Article Cite This: Mol. Pharmaceutics 2018, 15, 472−485
Ratio-Dependent Synergism of a Doxorubicin and Olaparib Combination in 2D and Spheroid Models of Ovarian Cancer Sina Eetezadi,† James C. Evans,† Yen-Ting Shen, Raquel De Souza, Micheline Piquette-Miller, and Christine Allen* Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada
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ABSTRACT: Ovarian cancer is the fourth leading cause of death in women in developed countries. Even though patients with the most lethal form of the disease (HGSOC; high grade serous ovarian cancer) respond well to initial treatment, they often relapse with progressively resistant disease. Inhibitors of the poly(ADP-ribose) polymerase (PARP) enzymes are a relatively new class of molecularly targeted small molecule drugs that show promise in overcoming resistance. The present study explores the combination of a DNA damaging agent, doxorubicin (DOX), with the PARP inhibitor, olaparib (OLP), in order to achieve optimal synergy of both drugs in serous ovarian cancer. This drug combination was evaluated and optimized in 2D monolayers and 3D multicellular tumor spheroids (MCTS) using a genetically and histologically characterized panel of nine OC cell lines with or without BRCA1 or BRCA2 mutations. Combination index (CI) values of DOX and OLP were determined using the Chou and Talalay method. The potency of this drug combination was found to rely heavily on the molar ratios at which the two drugs are combined. In general, MCTS growth inhibition was reflective of the patterns predicted by the CI values obtained in monolayers. Promising combination ratios identified in this study warrant further preclinical and clinical investigation. KEYWORDS: drug combination, PARP inhibitor, serous ovarian cancer, synthetic lethality, doxorubicin, olaparib, multicellular tumor spheroids, BRCA, combination index
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INTRODUCTION Ovarian cancer (OC) is the fourth leading cause of death in women of developed countries, with dismal survival improvements achieved in the past three decades.1,2 While the median survival rate for OC as a whole is 40 to 50% at 10 years postdiagnosis, it falls significantly to 21% and less than 6% when the disease is diagnosed at stages III and IV, respectively, as occurs in the majority of cases.1 About 90% of OCs are epithelial ovarian cancer (EOC), and between 60 and 70% of EOCs are high-grade serous ovarian cancer (HGSOC), the most common and deadliest form of the disease that is widely associated with p53 gene mutations.3−5 As a consequence of the initially asymptomatic or ambiguously symptomatic nature of EOC, most patients present with advanced, metastatic disease at diagnosis, which is currently treated by tumor cytoreductive surgery prior to carboplatin and paclitaxel adjuvant chemotherapy.3,6,7 HGSOC patients initially respond well to this regimen (response rate (RR) > 80%), but as many as 75% relapse, often within 18 months, with progressively resistant disease.4,8 Once platinum resistance occurs, selection of the most appropriate treatment for further management of the disease remains a challenge.1,9 Pegylated liposomal doxorubicin (PLD, Doxil/Caelyx) is predominant in the treatment of OC recurrence, however it is associated with low RRs of 9−16%.10,11 © 2017 American Chemical Society
Inhibitors of the poly(ADP-ribose) polymerase (PARP) enzymes are a class of molecularly targeted small molecule drugs that show promise in the setting of resistant recurrence. The first PARP inhibitor to be clinically assessed is olaparib (OLP, Lynparza), which has been approved by the FDA for refractory, advanced OC associated with germline breast cancer susceptibility gene (BRCA) mutations.12 In August 2017, Lynparza was additionally approved by the FDA as a maintenance therapy for women with recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer who have achieved complete or partial response to platinum-based chemotherapy, irrespective of their BRCA status.13 Also in 2017, another PARP inhibitor, niraparib (Zejula), was approved by the FDA for maintenance therapy for adults with recurrent EOC that is responsive (either complete or partial) to platinum-based chemotherapy.14 PARP inhibitors capitalize on deficiencies or aberrations in DNA damage repair pathways that are present in about half of all HGSOC.1,6,15,16 About 51% of HGSOC display deficiencies in homologous recombination repair (HRR), the high-fidelity DNA double-strand break Received: Revised: Accepted: Published: 472
September 25, 2017 December 17, 2017 December 28, 2017 December 28, 2017 DOI: 10.1021/acs.molpharmaceut.7b00843 Mol. Pharmaceutics 2018, 15, 472−485
Article
Molecular Pharmaceutics
histopathological origin and genomic characteristics are unknown for the majority of these lines, limiting their utility in accurately representing aspects of interest of the disease.35,36 For this study, multiple cell lines were carefully selected, based on data from three recent large-scale cell line characterization studies, to histologically and molecularly represent an array of serous and high-grade serous OCs, including HRR-deficient variations.35−37 The potential of each cell line to form multicellular tumor spheroids (MCTS) using an established liquid-overlay technique was assessed.38 MCTS are spherical, 500 μm structures composed of an outer region of proliferative cells surrounding intermediate regions of quiescent cells.39,40 MCTS are clinically representative 3D models of metastatic cell spheroids that form in advanced disease states as well as microscopic residual disease that remains after cytoreductive surgery in HGSOC.41,42 In particular, to the best of our knowledge, MCTS of BRCA mutated cell lines have not been reported in the literature thus far. In this study, combination effects of DOX and OLP at ten different molar ratios were evaluated in 2D and 3D cell models of the selected ovarian cancer cell lines using the well-established Chou and Talalay method to evaluate treatment synergy.43 In order to ascertain the mechanism of action of this drug combination, the γH2AX assay was used to quantify DNA DSBs.
