Genome-Wide CRISPR/Cas9 Screens in a 3D Tumor Spheroid Model Identify Tumor Suppressor Genes in Breast Cancer

Abstract

Compared with other breast cancer subtypes, triple negative breast cancer is characterized by poorer clinical outcomes, higher metastatic potential, and a lack of available targeted therapies. While previous studies have yielded valuable insights into the molecular basis of triple negative breast cancer, they have largely relied on in vitro 2D monolayer cancer cell cultures, which are unable to recapitulate many key aspects of in vivo tumors, including hypoxia and altered cell-cell interactions. Considering these issues, the present study aims to leverage in vitro 3D tumor spheroid models in combination with high-throughput CRISPR/Cas9 genetic screening in order to identify novel genes and pathways regulating triple negative breast cancer.

Utilizing a pooled CRISPR knockout library, we perform genome-wide screens in human triple negative breast cancer cells cultured under both 2D and 3D conditions. Strikingly, by using deep sequencing to investigate sgRNA dynamics, we identify a large number of positively selected genes with significantly enriched sgRNA representation in the 3D screens, which is not observed in the 2D screens. Computational and functional enrichment analyses not only support the characterization of many of these positively selected genes as tumor suppressors, but also suggest that the 3D screens more accurately capture relevant aspects of cancer genes and pathways compared to the 2D screens. Finally, we take initial steps towards the validation of selected candidate tumor suppressors.

Taken together, the findings from this study not only open opportunities for further molecular characterization of and development of potential targeted therapies for triple negative breast cancer, but also highlight the value and feasibility of using 3D tumor spheroid models for functional genomic investigations.