OVCAR8 was selected as a HGSC model, particularly since it is described as a Carboplatin-resistant OC cell line retrieved from a HGSC patient after a high-dose Carboplatin treatment (37). Parental OVCAR8 cell line was kindly provided by Doctor Francis Jacob, Gynecological Cancer Center and Ovarian Cancer Research, Department of Biomedicine, University Hospital Basel and University of Basel, Basel, Switzerland. Cells were grown in complete media, specifically, RPMI-1640 medium (ThermoFisher Scientific, Massachusetts, USA), supplemented with 10% (v/v) inactivated and filtered fetal bovine serum (FBS; Biowest, Nuaillé, France) and 1% (v/v) penicillin/streptomycin (ThermoFisher Scientific) and maintained at 37°C and 5% CO₂. OVCAR8 was authenticated using short tandem repeat profiling and regularly tested for the absence of mycoplasma.

PTX was purchased from Selleckchem (Houston, Texas, USA), dissolved in dimethyl sulfoxide (DMSO; AppliChem, Barcelona, Spain) and stored at −80°C, according to the manufacturer’s instructions. Immediately prior to use, an aliquot was diluted at required concentrations.

Two OVCAR8 PTX R variants were established in our laboratory from parental OVCAR8 by continuous (C) and pulse (P) exposure to a stepwise increasing PTX concentration (2 to 74.9 nM), for 3 months (details are shown in Figure 1A). The starting concentration used for PTX R induction corresponded to the IC10 value obtained from dose–response curves after exposing parental OVCAR8 to PTX (1.56 to 200 nM) for 48 h (Supplementary Figure 1A).

Initially, each OVCAR8 PTX R variant was maintained uninterruptedly in the presence of PTX for a continuous-selection strategy. After 1 month, two different approaches were adopted: (i) cells were exposed to PTX constantly (continuous-selection strategy) and (ii) cells were exposed to PTX, for 4 days followed by recovery in drug-free media for 4 days (pulse-selection strategy) for 2 months. Next, we tested if PTX R phenotype was stable in culture without drug. Hence, after 2 months of freezing cycles, cells were cultured in the absence of PTX medium supplementation (Figure 1B). Preston Blue (PB) assay was used to evaluate cell viability to PTX at different time points in order to confirm the maintenance of IC50 values after freezing cycles and persistent cultures.

To determine cell viability to PTX, a resazurin-based assay—Presto Blue (PB)—was performed, according to the manufacturer’s instructions. Briefly, 2 × 10³ cells/well were seeded into a 96-well plate in complete media and incubated at 37°C and 5% CO₂. After 48 h, cells were treated with increasing concentrations of PTX (1.56 to 200 nM) and incubated under the same conditions. After 48 h, 50 μl of PrestoBlue™ Cell Viability Reagent 1× (ThermoFisher Scientific) was added and incubated for 45 min at 37°C and 5% CO₂, protected from light. Fluorescence was measured (560 nm excitation/590 nm emission) using a Bio Tek SynergyTM 2 multi-mode microplate reader (BioTek, Vermont, USA). Treated cells were compared with control cells (considered 100% viable) containing 1% (v/v) of the vehicle (DMSO). The IC50 for parental OVCAR8 and PTX R variants was used to determine fold resistance, according to the following formula (38, 39):

To evaluate cell proliferation, MTT [3-(4.5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] assay was performed as previously described (40). Briefly, 1 × 10³ cells/well were seeded into a 96-well plate in complete media and incubated at 37°C and 5% CO₂ up to 6 days. At each time point, 500 µg/ml of MTT dye reagent (Sigma-Aldrich, Missouri, USA) was added and incubated at 37°C and 5% CO₂ for 3 h. Formazan crystals were dissolved in 100 µl of DMSO. Absorbance was measured (570 nm wavelength) using a Bio Tek SynergyTM 2 multi-mode microplate reader.

To differentiate viable and non-viable cells, trypan blue assay was performed. Cells at 80% confluence were diluted and mixed with a 0.4% trypan blue solution (ThermoFisher Scientific), placed in a Neubauer chamber, and counted in five counting grid squares, under a Leica DMi1 inverted phase contrast microscope (Leica Microsystems, Wetzlar, Germany), at 50× magnification.