(DSB) repair pathway, 15−20% of which display germline or somatic mutations in the BRCA1 or BRCA2 genes.15,17,18 Through active recruitment of repair proteins, the PARP1 enzyme plays a critical role in base excision repair, which is the predominant mechanism in the repair of DNA single strand breaks (SSBs).19−21 Upon catalytic inhibition by a PARP inhibitor, PARP1 becomes trapped at SSB sites, which leads to stalling of the replication fork and subsequent formation of SSBs.22 Additionally, PARP inhibitors hinder the role of PARP1 in DSB repair given that PARP1 is reported to aid in the recruitment of HRR proteins.19 On its own, PARP inhibition is relatively nontoxic to cells with intact HRR capabilities. However, synthetic lethality occurs upon PARP inhibition in cells with deficiencies in HRR due to BRCA mutations20,21 or the “BRCAness” phenotype, which renders cells more susceptible to DNA damage.15,19 PARP inhibitors have shown promise clinically in both BRCA-deficient and -proficient HGSOCs.23−25 In a phase II trial involving recurrent, platinumsensitive HGSOC patients, it was found that the inclusion of the PARP inhibitor olaparib into the paclitaxel−carboplatin standard regimen significantly improved progression-free survival, especially in the patient subpopulation with BRCA mutations.26 Due to heightened susceptibility to SSB and compromised repair capabilities, the combination of PARP inhibitors with DNA-damaging agents has the potential to synergistically affect HGSOC cells. The combination of a PARP inhibitor with doxorubicin (DOX) is particularly promising in HGSOC. DOX inhibits DNA polymerase activity and topoisomerase II, and induces free radical formation, causing DSBs and SSBs.11 Further, DOX shows enhanced efficacy in HRR-deficient OC, as BRCA mutation carriers treated with PLD show greater RR and longer overall survival (OS) compared to sporadic cases.10,11 Moreover, PARP inhibitors have been shown to enhance DOX antitumor activity in p53-deficient cancers,27,28 which is promising for HGSOC, as 90−100% of cases show p53 mutations.15,16,29 Further, PARP inhibitors increase topoisomerase II expression, thereby specifically potentiating the action of DOX.30 Yet, in clinical practice, dose limiting toxicities have been a major hurdle for such combinations, and have resulted in the need to halt a number of clinical trials and/ or to reduce drug doses midtrial.6,31 Generally, combination therapies have been designed with the assumption that maximal therapeutic effect is achieved when the maximum tolerated dose (MTD) of each drug is employed. However, it has been shown that the effect of drug combinations depends not only on the nature of each drug’s distinct mechanism of action but also on the molar ratios at which they have been combined.32,33 For example, a given drug may induce a strong cell defense response that in turn reduces the therapeutic effect of a second drug when given in combination. Hence, the choice of ratio is important; while certain ratios are synergistic, others can be antagonistic.34 Based on the potential of combining PARP inhibitors with DNA damaging agents, it was hypothesized that, by evaluating a range of specific molar ratios of the PARP inhibitor OLP with the DNA-damaging agent DOX, it would be possible to maximize the therapeutic index of this drug combination by identifying synergistic ratios of the two drugs. To assess this strategy in a setting representative of clinical disease presentation, we sought to employ in vitro models encompassing the heterogeneity of EOC. About 100 OC cell lines are currently available through public cell banks;35 however, the
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MATERIALS AND METHODS Materials. DOX was purchased from Polymed (Houston, TX, USA). OLP was obtained from Tongchuang Pharma, Suzhou Co. Ltd. (China). Acetic acid, P-nitrophenyl phosphate, Triton X-100, G-418 disulfate salt, sodium pyruvate, and sodium acetate were purchased from Sigma-Aldrich (Oakville, ON, Canada). MCDB 105 and DMEM cell media were obtained from Sigma-Aldrich, while all other cell media, sterile phosphate buffered saline (PBS, pH = 7.4), CellMask Green plasma membrane stain, and fetal bovine serum (FBS) were obtained from Life Technologies (Burlington, ON, Canada). MEGM Mammary Epithelial Cell Growth Medium Kit was purchased from Lonza (Mississauga, ON, Canada). UWB1.289, UWB1.289+BRCA1, OV-90, and SKOV3 cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). PEO1, PEO4, and COV362, originally from the European Collection of Cell Cultures (ECACC, Public Health England; Salisbury, U.K.), were purchased through Sigma-Aldrich. HEYA8 was obtained from the M. D. Anderson Cancer Center (Houston, TX, USA). OVCAR8 was obtained from the Biological Testing Branch of the National Cancer Institute (NCI; Frederick, MD, USA). Cell Culture. All cell lines were cultured as monolayers in the medium recommended by the supplier and maintained in a dedicated cell culture incubator at 37 °C, 5% CO2 atmosphere, and 90% relative humidity. UWB1.289 and UWB1.289+BRCA1 were maintained in a 1:1 mixture of RPMI-1640 and MEGM including the five components of the MEGM Mammary Epithelial Cell Growth Medium Kit and 3% FBS. UWB1.289+BRCA1 growth medium was additionally supplemented with 200 μL/mL G-418 to maintain BRCA1 expression. HEYA8 and OVCAR8 were grown in RPMI-1640 medium with 10% FBS. PEO1 and PEO4 were maintained in RPMI-1640 medium with 10% FBS and additionally supplemented with 2 mM sodium pyruvate. COV362 was cultured in DMEM and SKOV3 in McCoy’s 5a medium, both supplemented with 10% FBS, and OV-90 was grown in a 1:1 mix of MCDB 105 and Medium 199 supplemented with 15% 473
DOI: 10.1021/acs.molpharmaceut.7b00843 Mol. Pharmaceutics 2018, 15, 472−485
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through to the end of the experiment (5000 cells/well for OV90 and COV362; 4000 cells/well for PEO1 and PEO4; 3000 cells/well for UWB1.289, UWB1.289+BRCA1, and SKOV3; 1000 cells/well for OVCAR8 and HEYA8 cell lines). Following treatment, cells were washed with PBS prior to assessment of viability using the APH assay. In brief, cells were incubated with 100 μL of freshly prepared reaction buffer (sodium acetate buffer at pH 5.5 containing 1% Triton X supplemented with 2 mg/mL p-nitrophenyl phosphate) for 2 h at 37 °C. Cell viability was determined by measuring the UV absorbance at 485 nm using an automated 96-well plate reader (Synergy 2 from BioTek; Winooski, VT, USA) following addition of 10 μL of 1 M sodium hydroxide to each well. Average absorbance (A) for each drug concentration of three independent experiments performed on different days was expressed relative to controls as follows:
FBS. Penicillin/streptomycin was added to all media at a concentration of 100 units/mL penicillin and 100 μg/mL streptomycin. Cell passage number was maintained below 25. Cell Line Authentication. Short tandem repeat (STR) analysis was performed by The Centre for Applied Genomics at The Hospital for Sick Children (Toronto, ON, Canada) to verify the authenticity of cell lines employed here. To this end, the GenePrint 10 System (Promega Corporation; Madison, WI, USA) was used, following the manufacturer’s instructions. Briefly, the kit employs an STR multiplex assay that amplifies nine loci (the ANSI Standard (ASN-0002) recommends eight) and the Amelogenin gender-determining marker in a single PCR amplification. To prepare each reaction, 10 ng of genomic DNA was incubated with a master mix (5 μL of GenePrint 10 5× Master Mix, 5 μL of GenePrint 10 5× Primer Pair Mix, water for a 25 μL total volume). The cycling conditions were 96 °C for 1 min; 30 cycles of 94 °C for 10 s, 59 °C for 1 min, 72 °C for 30 s; 1 cycle of 60 °C for 10 min and 4 °C maintenance. Samples were run on an AB 3130 Genetic Analyzer using POP7, Promega’s internal lane standard 600, dye set F and analyzed in GeneMapper v3.7. Since allelic nomenclature for the 10 loci is standardized worldwide, the obtained STR profiles were compared to those made available by each cell line’s corresponding repository or previous publications (S1 Table). BRCA1 and BRCA2 Mutation Sequencing. Regions pertaining to BRCA1 and BRCA2 gene variants of interest were amplified for Sanger sequencing by The Centre for Applied Genomics at The Hospital for Sick Children (Toronto, ON, Canada) to confirm BRCA mutation status of relevant cell lines. Primer sequences are outlined in S2 Table. Briefly, each 25 μL PCR reaction was prepared with 2 mM dNTPs (2.5 μL), 10× PCR buffer II (2.5 μL; Life Technologies), 25 mM MgCl2 (1.3 μL), AmpliTaq DNA polymerase (0.25 μL; Life Technologies), 5 M betaine (5 μL), water (10.45 μL), 10 μM primers (1 μL each), and genomic DNA (1 μL; normalized to 50−100 ng/μL). Samples were amplified by PCR using the following cycling conditions: 30 cycles of 94 °C for 10 s, 60 °C for 30 s, and 72 °C for 1 min. Agarose gel was used to visualize the PCR products, which were purified using the AxyPrep PCR Clean-up Kit (Axygen Biosciences). A total of 50 ng of purified product was Sanger sequenced from the forward primer using BigDye3.1 chemistry on a ABI3730XL capillary DNA analyzer (Life Technologies). Doubling Time. For evaluation of cell proliferation rates, subconfluent cells were harvested, seeded onto 6-well plates at a seeding density of 100,000 cells per well, and allowed to adhere overnight. Over a 6-day period, cells were detached at selected time points with TrypLE Express cell dissociation solution and counted using a Countess II FL automated cell counter (Life Technologies). Cell doubling time was determined by nonlinear regression based on an exponential growth equation that was fit to the cell counts at each time point using GraphPad Prism software. Monolayer IC50 Evaluation. Monolayer cell cytotoxicity for DOX, OLP, and all drug combinations was determined using the acid phosphatase (APH) assay based on a method previously published by our group.38 Briefly, subconfluent cells were harvested, seeded onto 96-well plates, and allowed to adhere overnight prior to incubation for 72 h using ten 1:4 serial dilutions of single drug or drug combinations (n = 6 wells per dilution). Seeding density was determined separately for each cell line so that the control was maintained subconfluent
viability [%] =
(A treatment − A media ) (Acontrol − A media )
The concentration that kills 50% (IC50, or fraction affected (Fa) = 0.5) and 75% (Fa = 0.75) of cells was determined by fitting the data to the Hill equation using GraphPad Prism software. Determination of Combination Index (CI) Values. CI values were determined according to a widely used method established by Chou and Talalay.44,45 Briefly, to determine each CI value, the following monolayer cytotoxicity studies were conducted: (1) DOX as a single drug; (2) OLP as a single drug; and (3) DOX−OLP combinations at a series of specific molar ratios. IC50 values that resulted from the drugs when used in combination were between 50 μM and 5 nM for both drugs, depending on the cell line and drug ratio used. For each experiment, starting concentrations were chosen such that the IC50 value was the intermediate concentration employed in the ten serial dilutions chosen, as outlined above. For all experiments, Fa = 0.5 and Fa = 0.75 molar drug concentrations were determined, as outlined above. CI values were then calculated for each combination and for both effect levels based on the formula below for mutually nonexclusive drugs as established by Chou and Talalay, where D SD is the concentration of DOX that kills 50% of cells (for Fa = 0.5) or 75% of cells (for Fa = 0.75), DSO is the concentration of OLP at Fa = 0.5 or Fa = 0.75, DCD is the concentration of DOX in combination with OLP at Fa = 0.5 or Fa= 0.75, and DCO is the concentration of OLP in combination with DOX at Fa = 0.5 or Fa = 0.75: CI =
DCD D D D + CO + CD CO DSD DSO DSDDSO
Based on this method, CI values are indicative of strong synergism (3.3).44 Microsoft Excel was used to generate a tricolor system based on these values, where strong synergism is represented by green, additive effect by yellow, and strong antagonism by red. The software interpolates the color of each value in between these constraints accordingly. γ-H2AX Determination. In order to quantify the level of DNA DSBs, 1 × 105 UWB1.289 and UWB1.289+BRCA1 cells were seeded on 18 × 18 mm glass coverslips in 6-well plates. Cells were treated for 72 h with either (a) DOX, (b) OLP, (c) a synergistic ratio of DOX:OLP (i.e., 50:1), or (d) an antagonistic ratio of DOX:OLP (1:2). As DOX is the more 474
DOI: 10.1021/acs.molpharmaceut.7b00843 Mol. Pharmaceutics 2018, 15, 472−485
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Figure 1. Single-drug IC50 values. IC50 values for cell lines and MCTS (OVCAR8, OV90, and HEYA8 cells), grouped as BRCA deficient or proficient, following treatment with DOX (left) or OLP (right). When the IC50 values for BRCA-deficient (i.e., mutated or methylated) cell lines are considered together and compared to values for the BRCA-proficient cell lines, no significant difference is identified (n = 3).
Initial cell seeding density was chosen such that MCTS reached a diameter of about 500 μm after 7 days. Prior to combination studies, MCTS were treated with DOX (2 μM, 1 μM, and 0.2 μM) for 72 h to determine a drug concentration sufficient to inhibit MCTS growth while still allowing MCTS to remain structurally intact, such that assessment of different molar combinations of DOX with OLP would be possible. MCTS were incubated for 72 h with single drug or drug combinations at selected molar ratios (n = 6 per treatment group for all studies). Following the 72 h treatment period, MCTS were washed with medium. Every other day thereafter, 50% of medium was replaced prior to imaging for MCTS volume determination, as described above. Statistical Analysis. Results are reported as mean of at least 3 independently conducted experiments ± standard deviation (SD). Differences between treatments were compared using one-way ANOVA’s F-test followed by post hoc analysis using Bonferroni correction, with significance assigned at p ≤ 0.05.
potent drug in the combination (Figure 1), the respective IC50 for DOX was used as the DOX concentration in the DOX, synergistic, and antagonistic ratios. For the combination groups, the OLP concentration was determined relative to the DOX. Finally, the concentration of OLP in the OLP group was determined from the highest concentration of OLP used in the combination groups. Cells were incubated with 10 μM 5ethynyl-2′-deoxyuridine (EdU) for 30 min prior to fixation in order to exclude those cells in the S phase that harbor endogenous DSBs. Cells were fixed at room temperature for 20 min in 4% paraformaldehyde/0.2% Triton X-100 (pH 8.2). Click-iT EdU Alexa Fluor 647 kit was used to stain S phase cells that had incorporated EdU. γ-H2AX foci were stained using Antiphospho Histone H2AX clone JBW301 (1:100) (Millipore, Billerica, MA) overnight at 4 °C. 4′6-Diamidino-2-phenylindole (DAPI; Invitrogen) was used for nuclear staining. Images were acquired using the Zeiss LSM 710 confocal/two-photon microscope using a 633/1.4 oil immersion objective lens. For each replicate, at least 20 nuclei were acquired using ZEN black software (Oberkochen, Germany). Samples were analyzed using ImageJ software (NIH, Bethesda, MD). MCTS Growth Studies. MCTS establishment was attempted for all cell lines using a method previously reported by our group.46 Subconfluent cells were seeded onto nonadherent 96-well round-bottomed Sumilon PrimeSurface spheroid plates (MS-9096U; Sumitomo Bakelite, Tokyo, Japan) containing the recommended complete growth medium for each cell line and incubated for 10 days at 37 °C, such that each well contained a single MCTS. Images were taken using a light microscope with a 10× objective lens (VistaVision; VWR, Mississauga, ON, Canada) connected to a digital camera (DV2B; VWR). The volume of each MCTS was determined according to a published method.46 Briefly, the 2D crosssectional area was measured using an automated image analysis macro developed for use with the ImageJ software package (Version 1.48 V), and volumes were calculated assuming spherical shape. Growth curves were fit to the Gompertz growth equation of tumor growth.46 For morphology studies, MCTS were incubated for 1 h with 1× CellMask Green plasma membrane stain (Life Technologies) in PBS and imaged using a Zeiss LSM700 confocal microscope (Carl Zeiss AG; Oberkochen, Germany) with a FITC filter (Ex 522 nm, Em 535 nm). MCTS Growth Inhibition Studies. MCTS of OVCAR8, OV-90, and HEYA8 cell lines were grown as described above.
