High‐throughput CRISPR‐mediated 3D enrichment platform for functional interrogation of chemotherapeutic resistance

Abstract

Cancer is a disease of somatic mutations. These cellular mutations compete to dominate their microenvironment and dictate the disease outcome. While a therapeutic approach to target-specific oncogenic driver mutations helps to manage the disease, subsequent molecular evolution of tumor cells threatens to overtake therapeutic progress. There is a need for rapid, high-throughput, unbiased in vitro discovery screening platforms that capture the native complexities of the tumor and rapidly identify mutations that confer chemotherapeutic drug resistance. Taking the example of the CDK4/6 inhibitor (CDK4/6i) class of drugs, we show that the pooled in vitro CRISPR screening platform enables rapid discovery of drug resistance mutations in a three-dimensional (3D) setting. Gene-edited cancer cell clones assembled into an organotypic multicellular tumor spheroid (MCTS), exposed to CDK4/6i caused selection and enrichment of the most drug-resistant phenotypes, detectable by next-gen sequencing after a span of 28 days. The platform was sufficiently sensitive to enrich for even a single drug-resistant cell within a large, drugresponsive complex 3D tumor spheroid. The genome-wide 3D CRISPR-mediated knockout screen (>18,000 genes) identified several genes whose disruptions conferred resistance to CDK4/6i. Furthermore, multiple novel candidate genes were identified as top hits only in the microphysiological 3D enrichment assay platform and not the conventional 2D assays. Taken together, these findings suggest that including phenotypic 3D resistance profiling in decision trees could improve discovery and reconfirmation of drug resistance mechanisms and afford a platform for exploring noncell autonomous interactions, selection pressures, and clonal competition.

A CRISPR Path to Finding Vulnerabilities and Solving Drug Resistance: Targeting the Diverse Cancer Landscape and Its Ecosystem

“…Slc39a6 was further validated to play a role in resistance to both CDK4/6 inhibition and fulvestrant, demonstrating the utility of a more physiologically relevant culture system in identifying novel resistance mechanisms. [ 132 ] Although not all of these highlighted genetic vulnerabilities are directly therapeutically targetable yet, CRISPR screens can effectively contribute to our understanding of the epigenetic drivers of tumor immunosuppression, broad therapeutic resistance and importantly, aid identification of novel predictive biomarkers of treatment response as well as new therapeutic strategies in the near future.…”

Progress of 3D Organoid Technology for Preclinical Investigations: Towards Human In Vitro Models

“…The ultra-low attachment (ULA) plate is a spheroid plate with a chemical coating surface minimizing protein and cell attachment to wells. It is an upgraded multiple-well version (96 or 384 wells) of the liquid overlay method in producing a single spheroid/organoid and is commercially available [3,45]. Rather than ready-to-use commercial plates, many studies developed their own ULA 96 well plates coated with various hydrophilic polymer surfaces such as agar or agarose [46,47] and hydrolysed poly(vinyl alcohol) [48].…”

Research Progress with Luteolin as an Anti-Tumor Agent

“…Although chemoradiotherapy is the main strategy of tumor treatment, chemotherapeutic drugs have extremely serious cytotoxic effects and produce drug resistance. 2 Research on traditional Chinese medicine confirmed that many herbal preparations have satisfactory clinical and therapeutic effects on tumors. Therefore, in order to better develop and design antitumor drugs, more and more research focuses on natural drugs.…”