All conditions were arrayed in a Cell MicroArray (CMA) block designed and constructed as previously described (41, 42). Briefly, 2 × 10⁵ cells/well were seeded into six-well plates and incubated at 37°C and 5% CO₂. After 48 h, cells were treated with 10 nM PTX and incubated under the same conditions. After 48 h, cells were scraped from culture dishes and fixed with 10% (v/v) neutral-buffered formalin (AppliChem). Cells were re-suspended in liquefied HistoGel™ (ThermoFisher Scientific), according to the manufacturer’s instructions, followed by standard histological processing and paraffin embedding. Finally, CMA block was constructed and sectioned with a microtome (43). To perform immunocytochemistry, slides were deparaffinized and hydrated. Next, heat-induced (98°C) antigen retrieval was performed with citrate buffer solution (1:100 at pH 6.0; ThermoFisher Scientific) or ethylenediamine tetraacetic acid (EDTA; 1:100; ThermoFisher Scientific) for 40 min. Endogenous peroxidase activity was blocked with hydrogen peroxide solution 3% (v/v) (ThermoFisher Scientific) for 10 min. Slides were incubated with a specific primary antibody (conditions described in Supplementary Table 1) and detected using a secondary antibody with horseradish peroxidase (HRP)-labeled polymer (Dako REAL™ EnVision™ Detection System Peroxidase/DAB+, Rabbit/Mouse) for 30 min. Visualization of the reaction was performed using diaminobenzidine according to the manufacturer’s instructions. Finally, nuclear staining with hematoxylin was performed and slides were dehydrated, clarified, and coverslipped using a permanent mounting medium for optical microscope analysis.

Immunocytochemistry results were evaluated by two independent observers (MN and SR) that register the staining pattern (nuclear, cytoplasm, or membrane) and the percentage of cells stained (0%, 1%–10%, 11%–25%, 26%–50%, 51%–75%, and 76%–100%).

To perform a qualitative assessment of cell ultrastructure, an electron microscopy analysis was performed. Briefly, for transmission electron microscopy and semithin section analysis, cultured cells were grown until 80% confluence, washed three times with phosphate buffer saline (PBS, ThermoFisher Scientific), gently scraped from culture dishes and fixed overnight in a glutaraldehyde (2.5%), paraformaldehyde (2%), and 0.1 M sodium cacodylate buffer (1:1) solution, at 4°C. After fixation, cells were pelleted by centrifugation and washed three times in 0.1 M sodium cacodylate buffer for 5 min. A post-fixation in 0.1 M sodium cacodylate buffered 2% osmium tetroxide was performed for 2 h at room temperature (RT). Then, cells were washed three times in distilled water for 5 min. The samples were incubated with 1% uranyl acetate for 30 min at RT, followed by three washes in distilled water for 5 min. Finally, cell pellet was imbedded in Histogel™, dehydrated in ethanol, and embedded in an epoxy resin. Ultrathin sections were stained and observed in a JEOL 100CXII transmission electron microscope (JEOL, Tokyo, Japan) operated at 80 kV, and images were digitally recorded using a CCD digital camera Orius 1100W (JEOL).

To evaluate cell apoptosis and cell cycle distributions, Annexin V-FITC apoptosis detection kit™ (ThermoFisher Scientific) and propidium iodide (PI) staining were used, according to the manufacturer’s instructions. Briefly, 2 × 10⁵ cells/well were seeded into six-well plates and incubated at 37°C and 5% CO₂. After 48 h, cells were treated with 10 nM PTX and incubated under the same conditions.

For apoptosis analysis, after 48 h, floating and adherent cells were collected using a cell dissociation buffer enzyme-free in PBS, pelleted by centrifugation (800 g, 5 min), filtrated through a 70-μm filter to obtain single-cell suspensions, and suspended in 195 µl of binding buffer. Next, 5 µl of Annexin V-FITC was added and incubated for 10 min, protected from light. Cells were washed, resuspended in 190 µl of binding buffer, and 10 µl of PI (20 μg/ml) was added and cells were incubated for 1 min, protected from light.

For cell cycle, following 24 h, floating and adherent cells were collected, pelleted by centrifugation, and resuspended in PBS. Cells were fixed with 70% (v/v) cold ethanol and incubated at 4°C for 30 min. Next, cells were pelleted by centrifugation, resuspended in PBS containing PI (250 µg/ml), and RNase A (100 µg/ml) and incubated at 4°C for 15 min, protected from light.

