Chemical biology fluorescent tools for in vitro investigation of the multidrug resistant P-glycoprotein (P-gp) expression in tumor cells.

Selective P-glycoprotein (P-gp)-targeted fluorescent conjugates are desirable tools to investigate the role of P-gp, a protein strongly implicated in mediating multidrug resistance and a major cause of chemotherapy failure. Herein, we report the development of 25 novel fluorescent small-molecule conjugates with varying physicochemical and optical properties, and their biological evaluation in a cell model as P-gp targeted constructs. This investigation revealed relationships between molecular structure and cell behavior and uncovered the capacity of conjugates with varying fluorophores to selectively target P-gp. Sulfocyanine 3 labeled conjugates (5, 10, 24, 29, 34) showed a particular intracellular staining pattern. Other conjugates bearing a boron dipyrromethene (BODIPY) core (3, 8, 13, 22, 27 (BODIPY FL), 12 (BODIPY 564/570) and 4, 9 (BODIPY 650/665)) or a 7-nitrobenz-2-oxa-1,3-diazole (NBD) core (11, 30) showed potential for global P-gp direct detection and quantification. These fluorescent conjugates holds key advantages over existing methods for drug resistance evaluation with regards to P-gp expression and could be used as innovative tools in preclinical assays and clinical diagnosis.

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.

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 R2 = 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.

The New Serum-Free OptiPASS® Medium in Cold and Oxygen-Free Conditions: An Innovative Conservation Method for the Preservation of MDA-MB-231 Triple Negative Breast Cancer Spheroids.

Cancer spheroids are very effective preclinical models to improve anticancer drug screening. In order to optimize and extend the use of spheroid models, these works were focused on the development of a new storage concept to maintain these models in the longer term using the Triple-Negative Breast Cancer MDA-MB-231 spheroid models. The results highlight that the combination of a temperature of 4 °C and oxygen-free conditions allowed the spheroid characteristics of OptiPASS® serum-free culture medium to preserve the spheroid characteristics during 3-, 5- or 7-day-long storage. Indeed, after storage they were returned to normal culture conditions, with recovered spheroids presenting similar growth rates (recovery = 96.2%), viability (Live/Dead® profiles) and metabolic activities (recovery = 90.4%) compared to nonstored control spheroids. Likewise, both recovered spheroids (after storage) and nonstored controls presented the same response profiles as two conventional drugs, i.e., epirubicin and cisplatin, and two anti-PARP1 targeted drugs-i.e., olaparib and veliparib. This new original storage concept seems to induce a temporary stop in spheroid growth while maintaining their principal characteristics for further use. In this way, this innovative and simple storage concept may instigate future biological sample preservation strategies.

The New Synthetic Serum-Free Medium OptiPASS Promotes High Proliferation and Drug Efficacy Prediction on Spheroids from MDA-MB-231 and SUM1315 Triple-Negative Breast Cancer Cell Lines.

Triple-negative breast cancers are particularly aggressive. In vitro cultures are one of the major pathways for developing anticancer strategies. The effectiveness and reproducibility of the drug screenings depend largely on the homogeneity of culture media. In order to optimize the predictive responses of triple-negative breast cancer 3D cell culture models, these works were focused on the development of SUM1315 and MDA-MB-231 cell lines in OptiPASS medium, a new serum-free formulation (BIOPASS). In monolayer cell culture, OptiPASS medium was more suitable for MDA-MB-231 than SUM1315 cell line but maintained cell phenotype and allowed sufficient proliferation. For spheroids produced in OptiPASS, the size monitoring showed a 1.3 and 1.5-fold increase for MDA-MB-231 and SUM1315 cell lines, respectively and viability/mortality profiles were maintained. Spheroids drug sensitivity thresholds were also improved allowing quicker high throughput drug screenings. These results showed the suitability of OptiPASS for 2D and 3D cell cultures of these two triple-negative breast cancer cell lines, with reproducibility of spheroid formation superior to 98%. This opens the way to the common use of this synthetic medium in future preclinical breast cancer research studies.

Low-Dose and Long-Term Olaparib Treatment Sensitizes MDA-MB-231 and SUM1315 Triple-Negative Breast Cancers Spheroids to Fractioned Radiotherapy.