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RESULTS Properties of the HGSOC Cell Panel. Based on three large-scale cell line characterization studies,35,36,47 nine ovarian cancer cell lines were selected to represent EOC, particularly the HGSOC subtype (S3 Table). Additional criteria were BRCA1 or BRCA2 mutation status and availability of a corresponding BRCA-proficient cell line. BRCA was chosen as the most relevant genomic mutation because, up until August 2017, OLP was only approved for treatment of patients with BRCA mutations. Although not considered to be of serous histology, SKOV3 was employed as an additional, wellcharacterized, BRCA-proficient cell line. Cell line authentication was performed on the basis of STR analysis (S1 Table). UWB1.289 is derived from a tumor of papillary serous histology, the most common form of OC.48 This cell line’s BRCA1 wild-type allele is absent as the gene is mutated within exon 11. UWB1.289 was transfected with a BRCA1 construct to restore BRCA1 function, creating the UWB1.289+BRCA1 cell line.48 PEO1 and PEO4 were derived from the ascites of the same patient before and after development of resistance to platinum-based chemotherapy, respectively.49 Subsequent analysis revealed that PEO1 had a BRCA2 loss-of-function mutation, while PEO4 showed a secondary mutation that caused functional restoration of BRCA2 protein, causing PEO4 to be more resistant to platinum or PARP inhibitors than 475
DOI: 10.1021/acs.molpharmaceut.7b00843 Mol. Pharmaceutics 2018, 15, 472−485
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Molecular Pharmaceutics
Figure 2. Combination index values for DOX and OLP at different molar ratios. Summary of CI values for the combinations of DOX and OLP following 72 h incubation in nine ovarian cancer cells lines at the indicated molar ratios required for Fa = 0.5 (A) and Fa = 0.75 (B). Each CI value was calculated and a heat map was generated as outlined in Materials and Methods on the basis of three independent IC50 experiments performed on different days for DOX, OLP, and each combination ratio (n = 3).
PEO1.50 COV362 is another cell line with BRCA1 loss-offunction mutation which was originally described as being derived from a tumor of endometrioid subtype,51 with later analysis qualifying it as HGSOC.35,36 OVCAR8 was derived from a platinum-refractory patient who showed disease progression even after receiving high doses of platinum.52 BRCA1 promoter methylation was later identified in this cell line.53 This epigenetic modification results in lack of BRCA1 mRNA expression and has been clinically reported in 8.1%54 to 13.3%55, of EOCs and in 14.8% of HGSOC cases.56 OV-90, derived from previously untreated ovarian malignant ascites, forms tumors of serous histology and expresses functional BRCA1 and BRCA2.57 HEYA8 was derived from a mouse xenograft tumor after three passages of the patient-derived cells in immunocompromised mice.58,59 The original xenografted tumor was described under the name HX-62 as a serous ovarian cystadenocarcinoma derived from peritoneal metastasis.53,60 SKOV3 was established in 1973 and is one of the most widely used ovarian cancer cell lines based on publication records.36,61 SKOV3 has been characterized as being of either serous or clear cell histotype.35 While HEYA8 expresses wild-type p53,62 all other cell lines employed here have been reported to have a p53 mutation, a common trait of HGSOC.35,36,63 BRCA status was confirmed by sequencing regions of BRCA1 and/or BRCA2 gene variants of interest for UWB1.289, UWB1.289+BRCA1, PEO1, PEO4, and COV362 cells (S4 Table). The doubling time (T2) of all cell lines was evaluated, since previous studies on a large panel of ovarian cancer cell lines have shown that doubling time significantly correlates with in vitro sensitivity to DOX and other drugs, such as taxanes and platinum derivatives.35 The cell line with the highest proliferation rate was HEYA8 (16 h), while COV362 had the lowest rate (99 h). T2 appeared to be unrelated to BRCA mutation status. For instance, the BRCA1-restored UWB1.289+BRCA1 cell line displayed a T2 that was 30% shorter than that of UWB1.289. Conversely, T2 for the
BRCA2-restored cell line PEO4 was 31% faster than for PEO1, which are cells derived from the same patient at different stages of the disease.49 Cell Monolayer Viability Studies of DOX and OLP Treatment. All cell lines were treated with DOX or OLP over 72 h for determination of IC50 values. In general, the DNA damaging agent DOX was observed to be approximately 3 orders of magnitude more cytotoxic than OLP. Consideration of the IC50 values with respect to BRCA status revealed that BRCA1 or BRCA2 deficiencies are not sufficient to predict sensitivity to OLP or DOX (Figure 1). For this analysis, cell lines were categorized as “BRCA proficient” and “BRCA deficient” according to S4 Table, and for the purpose of this analysis, OVCAR8 (light orange) was classified as “BRCA deficient”. As shown in Figure 1, cell lines that are BRCA deficient showed a very narrow distribution of IC50 values for both drugs, whereas BRCA-proficient cells showed a more broad distribution in IC50 values, especially for OLP. It may be that some of the BRCA-proficient cell lines harbor other deficiencies in HRR that make them more sensitive to DOX or OLP. Additionally, for HEYA8, the relatively low doubling time of 16 h may be contributing to its very high sensitivity to DOX as well as OLP. When the IC50 values for each group were considered together, comparison between the two groups found no statistically significant difference. However, the BRCA-proficient PEO4 (purple) and UWB1.289+BRCA1 (dark blue) were indeed two to three times more resistant to OLP than their BRCA-deficient counterparts PEO1 and UWB1.289, respectively. DOX−OLP Combination Studies. Following the assessment of single-drug cytotoxicity, DOX and OLP combination treatments at 10 different molar ratios were evaluated over a 72 h period. For each ratio, three independent experiments were performed on different days, and the molar drug concentrations required to kill 50% (Fa = 0.5) and 75% (Fa = 0.75) of cells were determined. The median-effect algorithm based on the widely used method established by Chou and Talalay was 476
DOI: 10.1021/acs.molpharmaceut.7b00843 Mol. Pharmaceutics 2018, 15, 472−485
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Molecular Pharmaceutics
