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A high throughput proliferation and cytotoxicity assay for co-cultured isogenic cell lines
Syed Ahmad1, Kris C Wood2, John E Scott1
1Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise (BRITE), North Carolina Central University, Durham, NC, United States.
Abstract:
PTEN is a well-known tumor suppressor that is inactivated or suppressed at a high frequency in cancer. We sought an assay to screen compounds for ones that differentially inhibited proliferation or induced cytotoxicity in PTEN mutated cancer cells. We employed the isogenic pair of cell lines MCF10-A breast cell line (wild type, WT) and the same cell line with PTEN knocked out (KO) by CRISPR. We sought an assay where these PTEN WT and KO isogenic cell lines were co-cultured in the same well for compound testing. The KO cell line, but not the WT, was tagged with the red fluorescent protein mKate2. We employed a real time microscopic imaging instrument to identify cell populations in co-culture based on red fluorescence to obtain a cell count for each cell line. To acquire cytotoxicity data for each population, the dye CellTox Green was added to the media. To assess the assay, we determined the concentration response of paclitaxel. In order to assess the potential for screening, we performed mock screening in 384-well plate format. Thus, we developed a high throughput co-culture cell cytotoxicity and proliferation assay method that could be employed for any pair of cell lines to identify selective compounds.
Insights
This study developed a novel co-culture assay to screen for compounds targeting cancer cells with PTEN mutations. The assay uses fluorescent markers and real-time imaging to differentiate and quantify cell populations, enabling selective compound identification.
Area of Science:
- Oncology
- Molecular Biology
- Biotechnology
Background:
- Phosphatase and tensin homolog (PTEN) is a critical tumor suppressor frequently inactivated in various cancers.
- Developing targeted therapies requires assays that can specifically identify compounds effective against PTEN-deficient cancer cells.
Purpose of the Study:
- To create a high-throughput co-culture assay for screening compounds that selectively inhibit proliferation or induce cytotoxicity in PTEN-mutated cancer cells.
- To establish a method applicable to any isogenic cell line pair for identifying selective anti-cancer agents.
Main Methods:
- Utilized MCF10-A breast cell lines, one wild-type (WT) and one PTEN knockout (KO) via CRISPR.
- Co-cultured WT and mKate2-tagged KO cells, enabling real-time microscopic identification and cell counting based on red fluorescence.
- Incorporated CellTox Green dye to measure cytotoxicity in each distinct cell population.
Main Results:
- Successfully developed and validated a co-culture assay capable of differentiating and quantifying WT and PTEN-KO cells.
- Demonstrated the assay's ability to assess compound effects on proliferation and cytotoxicity in a mixed-cell environment.
- Confirmed assay performance through paclitaxel concentration-response analysis and mock screening in a 384-well format.
Conclusions:
- The developed high-throughput assay enables the identification of compounds with selective activity against PTEN-deficient cancer cells.
- This versatile co-culture method can be adapted for screening against various genetic alterations in cancer research.
- The assay facilitates the discovery of novel targeted cancer therapies.
