PII-042 - DEVELOPMENT OF A NOVEL FLUORESCENCE-BASED APPROACH FOR SOLUTE CARRIER-FOCUSED CRISPR/CAS9 KNOCKOUT SCREENING IN BREAST CANCER CELLS

Y. Xu¹, D. Rane², D. Palmieri², M. Nepal³, A. Sparreboom², B. Peterson², S. Hu²; ¹The Ohio State Univerity, OH, USA, ²The Ohio State University, ³The Ohio State University, OH, United States.

Background: Paclitaxel is among the most widely used anticancer drugs and is known to cause a dose limiting peripheral neurotoxicity via a solute carrier (SLC) transporter, OATP1B2, in the dorsal root ganglion. However, the roles of SLCs in paclitaxel uptake by breast cancer cells remain largely unknown. The traditional endpoint of CRISPR/Cas9 library screening evaluated by cytotoxicity may not directly reveal the process of transmembrane influx. The main purpose of this study is to develop an alternative fluorescence-based approach for SLC-focused screening and identify solute carrier(s) responsible for paclitaxel uptake in cancer cells.

Methods: Uptake studies of a novel fluorescent probe 6FC-Taxol (0.1 µM), a derivative of paclitaxel linked to a drug-like fluorophore related to Pacific Blue, were optimized with unlabeled paclitaxel by confocal microscopy and flow cytometry in various breast cancer cell lines. Cell lines with the greatest decrease in fluorescence were selected for cellular competition binding and cytotoxicity MTT assay before optimized further for CRISPR/Cas9 library edition and high-throughput cell sorting.

Results: Confocal microscopy assay revealed MDA-MB-231 and MDA-MB-468 cells with greatest reduction of fluorescence intensity of 6FC-Taxol upon co-incubation with unlabeled paclitaxel. 6FC-Taxol showed similar cytotoxicity (IC50=77-171 nM) compared to paclitaxel towards these cell lines. Cellular competition binding assays and uptake assays demonstrated comparable inhibitory potency between 6FC-Taxol and paclitaxel with similar lower nanomolar IC50. Finally, conditions for cell sorting using 6FC-Taxol were optimized to obtain maximal separation of fluorescent cell clusters which can be used to distinguish uptake of 6FC-Taxol with and without inhibitors in both cell lines, with over a 4-fold difference in intracellular 6FC-Taxol fluorescence.

Conclusion: The novel fluorescent probe 6FC-Taxol was validated as a sensitive probe for CRISPR/Cas9 knockout screening in breast cancer cell lines MDA-MB-231 and MDA-MB-468 with optimal inhibition profiles. Collectively, we developed a novel fluorescence-based approach with the potential to fill a knowledge gap identifying uptake transporter for the important anti-cancer drug paclitaxel in cancer cells.