effects. In summary, the data suggests that synergism of the DOX:OLP combination is drug-ratio dependent and more pronounced with higher Fa, but cannot be predicted from BRCA status or sensitivity to single-drug treatments. In order to ascertain the mechanism of action of the DOX and OLP combination, the level of DSBs induced by the combination was assessed in two cell lines with varying BRCA status (i.e., UWB1.289 and UWB1.289+BRCA) using the γH2AX assay (Figure 3). For the OLP group, the levels of
employed to calculate the CI for each ratio at both effect levels (i.e., Fa = 0.5 and Fa = 0.75) as outlined in Materials and Methods.64 The CI equation was used to generate CI values, which categorize the effect of the drug−drug combination at specific ratios as synergistic, additive or antagonistic, as described in Materials and Methods. A synergistic effect implies that the two drugs are more effective together than what would be expected from adding the effects of each drug when used separately. 43,44 In the case of OLP+DOX combinations, it is important to evaluate combination effects at different effect levels (i.e., Fa) since dose−response curves for both DOX and OLP are not linear. Consequently, the cytotoxicity of these drugs changes incrementally with dose, and the relative cytotoxicity contribution of each drug will differ with varying Fa.43,65 For cancer chemotherapy, higher effect levels of Fa = 0.8, for instance, are commonly used and more relevant for treatment, where complete cancer cell eradication is the goal.44,66 The CI results are presented in a “heat map” for each ratio of DOX:OLP at both Fa levels (Figure 2A,B). Overall, the combination of OLP and DOX resulted in synergistic effects when the molar concentrations of each drug differed from each other to a greater extent, such as DOX:OLP at 1:100, 1:50, 50:1, and 100:1. Taking this into consideration, we can extrapolate from the data that there needs to be a minimum difference in the molar ratios of these 2 drugs in order to observe synergism across the cell lines (based on the average CI values). As it is not feasible to determine the CI values for every possible permutation of molar ratios of DOX:OLP, we have identified from the data presented here that the magnitude of the molar ratio should be greater than 1:10 or 10:1. We have observed that once these ratios have been achieved, there was very little difference, with respect to CI values between 1:100 and 1:50 (CI values of 0.81 vs 0.83 respectively) and 50:1 and 100:1 (0.78 vs 0.74 respectively). More equimolar ratios of 1:2, 1:5, and 1:10 as well as the inverse resulted in an additive effect. For Fa = 0.75, a trend toward more synergy was generally observed in comparison to Fa = 0.5, with the exception of the 50:1 and 100:1 ratios (Figure 2B). Notably, OLP concentrations required when in combination with DOX were 10- to 1000-fold below the IC50 values of OLP as a single agent. Neither BRCA status nor sensitivity to DOX or OLP singledrug treatment appeared to be predictive of DOX−OLP synergism. For example, although the BRCA1-deficient cell line UWB1.289 was more sensitive to single-drug treatments by a factor of three to four in comparison to UWB1.289+BRCA1, the CI values for UWB1.289 were lower than for UWB1.289+BRCA1 at certain ratios (1:100, 1:50, 10:1, 50:1, and 100:1) but not others (1:10, 1:5, 1:2, 2:1, and 5:1). CI values for the BRCA2-deficient cell line PEO1 were generally lower than for its BRCA2-proficient counterpart PEO4, except for the 1:50 and 100:1 ratios. Furthermore, while HEYA8 and PEO1 are both quite sensitive to DOX, most DOX:OLP ratios resulted in synergism in the former cell line and an additive effect or antagonism in the latter. Conversely, DOX−OLP combinations for UWB1.289+BRCA1, the most DOX-resistant cell line, were found to be very effective, especially at Fa = 0.75. As was the case with DOX, single-drug OLP sensitivity did not correlate with combination outcomes. While PEO4 showed the strongest resistance to single-drug OLP as well as overall antagonistic effects in response to DOX−OLP combinations, OVCAR8 was highly sensitive to single-drug OLP but the DOX−OLP combination did not yield particularly synergistic
Figure 3. γH2AX assay following the treatment of cells with DOX:OLP combination. The number of γH2AX foci per cell was determined in UWB1.289 and UWB1.289+BRCA cells (A). Representative images for UWB1.289 (B) and UWB1.289+BRCA (C) cells treated with DOX, OLP, 50:1 and 1:2. DAPI channel indicates nuclear staining, with the green channel (i.e., γH2AX) indicating the formation of DSBs. * = relative to untreated control, ∧ = relative to 50:1 group, and # = relative to DOX (n = 3).
γH2AX foci were not significantly different from the levels observed in the untreated controls for both of the cell lines (p > 0.05). According to the CI values (Figure 2), the 50:1 ratio was synergistic in UWB1.289 and UWB1.289+BRCA cells. This data was supported by the γH2AX assay, where 50:1 resulted in significantly more (p < 0.001) DSBs compared to OLP (approximately 10- and 13-fold increases for UWB1.289 and UWB1.289+BRCA, respectively) and DOX (approximately 50% and 65% increases for UWB1.289 and UWB1.289+BRCA, respectively) as monotherapies. In the UWB1.289+BRCA cell line, there was no significant difference (p > 0.05) between the 50:1 and 1:2 DOX:OLP ratios. Again, this data is supported by the CI values, where both displayed strong synergy in this cell line (CI values of 0.36 and 0.70 respectively). For the UWB cell line, the 50:1 ratio had a significantly higher amount of DSBs 477