Fluorescence from both assays was assessed by BD FACS Canto™ II flow cytometer and data were analyzed by FlowJo software v10.0.7 (Ashland, Oregon, USA).

Live-cell imaging experiments were performed as previously described (44). Briefly, 1.2 × 10⁵ cells were seeded into LabTek II chambered cover glass (Nunc, New York, USA) containing 1 ml of culture medium and incubated at 37°C with 5% CO₂. After 24 h, cells were treated with 20 nM PTX in RPMI without phenol red and supplemented with 5% FBS. Images were captured at 5-min intervals up to 72 h under differential interference contrast (DIC) optics, with a 63× objective under an Axio Observer Z.1 SD inverted microscope, equipped with an incubation chamber at 37°C and 5% CO₂. Movies were generated from time-lapse images using ImageJ 1.4v software (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA). The mitosis duration and the number of cells arrested at mitosis, dead by apoptosis, or bypassing cytokinesis were scored.

To evaluate anoikis resistance, an aggregate formation assay was performed as previously described (40). Briefly, 1 × 10⁶ cells/well were seeded into polyHEMA (Poly2-hydroxyethyl methacrylate; Sigma-Aldrich)-coated plates and incubated at 37°C and 5% CO₂ up to 15 days. At each time point (5, 10, and 15 days), floating cells were collected and dissociated using trypsin, pelleted by centrifugation, and resuspended in PBS. Cell suspensions were incubated with DAPI (1 µg/ml) for 2 min. Fluorescence was assessed by BD FACS Canto™ II flow cytometer, and data were analyzed by FlowJo software v10.0.7.

To evaluate clonogenic capacity, 1 × 10³ cells/well were seeded into six-well plates and incubated at 37°C and 5% CO₂. Following 24 h, cells were treated with 10 nM PTX and incubated under the same conditions. After 48 h, media was replaced, and cells were allowed to recover for 8 days. Surviving colonies were fixed and stained as previously described (45). The percentage of cell survival was determined according to the following formula:

To analyze migration capacity, cell monolayers at confluence cultured in six-well plates were serum starved overnight and incubated at 37°C and 5% CO₂. Then, monolayers were washed twice with PBS and scratched in half with a sterile 200-µl pipette tip. To monitor cell migration, a phase contrast microscopy image was taken at 0, 24, and 48 h. The quantification of wound closure was performed by measuring the unmigrated remaining area using ImageJ 1.4v software.

Total RNA was extracted from cells with PureZOL™ RNA Isolation Reagent (Bio-Rad Laboratories, California, USA), according to the manufacturer’s instructions. cDNA synthesis was performed with iScript™ cDNA Synthesis Kit (Bio-Rad), using total cell RNA as template, following supplier’s instructions. For real-time PCR, cDNA was amplified using iQ™ SYBR Green Supermix Kit (Bio-Rad). Primers for P-gp were as follows: forward: 5’-GCCAAAGCCAAAATATCAGC-3’ and reverse: 5’-TTCCAATGTGTTCGGCATTA-3’; GAPDH: forward: 5’-ACAGTCCAGCCGCATCTTC-3’ and reverse: 5’-GCCCAATACGACCAAATCC-3’; and β-Actin, used as housekeeping gene: forward: 5’-AAT CTG GCA CCA CAC CTT CTA-3’ and reverse 5’-ATA GCA CAG CCT GGA TAG CAA-3’. Data were acquired with CFX Manager™ Software v1.0 (Bio-Rad) and results were analyzed according to ΔCT.

Total cell protein extracts and Western blotting were performed as previously described (46). Membrane was incubated with primary (Supplementary Table 1) and secondary antibodies [anti-rabbit horseradish peroxidase (Vector Laboratories, Burlingame, USA) and anti-mouse horseradish peroxidase (Sigma-Aldrich), diluted at 1:1,000 or 1:4,000, respectively]. The protein signal intensity was quantified using ImageJ 1.4v software and normalized against α-tubulin expression levels.

