Abstract 2931: Utilizing an extracellular matrix-embedded 3D tumor model of NSCLC to identify novel cancer dependencies via genome-scale CRISPR screen

Abstract

Abstract INTRODUCTION: Non-small cell lung cancer (NSCLC) is a prevalent and deadly cancer type. Despite advances in targeted therapies in NSCLC, one-third of adenocarcinomas lack actionable oncogenes, limiting targeted therapy options. To address these challenges, CRISPR screening has emerged as a tool to unveil cancer dependencies. However, current genome-scale studies rely heavily on 2D monolayer cancer models that fail to replicate in vivo tumor biology due to altered gene/protein expressions, absence of cell-extracellular matrix (ECM) signaling, and rewired metabolic pathways. To overcome these limitations, we developed a high-throughput 3D extracellular matrix (ECM) based hydrogel culture platform suitable for conducting genome-scale CRISPR screening. Leveraging this platform, we performed genome-wide CRISPR loss of function screens in 3D tumor models in ECM (3D-ECM). These results were compared with the screening outcomes from 3D cultured cells lacking an extracellular matrix (3D-spheroids) and traditional 2D monolayer (2D-monolayer) cells to identify new genes that might be crucial in driving NSCLC tumor cells. METHODS: Three NSCLC cell lines (NCI-H358, PC-9, and EKVX) were utilized for CRISPR screens. A genome-wide CRISPR library containing 60,000 sgRNAs targeting 20,000 genes was prepared. To create genome-scale 3D tumor models in ECM, cells were encapsulated in sub-millimeter microcapsules made of alginate and an extracellular matrix protein-hydrogel mix. Encapsulated cells were cultured for up to 4 weeks. 3D-spheroids were cultured on ultra-low attachment plates in viscous growth media and split every 3-4 days over 4 weeks. RESULTS: Cancer cells' proliferation and metabolic activity notably increased in 3D-ECM compared to 3D-spheroids. The CRISPR screens in 3D-spheroids and 3D-ECM revealed numerous positive growth phenotypes in contrast to the conventional 2D monolayer culture model. Specifically, we found 79 additional genes exhibiting positive-growth phenotype in 3D-ECM. Moreover, prominent oncogenic drivers, such as KRAS and EGFR were ranked considerably higher in both 3D-ECM and 3D-Spheroids compared to the 2D-monolayer. Interestingly, the pathway-enrichment analysis revealed that Notch signaling (CREBBP, EP300, APH1A), FoxO signaling (STK11, TGFBR2, MAPK14), and Citrate cycle signaling (MDH2, SDHC, PDHA1) were enriched in 3D-ECM when compared to 3D-spheroids. CONCLUSION: Our results reveal significant distinctions between the 2D monolayer model, the 3D spheroid model lacking extracellular matrix, and the 3D tumor model incorporating ECM. These differences underscore the importance of conducting CRISPR screens with 3D tumor models within the extracellular matrix to unveil novel cancer dependencies. Citation Format: Pranay Agarwal, Soon Youn Choi, Xin Liu, Jina Song, Maya Tureez, Un Jae Baek, Aashka Mehta, Kyuho Han, Hong-Pyo Lee. Utilizing an extracellular matrix-embedded 3D tumor model of NSCLC to identify novel cancer dependencies via genome-scale CRISPR screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2931.