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shapes and organized in an irregular fashion. Finally, OV-90 MCTS showed the most heterogeneous structure, with areas of very tightly packed cells interspersed with voids in other areas. Single OV-90 cells appeared to be more uniform than OVCAR8 cells in MCTS, but were of various sizes, in contrast to HEYA8. MCTS Growth and DOX Sensitivity Studies. The growth patterns of OVCAR8, OV-90, and HEYA8MCTS were assessed for 13 days following seeding of 1000, 3000, and 5000 cells per well. Growth curves were fit to the Gompertz equation for tumor growth (Figure 5A,C,E).38 All MCTS showed a characteristically sigmoidal growth pattern, which is typical of in vivo tumor growth.38,69 The instantaneous growth rate was calculated based on the Gompertz growth equation parameters, which enables comparison of growth rates between cell lines (S5 Table). Akin to T2 in monolayer cell studies, HEYA8 showed the fastest instantaneous growth rate of the cell lines assessed, followed by OVCAR8 and OV90. In comparison to OVCAR8 and HEYA8MCTS, greater size variations were observed among OV90 MCTS within a single treatment group. Subsequent studies were conducted with initial cell seeding numbers of 3000 cells/well for OVCAR8 and OV-90, and 1000 cells/well for HEYA8. This yielded a diameter of about 500 μm after 7 days of growth for all MCTS. MCTS were used to determine the DOX concentration needed for subsequent DOX:OLP combination studies (Figure 5B,D,F). Specifically, the goal was to identify a DOX concentration that resulted in MCTS growth inhibition while preserving the structural integrity of the spheroids. For this purpose, MCTS were treated with DOX concentrations of 2 μM, 1 μM, and 0.2 μM for 72 h prior to washing and imagebased volume determination. Given the heightened resistance of MCTS to drug treatments, these concentrations are 2- to 200-fold greater than the IC50 for DOX in cell monolayers (Figure 1).38 OVCAR8MCTS proved to be the most resistant to treatment, where only concentrations of 1 and 2 μM significantly inhibited MCTS growth starting on day 5 after treatment initiation (Figure 5B). At the concentrations evaluated, all OVCAR8MCTS remained structurally intact. OV-90 MCTS, on the other hand, lost structural integrity immediately following treatment with the higher doses, but remained intact with a 0.2 μM dose, which induced significant growth inhibition (Figure 5D). For HEYA8MCTS, drug effect was immediately observed at all doses (Figure 5F). At the highest DOX dose of 2 μM, structural integrity of the MCTS was lost 5 days after initiation of treatment. With 1 μM DOX, spheroid integrity was maintained, but the MCTS decreased to small nodules. With a 0.2 μM dose, the remaining nodules were larger and structurally sound, which was more desirable for the purposes of the combination studies. MCTS DOX and OLP Combination Studies. Combination studies were conducted using a fixed dose of DOX that resulted in a significant reduction in MCTS volume while maintaining their structural integrity. For OVCAR8, 1 μM DOX was used, while 0.2 μM DOX was selected for OV-90 and HEYA8MCTS. Corresponding concentrations of OLP that resulted in molar ratios of 1:2 and 1:100 as well as 2:1 and 100:1 DOX:OLP were employed as treatments for 72 h prior to washing and imaging of the MCTS over a 10 day period. When compared to the IC50 values for DOX as a single agent in cell monolayers, the DOX concentrations required to induce growth inhibition in the MCTS combination studies were 20fold higher for OVCAR8MCTS (0.05 μM vs 1 μM) and
relative to 1:2, which also displayed significantly lower levels of γH2AX foci relative to DOX alone (p < 0.001). MCTS Model Development and Morphology Studies. For further assessment of the DOX−OLP combination effects in 3D cell culture, growth of each cell line as MCTS was attempted using a previously established liquid-overlay method.38 3D cell cultures based on MCTS with diameters of around 500 μm can be used as in vitro models of microscopic ovarian cancer residual disease, which occurs in patients after optimal cytoreductive surgery.42 For establishment of MCTS, cells were seeded onto nonadherent 96-well plates and growth was assessed over 10 days using a light microscope (S6 Figure). In order to qualify as a suitable 3D cell culture model, cell lines were required to form a coherent spheroid structure that grows in volume over the 10-day time frame. These criteria ensured that the MCTS differed fundamentally from simple cell aggregates that form shortly after incubation (1−2 days). All cell lines were evaluated for their ability to form MCTS, finding that only OVCAR8, OV-90, and HEYA8 were capable of forming MCTS that complied with the required criteria outlined above. SKOV3 and PEO4 initially appeared to form MCTS, but these were not sufficiently stable to withstand the required experimental manipulation. Additionally, SKOV3 aggregates did not increase in volume over time. The remaining cell lines formed a layer of cells at the bottom of each well, failing to form MCTS. Interestingly, unexpected differences were observed within each of the two cell line pairs. Despite the fact that the PEO1 and PEO4 cell lines were derived from the same patient, they resulted in very distinct morphologies in vitro. PEO1 cells appeared not to aggregate and simply accumulated at the bottom of the well, whereas PEO4 cells aggregated and grew in volume, but were not stable enough to be used as MCTS models. In contrast, the UWB1.289 and UWB1.289+BRCA1 cell line pair showed very similar structure to each other, with monolayers outgrowing the perimeter of the microscope objective. Cellular organization was assessed for OVCAR8, OV-90, and HEYA8MCTS using confocal microscopy following staining of cell membranes with CellMask Green fluorescent stain (Figure 4). Cellular organization is an important characteristic of
Figure 4. Representative confocal images of cellular organization of OVCAR8, OV-90, and HEYA8MCTS. MCTS were incubated for 1 h with 1× CellMask Green plasma membrane stain in PBS, and the fluorescent stain was detected using confocal microscopy. Scale bars represent 100 μm.
MCTS, as cellular packing density influences the diffusion of drugs into the MCTS core and significantly affects treatment outcomes.67,68 The three cell lines displayed distinct patterns of cellular organization upon MCTS formation. HEYA8MCTS showed a densely packed, homogeneous pattern of similarly sized cells throughout the entire structure. OVCAR8MCTS were also densely packed, but cells were of different sizes and 478
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Figure 5. MCTS growth curves and DOX growth inhibition. MCTS volume (left panels) and growth inhibition upon DOX treatment (right panels) for OVCAR8 (A, B), OV-90 (C, D), and HEYA8 (E, F) ovarian cancer cell lines. MCTS volume as a function of time is shown on a log plot fit to the Gompertz equation of tumor growth following seeding of 1000, 3000, and 5000 cells per well (A, C, E). The dotted line indicates a diameter of 500 μm, which was chosen as baseline for further studies. MCTS growth inhibition was assessed following treatment with DOX for a 72 h period (B, D, F). Every other day, 50% of the medium was replaced prior to imaging and determination of MCTS volume (n = 3).