To evaluate P-gp functional activity, a total of 1 × 10⁵ cells of OVCAR8 and OVCAR8 PTX R were seeded into six-well plates and incubated at 37°C and 5% CO₂. After 24 h, 1 µM of the fluorescent P-gp substrate Rhodamine 123 (RH-123; Sigma-Aldrich) was added, in the presence or absence of 20 µM Verapamil (Sigma-Aldrich)—P-gp inhibitor—and incubated under the same conditions. After 1 h, cells were harvested and washed twice with ice-cold PBS, and cell pellet was gently re-suspended in ice-cold PBS for analysis. The mean fluorescence intensity (MFI) was assessed by BD Accuri C6™ II flow cytometer (BD Biosciences, California, USA), and data were analyzed by BD Accuri C6 Plus Software, version 1.0.27.1 (BD Biosciences).

To determine cell viability to PTX after Verapamil incubation, a Sulforhodamine B (SRB) assay was performed. Briefly, 5 × 10⁶ cells/well were seeded in 96-well plates and incubated at 37°C and 5% CO₂. After 24 h, cells were treated with 10-fold serial dilutions of PTX, in the presence or absence of 10 µM Verapamil and incubated under the same conditions. In parallel, cells were treated with equivalent amount of vehicle (DMSO) up to 0.25% concentration. Then, cells were fixed with 50% (m/v) trichloroacetic acid (Merck Millipore, Darmstadt, Germany) for 1 h, washed with distilled water, and stained with 0.1% (m/v) SRB in acetic acid (Sigma-Aldrich) for 30 min at RT. After washing with 1% (v/v) acetic acid aquae solution (Merck Millipore), plates were left to dry at RT followed by SRB complex solubilization with 10 mM Tris-Base buffer (Sigma-Aldrich) for 30 min. Absorbance was measured (515 nm wavelength) using a Bio Tek SynergyTM 2 multi-mode microplate reader. The IC50 of PTX, in the presence or absence of Verapamil, was determined as described above in Section 2.3.

All assays were done in triplicate with at least three independent experiments. Data were expressed as mean ± standard deviation (SD), statistical analysis was carried out in GraphPad Prism Software Inc. v6 using ordinary one-way or two-way ANOVA followed by Tukey’s or Šídák’s multiple comparison test, and values of *<0.05; **<0.001; ***<0.0005; ****<0.0001 were considered statistically significant.

OVCAR8 and OVCAR8 PTX R cell lines were treated with increasing concentrations of PTX, and cell viability was evaluated by PB assay. Cell viability of OVCAR8 cell line exposure to 10 nM PTX was 51.52% ± 7.69%, whereas for OVCAR8 PTX R C and OVCAR8 PTX R P variants, it was 89.16% ± 6.62% and 86.79% ± 4.25%, respectively (Figure 1B). A significant increase in IC50 PTX was observed for OVCAR8 PTX R C (128.97 ± 6.48 nM, p < 0.001) and OVCAR8 PTX R P (152.80 ± 6.51 nM, p < 0.001) variants when compared to OVCAR8 cell line (10.51 ± 1.99 nM), demonstrating a 12.27-fold and 14.54-fold increase in resistance, respectively (Supplementary Figure 1B).

In order to assess the stability of acquired resistance in both OVCAR8 PTX R variants, sensitivity to PTX was assessed after two continuous months in culture and after cryopreservation cycles, without PTX medium supplementation. Our results show that OVCAR8 PTX R variants have a long-term stability without drug supplementation and retain their resistant phenotype following freeze/thawing cycles (Figure 1B and Supplementary Figure 1B).

Additionally, we evaluated if OVCAR8 PTX R variants increased resistance to Carboplatin, since parental OVCAR8 cell line was described as a Carboplatin chemoresistant model. Our results show no significant differences in IC50 for Carboplatin concentrations in OVCAR8 PTX R C (125.74 ± 3.60 nM) and OVCAR8 PTX R P (151.26 ± 10.17 nM, p < 0.05) when compared to OVCAR8 cell line (127.49 ± 4.76 nM) (Figure 1C).

To further evaluate the PTX R phenotype, we performed cell proliferation, apoptosis, and viability assays. By MTT assay, no significant increase in proliferation was observed in OVCAR8 PTX R variants compared to OVCAR8 cell line, at 120 h in culture (Figure 1D). Accordingly, by immunocytochemistry, no significant differences were observed in proliferation (75%–100% of Ki67 staining) and apoptosis (1%–10% of cleaved-PARP staining) for OVCAR8 PTX R variants when compared to OVCAR8 cell line after 48 h in culture (Figure 1E). Trypan blue exclusion assay for assessing viability indicated no significant differences between OVCAR8 PTX R variants (both 97.50% viable) when compared to OVCAR8 cell line (97.50% viable) (Figure 1F).

