the process of human CRC metastasis.
Citation Format: Zvi Cramer, Xin Wang, Nicolae Adrian Leu, Keara Monaghan, Joshua H. Rhoades, Yuhua Tian, Joshua Rico, Diego Mendez, Ricardo Petroni, Melissa S. Kim, Ning Li, Christopher J. Lengner, M. Andres Blanco. In vitro and in vivo CRISPR screens in colorectal cancer organoids identify suppressors of metastasis [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr PR004.