LightSpot(®)-FL-1 Fluorescent Probe: An Innovative Tool for Cancer Drug Resistance Analysis by Direct Detection and Quantification of the P-glycoprotein (P-gp) on Monolayer Culture and Spheroid Triple Negative Breast Cancer Models

SIMPLE SUMMARY: Tumoral drug resistance is mainly caused by multidrug resistance transporters (MDR), such as the P-gp, which presents high clinical interest. For this reason, the P-gp-mediated drug resistance diagnosis may be very relevant for optimizing anticancer treatment efficacy. However, the lack of effective analytical tools limits this clinical diagnostic approach. Therefore, our group has developed LightSpot(®)-FL-1, a new cell-permeant fluorescent probe able to specifically localize and quantify the P-gp inside unicellular, monolayer, and cellular mass models. The application of this innovative tool was firstly demonstrated in the preclinical field, using five triple-negative breast cancer (TNBC) cell models. The comparison between classical anti-P-gp immunostaining and LightSpot(®)-FL-1 P-gp staining highlighted a strong similarity with P-gp localization and expression level quantification. LightSpot(®)-FL-1 P-gp detection and quantification, using several fluorescence imaging methods, are easy, direct, and cost-effective and are, therefore, very promising for future clinical diagnosis development. ABSTRACT: P-gp is the most widely studied MDR protein conferring cellular resistance to many standard or targeted therapeutic agents. For this reason, P-gp chemoresistance evaluation, established before or during chemotherapy, can be very relevant in order to optimize the efficacy of treatments, particularly for aggressive tumoral subtypes such as triple-negative breast cancer (TNBC). In this context, our team developed an innovative cell-permeant fluorescent probe called the LightSpot(®)-FL-1, which is able to specifically localize and quantify the P-gp in cells or cell masses, as evidenced on different TNBC cell models. First, flow cytometry analysis showed LightSpot(®)-FL-1 cell penetration and persistence in time, in TNBC cells. Then, LightSpot(®)-FL-1 staining was compared to anti-P-gp immunostaining by fluorescence microscopy on five TNBC cell lines. Results showed a clear similarity of P-gp localization and expression level, confirmed by Pearson’s and Mander’s colocalization coefficients with 92.1% and 100.0%, and a strong correlation coefficient of R(2) = 0.99. In addition, the LightSpot(®)-FL-1 staining allowed the quantification of a P-gp induction (33% expression increase) following a 6-hour spheroid model exposure to the anti-PARP Olaparib. Thus, the new LightSpot(®)-FL-1 cell-permeant probe, targeting P-gp, appears to be an effective tool for drug resistance evaluation in preclinical models and shows promising possibilities for future use in clinical diagnosis.