To assess ultrastructural alterations in OVCAR8 PTX R variants, we performed qualitative analysis of several transmission electron microscopy micrographs. No significant ultrastructural differences were observed between OVCAR8 and OVCAR8 PTX R cell lines, which revealed similar ultrastructure aspects such as numerous dense mitochondria, developed Golgi apparatus and rough endoplasmic reticulum, and multiple lysosomes (Figure 1G).

Levels of PTX-induced apoptosis were determined by the Annexin V-FITC apoptosis detection kit™ using flow cytometry. Upon 48 h incubation with 10 nM PTX, a significant (p < 0.0001) increase in PTX-induced apoptosis was observed on OVCAR8 cell line (37.01% ± 1.63%) when compared with the nontreated control (7.40 ± 2.48%) (Figure 2A). In contrast, after 10 nM PTX exposure, our results show a significant decrease (p < 0.0001) in PTX-induced apoptosis in OVCAR8 PTX R C and P variants (11.74% ± 4.58% and 13.38% ± 2.33%, respectively) when compared to OVCAR8 cell line (37.01% ± 1.63%) (Figure 2A).

Cell cycle distribution was assessed through PI staining by flow cytometry. After 10 nM PTX exposure, a significant decrease (p < 0.0001) in the percentage of cells in G1 phase (54.61 ± 3.32% vs. 27.84 ± 2.89%) and accumulation of cells in G2/M phase (18.77 ± 0.56% vs. 44.18 ± 4.75%) was observed in the OVCAR8 cell line when compared to control (Figure 2B). On the other hand, after 10 nM PTX exposure, OVCAR8 PTX R variants showed no significant changes in G1, S, and G2/M subpopulations when compared to control (Figure 2B). After 10 nM PTX exposure, a significant difference (p < 0.0001) in the percentage of cells in G1 phase was found in OVCAR8 PTX R C and P variants (48.92% ± 0.84% and 46.41% ± 2.62%, respectively) when compared to parental cell line (27.84% ± 2.89%) (Figure 2B). Likewise, a significant difference (p < 0.0001) in the G2/M population was found in OVCAR8 PTX R C and P variants (19.57% ± 1.94% and 20.82% ± 2.45%, respectively) when compared to the parental cell line (44.18% ± 4.75%) (Figure 2B). A significant increase (p < 0.001) was also observed in sub-G1 fraction (debris and apoptotic cells) for the OVCAR8 cell line (3.11% ± 0.91% vs. 7.60% ± 1.16%) and the OVCAR8 PTX R P variant (2.41% ± 0.79% and 7.15% ± 0.76%) when compared to controls (Supplementary Figure 2A) after exposure to 10 nM of PTX. OVCAR8 and OVCAR8 PTX R cell lines presented a similar proportion of polyploid (>4C) population (Supplementary Figures 2A, B).

To further characterize the behavior of OVCAR8 and OVCAR8 PTX R C cell lines, we analyzed mitosis duration and cell fate by live-cell imaging using time-lapse DIC microscopy. We found that the OVCAR8 PTX R C variant spent 55.33 ± 7.66 min in mitosis, from nuclear envelope breakdown to anaphase onset, similarly to what was observed in the OVCAR8 cell line that spent 39.35 ± 16.70 min (Figure 2C). Under 20 nM PTX exposure, the mitosis duration in OVCAR8 PTX R C variant is similar to that in untreated cells (33.00 ± 15.18 min) (Figure 2C) contrasting with the significant increase (p < 0.001) exhibited by the OVCAR8 cell line (331.29 ± 106.82 min). After 20 nM PTX exposure, the survival fate (outcome of cells delayed in mitosis under PTX exposure) analysis showed that 37.5% of parental OVCAR8 cells died in mitosis after mitotic arrest, and 62.5% of cells underwent normal cycling (Figure 2C). In contrast, OVCAR8 PTX R C variant undertook normal and multiple mitosis (Figure 2C).