HEYA8MCTS (0.01 μM vs 0.2 μM), but were similar for OV90 MCTS (0.15 μM vs 0.2 μM). The OLP concentration at the highest 1:100 DOX:OLP ratio was about 7-fold higher than the IC50 value of single-drug OLP in OVCAR8 monolayers (14 μM vs 100 μM) and significantly lower for HEYA8 (12.7 μM vs 2 μM) and OV-90 (54 μM vs 2 μM). Therefore, OLP likely plays a role as a sensitizer to DOX treatment rather than a particularly cytotoxic agent, an argument supported by the fact that MCTS growth was not influenced at these concentrations when using OLP alone (S7 Figure). In the MCTS, DOX:OLP combination treatment appeared to be more effective in comparison to DOX alone for OV-90 (1:100 on days 7 and 9, 100:1 on day 9) and HEYA8 (1:100 and 100:1 on day 7, and all ratios on day 9), but only in one instance for OVCAR8 (100:1, day 7) (Figure 6). Analogous to experiments in cell monolayers, higher ratios (1:100, 100:1) appeared to be more effective than lower ones (1:2, 2:1) in HEYA8MCTS (Figure 6B). For OV-90 MCTS, a very heterogeneous pattern was observed, where some MCTS
remained intact while others lost integrity within the same treatment group. This yielded a high level of variation in the resulting data, which perhaps prevented the detection of statistically significant differences between ratios. However, a trend toward greater efficacy in response to treatment with higher ratios (1:100 and 100:1) was still observed when compared to the lower ratios (1:2 and 2:1) (Figure 6C). A bar graph representing the MCTS volumes at T = 9 days is represented in S8 Figure. Collectively, the cytotoxic effects observed for DOX:OLP in comparison to DOX alone were comparable to what was observed in monolayer CI studies, yet appeared less pronounced in the more resistant MCTS model systems. Importantly, higher ratios (1:100 and 100:1) yielded greater efficacy than lower ratios (1:2 and 2:1) in HEYA8 and OV-90 MCTS, corroborating the trend observed in the monolayer CI studies. 479
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chemoresistant disease reports higher mutational burden and structural variants in recurrent disease than matched pretreatment tumors as a result of adaptation of the tumor genome in response to previous cycles of chemotherapy.70 In patients with originally BRCA-deficient disease, reversion mutations that restore BRCA function have been observed upon recurrence, although mutations do remain in a larger subset of patients with recurrent disease. 49,70 For this highly variable patient population, combination therapy using a DNA damaging agent and a molecularly targeted agent that can exploit HRR deficiencies appears to be a reasonable approach given the current state of knowledge.71 The combination of DOX and the PARP inhibitor, OLP (Lynparza), is particularly promising in this setting, since DOX lacks cross-resistance with platinum, and OLP is a clinically advanced PARP inhibitor.10−12 This report aimed to conduct a thorough preclinical evaluation of the conditions under which DOX and OLP can achieve synergistically beneficial outcomes in 2D and 3D in vitro cell model systems representative of recurrent and/or residual HGSOC. The nine ovarian cancer cell lines employed in this study reflect some of the challenges associated with the high heterogeneity in HGSOC in terms of BRCA status, proliferation rate, and cellular organization when in 3D cell culture. Consequently, a wide range of sensitivities to DOX and OLP administered individually or in combination were observed. Interestingly, cytotoxicity was not reliably predictable based on known cell line characteristics, such as BRCA status. Others studies have made similar observations.35,70 As a general observation across all cell lines, it was found that the combination effect of DOX:OLP in cell monolayers was predominantly additive for more equimolar drug ratios and synergistic when there were large differences in molar concentrations, such as 1:100, 1:50 or 50:1, and 100:1 (Figure 2). Given the inherent in vivo toxicity associated with high doses of DOX, it may be pertinent to use a DOX:OLP ratio in vivo that minimizes the dose of DOX, while still allowing for a synergistic response (i.e., 1:100 or 1:50). The data obtained also provides insight into the tremendous variability in drug sensitivity that is possible in cell lines with specific characteristics. For example, in cell monolayer studies with the OV-90 cell line, it was found that, despite having wild-type BRCA status and relatively high resistance to both drugs as single agents, synergy was observed for nearly all ratios of the DOX− OLP combination. OV-90 was also the only cell line in this study that was derived from ascites obtained from a patient with previously untreated HGSOC. Analysis of BRCA1 and BRCA2 expression in the panel of OC cell lines by Western blot revealed that OV-90 had a lower level of expression of BRCA1 relative to other BRCA1 expressing cell lines (i.e., OVCAR8, HeyA8 and UWB1.289+BRCA) (data not yet published). This low level of BRCA1 expression may account for the unexpectedly high levels of response observed with the DOX:OLP combination (Figure 2). The UWB1.289 and UWB1.289+BRCA1 cell line pair is illustrative of the restoration of BRCA1 status that can occur, for instance, following adaptation to chemotherapy. In this study, the BRCA1-restored cells were found to be 3- to 4-fold more resistant to both DOX and OLP monotherapy as compared to the parental, BRCA1-deficient cell line (Figure 1). However, the DOX−OLP combination appeared to be more synergistic in the BRCA1-restored cell line UWB1.289+BRCA1 (Figure 2). The PEO1 and PEO4 cell pair, derived from the same
Figure 6. MCTS combination treatment. Growth inhibition studies for OVCAR8 (A), HEYA8 (B), and OV-90 (C) MCTS in response to treatment with DOX:OLP at select molar ratios in comparison to DOX alone. The initial DOX concentration was chosen as described above to be 1 μM for OVCAR8MCTS and 0.2 μM for OV-90 and HEYA8MCTS. OLP concentrations were calculated for each molar ratio based on each DOX concentration. Following the 72 h treatment period, 50% of medium was replaced every other day prior to imaging and determination of MCTS volume. A # symbol represents significant differences between DOX and each ratio (#, p ≤ 0.05; ###, p ≤ 0.001) (n = 3).
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DISCUSSION The dismal survival rates for OC patients that have acquired resistance to platinum-based therapy present a demand for innovative treatment options. Recurrent OC is particularly challenging to treat, since the tumor genome seems to lack characteristic, exploitable drug targets. A recently published whole-genome characterization of 92 OC patients with 480
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nodule. None of the BRCA-mutated cell lines formed usable MCTS. It was observed that the effective dose of DOX in OV90 MCTS was similar to the IC50 value obtained for DOX in cell monolayers. In contrast, the effective concentrations of DOX in OVCAR8 and HEYA8MCTS were 20-fold greater than the respective IC50 values for the drug in cell monolayers. The sensitivity of the OV-90 MCTS to DOX may be explained by the heterogeneous structure within these spheroids as well as their loss of tumor spheroid integrity during treatment (Figure 4). The differences observed in the CI values obtained for the DOX:OLP combinations in cell monolayers did translate, to some extent, to comparable differences in the more treatment resistant MCTS. For the OVCAR8MCTS, the drug combination of DOX:OLP at all ratios tested did not appear to be substantially more effective in reducing MCTS volume than treatment with DOX alone, which is analogous to findings in the monolayer studies where an overall additive effect was observed for both Fa = 0.5 and Fa = 0.75. For HEYA8, strong synergy was observed at high molar ratios of DOX:OLP (1:100, 100:1) in both 2D monolayers and the 3D MCTS, and a similar trend was observed for OV-90 (Figure 6). Thus, results obtained from the monolayer CI, γH2AX, and MCTS growth inhibition studies successfully demonstrate the importance of selecting the appropriate ratio for a drug combination, and the significant impact that the optimal ratio can have in terms of enhancing the growth inhibitory effect of the two drugs. Collectively, the observations obtained in these studies illustrate that no singular characteristic of ovarian cancer cells can predict their sensitivity to a therapeutic agent. Cellular response to chemotherapy and/or molecular therapy is complex and is determined by an aggregate of the genetic, morphological, and