The Triple-Negative Breast Cancer subtype (TNBC) is particularly aggressive and heterogeneous. Thus, Poly-ADP-Ribose Polymerase inhibitors were developed to improve the prognosis of patients and treatment protocols are still being evaluated. In this context, we modelized the efficacy of Olaparib (i.e., 5 and 50 µM), combined with fractioned irradiation (i.e., 5 × 2 Gy) on two aggressive TNBC cell lines MDA-MB-231 (BRCAness) and SUM1315 (BRCA1-mutated). In 2D cell culture and for both models, the clonogenicity drop was 95-fold higher after 5 µM Olaparib and 10 Gy irradiation than Olaparib treatment alone and was only 2-fold higher after 50 µM and 10 Gy. Similar responses were obtained on TNBC tumor-like spheroid models after 10 days of co-treatment. Indeed, the ratio of metabolic activity decrease was of 1.2 for SUM1315 and 3.3 for MDA-MB-231 after 5 µM and 10 Gy and of only 0.9 (both models) after 50 µM and 10 Gy. MDA-MB-231, exhibiting a strong proliferation profile and an overexpression of AURKA, was more sensitive to the co-treatment than SUM1315 cell line, with a stem-cell like phenotype. These results suggest that, with the studied models, the potentiation of Olaparib treatment could be reached with low-dose and long-term exposure combined with fractioned irradiation.

Development and validation of a high-performance liquid chromatography method for the quantitation of intracellular PARP inhibitor Olaparib in cancer cells.

Olaparib is a potent PARP inhibitor in clinical use for cancer therapy. A bioanalytical assay was developed and validated for quantitation of intracellular level of olaparib in cells exposed to the drug. The assay involves an optimized and straightforward sample pretreatment with acetonitrile for olaparib solubilization, cell lysis and protein precipitation, and a high performance liquid chromatography (HPLC) method with ultraviolet detection. Several parameters in both the sample preparation and the detection steps were investigated. Optimal chromatographic conditions were achieved with a 5 µL injection on a Nova-Pak® C18 column (150 × 3.9 mm, 4 µm) using a mobile phase consisting of acetonitrile and ultra-pure water in gradient mode, at a constant 1.2 mL/min flow rate, at 35 °C. Detection was carried out at 254 nm and a diode array detector was used to insure purity of the olaparib peak. The method was validated according to Food and Drug Administration guidelines. Linearity, accuracy and precisions were satisfactory over the concentration range of 200-2000 ng/mL. Limits of detection and quantification for olaparib were 50 ng/mL and 200 ng/mL, respectively. Good stability was showed in three relevant analytical conditions. Finally, the validated analytical method was successfully used to estimate the intracellular level of olaparib in SUM1315 breast cancer cells. A significant difference was observed in intracellular drug level after 1 and 3 h incubations. This method permitting measurement of drug level in tumor cells would allow dosage optimization and improvement of treatment response predictions.

Development and cytotoxic response of two proliferative MDA-MB-231 and non-proliferative SUM1315 three-dimensional cell culture models of triple-negative basal-like breast cancer cell lines.

Triple-Negative Basal-Like tumors, representing 15 to 20% of breast cancers, are very aggressive and with poor prognosis. Targeted therapies have been developed extensively in preclinical and clinical studies to open the way for new treatment strategies. The present study has focused on developing 3D cell cultures from SUM1315 and MDA-MB-231, two triple-negative basal-like (TNBL) breast cancer cell lines, using the liquid overlay technique. Extracellular matrix concentration, cell density, proliferation, cell viability, topology and ultrastructure parameters were determined. The results showed that for both cell lines, the best conditioning regimen for compact and homogeneous spheroid formation was to use 1000 cells per well and 2% Geltrex®. This conditioning regimen highlighted two 3D cell models: non-proliferative SUM1315 spheroids and proliferative MDA-MB-231 spheroids. In both cell lines, the comparison of 2D vs 3D cell culture viability in the presence of increasing concentrations of chemotherapeutic agents i.e. cisplatin, docetaxel and epirubicin, showed that spheroids were clearly less sensitive than monolayer cell cultures. Moreover, a proliferative or non-proliferative 3D cell line property would enable determination of cytotoxic and/or cytostatic drug activity. 3D cell culture could be an excellent tool in addition to the arsenal of techniques currently used in preclinical studies.