To assess anoikis (cell detachment-induced apoptosis) resistance, an aggregate formation assay was performed and evaluated by flow cytometry. Our results for anoikis resistance demonstrate no significant differences in cell viability (DAPI negative cells) for OVCAR8 PTX R C (87.88% ± 3.29% and 78.20% ± 3.73%) and OVCAR8 PTX R P variants (89.96% ± 1.74% and 84.03% ± 4.97%) when compared to parental OVCAR8 cell line (94.32% ± 1.15% and 86.47% ± 1.66%), upon 5 and 10 days of culture, respectively (Figure 3A). Moreover, no significant differences were observed in the percentage of positive cells for Ki67 (76%–99%), BrdU (11%–25%), cleaved caspase 3 (1%–10%), and cleaved PARP (1%–10%) for OVCAR8 PTX R variants when compared to the parental OVCAR8 cell line (Figure 3B). Additionally, we demonstrate that aggregation capacity was partially affected, since different and irregular aggregates were formed in OVCAR8 PTX R cells when compared to OVCAR8 (Figure 3C).

To evaluate clonogenic capacity, we performed a colony-forming assay. Our results show that after 10 nM PTX exposure, a significant decrease (p < 0.0005) occurs in the number of colonies in the OVCAR8 cell line (274.78 ± 49.27 vs. 10.56 ± 8.59) when compared to the untreated control (Figure 3D). One the other hand, after 10 nM PTX exposure, no significant differences in colony formation capacity were observed in OVCAR8 PTX R C (294.22 ± 57.45 vs. 254.00 ± 58.68) and OVCAR8 PTX R P variants (241.44 ± 24.44 vs. 182.11 ± 44.29) when compared to untreated control (Figure 3D). In fact, the percentage of cell survival was significantly higher (p < 0.0005) in OVCAR8 PTX R C (85.80% ± 4.80%) and OVCAR8 PTX R P variants (74.92% ± 7.72%) when compared to the parental OVCAR8 cell line (3.98% ± 2.08%) (Figure 3D).

To evaluate migration capacity, we performed a wound healing assay. Our results display no significant differences on migration behavior for OVCAR8 PTX R C (32.48% ± 4.79%) and OVCAR8 PTX R P variants (25.27% ± 3.55%) when compared to the OVCAR8 cell line (27.63% ± 0.81%) at 48 h (Figure 3E).

P-gp encoded by MDR1 is abundantly expressed in cellular membrane, where it functions as a multidrug efflux pump that transports a wide range of structurally different substrates, including PTX (47). P-gp overexpression is the main mechanism of resistance to chemotherapeutics, being responsible for pumping the drug out of the cells, resulting in low intracellular concentration of the drugs and survival of cancer cells (48, 49). P-gp expression was evaluated at mRNA and protein levels, by qRT-PCR, Western blot, and immunocytochemistry. From qRT-PCR analysis, we found an increase in P-gp expression on OVCAR8 PTX R C (12.60 ± 2.90-fold increase) and OVCAR8 PTX R P variants (8.08 ± 4.00-fold increase) when compared to OVCAR8 cell lines (Figure 4A). This result was confirmed at the protein level by Western blot and immunocytochemistry that revealed a high percentage of cells positive for P-gp in OVCAR8 PTX R variants (76%–100% positive cells) when compared to the OVCAR8 cell line (negative/residual expression) (Figure 4A).

To confirm if P-gp upregulation is the chemoresistance mechanism in OVCAR8 PTX R variants, we determined the IC50 PTX in the presence and absence of Verapamil, a well-known P-gp inhibitor. We found that the IC50 of PTX decreased in OVCAR8 PTX R C (40-fold) and OVCAR8 PTX R P variants (33-fold) when Verapamil was added (Figure 4B). We also analyzed by flow cytometry the activity of P-gp through the evaluation of the mean fluorescence intensity (MIF) of cells exposed to RH-123, a known P-gp fluorescent substrate. The percentage of MIF is proportional to intracellular uptake and extracellular efflux of RH-123. We found that intracellular accumulation of RH-123 was very low for OVCAR8 PTX R C and OVCAR8 PTX R P variants (16.57% ± 8.46% and 6.99% ± 3.91%, respectively) when compared to the parental OVCAR8 cell line (considered 100%). Moreover, in the presence of Verapamil, the relative MIF significantly increased (p < 0.0005) for OVCAR8 PTX R C (89.73% ± 3.22%) and OVCAR8 PTX R P variants (83.14% ± 3.30%) when compared to control, demonstrating an accumulation of RH-123 due to P-gp pump inhibition (Figure 4C).