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Updated: Jun 26, 2026
Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms
Published on: December 9, 2015
Indirect CRISPR screening with photoconversion revealed key factors of drug resistance with cell-cell interactions
Keisuke Sugita1, Iichiroh Onishi1, Ran Nakayama1
1Department of Comprehensive Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan.
Abstract:
Comprehensive screenings to clarify indirect cell-cell interactions, such as those in the tumor microenvironment, especially comprehensive assessments of supporting cells' effects, are challenging. Therefore, in this study, indirect CRISPR screening for drug resistance with cell-cell interactions was invented. The photoconvertible fluorescent protein Dendra2 was inducted to supporting cells and explored the drug resistance responsible factors of supporting cells with CRISPR screenings. Random mutated supporting cells co-cultured with leukemic cells induced drug resistance with cell-cell interactions. Supporting cells responsible for drug resistance were isolated with green-to-red photoconversion, and 39 candidate genes were identified. Knocking out C9orf89, MAGI2, MLPH, or RHBDD2 in supporting cells reduced the ratio of apoptosis of cancer cells. In addition, the low expression of RHBDD2 in supporting cells, specifically fibroblasts, of clinical pancreatic cancer showed a shortened prognosis, and a negative correlation with CXCL12 was observed. Indirect CRISPR screening was established to isolate the responsible elements of cell-cell interactions. This screening method could reveal unknown mechanisms in all kinds of cell-cell interactions by revealing live phenotype-inducible cells, and it could be a platform for discovering new targets of drugs for conventional chemotherapies.
Insights
Researchers developed indirect CRISPR screening to identify supporting cells influencing cancer drug resistance. This method revealed key genes, offering new therapeutic targets for improving cancer treatment outcomes.
Area of Science:
- Cell Biology
- Genomics
- Cancer Research
Background:
- Assessing indirect cell-cell interactions in the tumor microenvironment is complex.
- Understanding supporting cells' roles in drug resistance requires advanced screening methods.
Purpose of the Study:
- To invent and validate an indirect CRISPR screening method for identifying genes in supporting cells that mediate drug resistance through cell-cell interactions.
- To uncover novel mechanisms of drug resistance and potential therapeutic targets.
Main Methods:
- Utilized indirect CRISPR screening with a photoconvertible fluorescent protein (Dendra2) in supporting cells.
- Co-cultured randomly mutated supporting cells with leukemic cells to induce drug resistance.
- Isolated drug-resistance-mediating supporting cells via green-to-red photoconversion and identified candidate genes.
Main Results:
- Identified 39 candidate genes involved in supporting cell-mediated drug resistance.
- Knockout of C9orf89, MAGI2, MLPH, or RHBDD2 in supporting cells decreased cancer cell apoptosis.
- Low RHBDD2 expression in clinical pancreatic cancer fibroblasts correlated with poor prognosis and negatively with CXCL12.
Conclusions:
- Established an indirect CRISPR screening platform for dissecting cell-cell interaction mechanisms.
- This method can reveal unknown biological processes and identify new drug targets for conventional chemotherapy.