physiological traits of a specific tumor. In this study, it was expected that the combination of DOX and OLP would result in overall synergistic effects, especially in the BRCA-deficient ovarian cancer cell lines. However, it was generally found that BRCA deficiency alone was not entirely predictive of a cell line’s sensitivity to the DOX:OLP combination. Others have reported that other known genomic mutations such as PIK3CA, ARID1A, or KRAS are also not sufficient to solely predict drug sensitivity.35,36,48 Further, some BRCA-proficient cell lines used here were found to be relatively sensitive to the combination. These cell lines may harbor other defects in HRR or related repair pathways that make them susceptible to PARP inhibition and DNA damage. As such, the evaluation of genomic markers predictive of response to treatment with DOX and OLP, alone or in combination, is still warranted. The full potential of DOX and OLP may have not yet been achieved in the clinic due to lack of optimized combination strategies. The identification and administration of these drugs at synergistic ratios may offer a solution to fully exploit this promising combination. In order to facilitate the controlled delivery of two drugs at such specific ratios, it may be prudent to formulate both drugs at a specific molar ratio into a nanoparticle. Optimization of such a system would allow for the maintenance of the synergistic DOX:OLP ratio at the tumor site. For example, CPX-351 (VYXEOS; Jazz Pharmaceuticals) is a liposomal formulation of cytarabine (CYT) and daunorubicin (DAN). CPX-351 is formulated at a molar ratio of CYT:DAN to 5:1. CPX-351 demonstrated superior outcomes in a phase III multicenter clinical trial in high risk AML patients.73 The preceding in vitro studies that assessed and determined optimal combination ratios of CYT:DAN using the Chou and Talalay
patient before and after chemotherapy, respectively, showed a more expected behavior, where restoration of BRCA2 in response to treatment yielded lower sensitivity to both single drugs, and a trend toward antagonism for the drug combination (Figure 2). The disparity in cell doubling time between the UWB1.289 and UWB1.289+BRCA1 cells (54 h vs 38 h) and the difference in the morphologies of the PEO1 and PEO4MCTS (S 6 Figure) suggest that there may be other differences beyond BRCA status between these cell lines. This calls into question the utility of these cell “pairs” for evaluation of effects that are assumed to be solely related to BRCA status. Furthermore, variations in proliferation rates have been identified as the most relevant associative factor to chemotherapy response in ovarian cancer cell lines in comparison to the expression of oncogenic protein markers,35 in that higher rates of proliferation render cells more susceptible to chemotherapeutics that target cell cycle mechanisms. This effect was indeed observed in the present study. Despite being BRCA wild-type, the HEYA8 cell line was found to be most sensitive to single-drug treatment with both DOX and OLP, possibly due to its short T2 of 16 h. As well, combination treatment resulted in overall synergistic effects in this cell line. Correspondingly, the BRCA1-deficient COV362 cell line, which had the slowest proliferation rate (T2 = 99 h), appeared to be relatively resistant to both DOX and the combination treatment. Determination of the γH2AX foci is a reliable biomarker for determining DSBs.72 A “synergistic” and “antagonistic” ratio was investigated for its ability to form γH2AX foci in UWB1.289 and UWB1.289+BRCA cells. It was shown that the combination of DOX and OLP at the synergistic DOX:OLP molar ratio of 50:1 resulted in significantly more γH2AX foci per cell than DOX and OLP as monotherapies in both cell lines (Figure 3). At the antagonistic 1:2 (DOX:OLP) molar ratio there were significantly fewer γH2AX foci in the BRCA-deficient cell line, UWB1.289, compared to both 50:1 and DOX treatments. This is interesting as the modus operandi for drug combination trials in the clinic tends to be the administration of both drugs at their MTD. Using this study as an example, the administration of two drugs at molar ratios that have not been optimized may actually be detrimental to the patient and offer a worse outcome compared to a monotherapy (in this case, DOX). It was surprising that OLP as a monotherapy did not induce a significant amount of γH2AX foci in both cell lines tested. This was particularly interesting, as the mechanism of action of OLP has been shown to be the induction of SSBs in DNA (due to stalling of PARP at the replication fork), which consequently leads to DSBs.22 These results may be explained by the fact that relative to DOX the IC50 of OLP is relatively high (several hundred fold difference between the two drugs for both of the cell lines). For this assay, the concentrations of OLP used may not have been sufficient to induce a significant response. In general, the results from the monolayer viability combination study (Figure 2) support those of the γH2AX assay (Figure 3) and perpetuate the necessity for the delivery of a specific molar ratio of the drug combination to the tumor site. Evaluation of drug sensitivity in MCTS can provide additional important insight, as the 3D structure enables evaluation of effects of tissue microenvironment, cellular organization, and drug penetration in addition to cytotoxicity. However, these effects depend on the nature of the MCTS and only manifest in MCTS that present as a coherent spheroid 481
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method reported that, while synergism was generally found for the great majority of cell lines studied, antagonism was prevalent in a small subset of cell lines.74 It is also noteworthy that the bolus dosing regimen employed in this study is unlike that used clinically, which includes twicedaily administration of OLP during chemotherapy and beyond as maintenance therapy. In future in vivo preclinical evaluations, it will be interesting and clinically important to determine the impact of OLP maintenance therapy following DOX−OLP combination treatment at synergistic ratios, and the impact of the combination dosing schedule, on treatment efficacy. The main findings from this study can be utilized by researchers in the drug combination field. The systematic approach highlighted in this study to treat cells with a wide range of molar ratios of drugs is a useful tool to identify synergistic combinations that may have performed poorly in clinical trials due to being administered at their respective MTDs. In addition we have determined the growth parameters of three OC cell lines (OV-90, OVCAR8, HEYA8, Figure 5) grown as MCTS. These can be used as platforms for assessing novel drug combinations with the hypothesis that MCTS will better recapitulate the microenvironment in vivo. Finally, we have also shown that certain DOX:OLP combinations are highly effective in inhibiting OC cell growth. These combinations could be applied to other BRCA-deficient cancer types (such as breast cancer).
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS C.A. and M.P.-M. acknowledge funding support from the U.S. Army D.O.D. S.E. was funded by the NSERC CREATE Biointerfaces Training Program and held an Ontario Trillium Scholarship. Y.-T.S. is funded by the Leslie Dan Faculty of Pharmacy Dean’s Fund. C.A. acknowledges GlaxoSmithKline for an endowed chair in Pharmaceutics and Drug Delivery. The authors acknowledge Dr. Tara Paton from the Genetic Analysis Facility at The Hospital for Sick Children, for providing technical expertise in the STR analysis and BRCA sequencing presented herein, and Dr. Tara Spence for critical review of the manuscript.
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(1) Jayson, G. C.; Kohn, E. C.; Kitchener, H. C.; Ledermann, J. A. Ovarian cancer. Lancet 2014, 384 (9951), 1376−88. (2) Berns, E. M.; Bowtell, D. D. The changing view of high-grade serous ovarian cancer. Cancer Res. 2012, 72 (11), 2701−4. (3) Vargas, A. N. Natural history of ovarian cancer. ecancer 2014, 8, 465. (4) Colombo, P. E.; Fabbro, M.; Theillet, C.; Bibeau, F.; Rouanet, P.; Ray-Coquard, I. Sensitivity and resistance to treatment in the primary management of epithelial ovarian cancer. Critical reviews in oncology/ hematology 2014, 89 (2), 207−16. (5) Ahmed, A. A.; Etemadmoghadam, D.; Temple, J.; Lynch, A. G.; Riad, M.; Sharma, R.; Stewart, C.; Fereday, S.; Caldas, C.; DeFazio, A.; Bowtell, D.; Brenton, J. D. Driver mutations in TP53 are ubiquitous in high grade serous carcinoma of the ovary. J. Pathol 2010, 221 (1), 49− 56. (6) Syrios, J.; Banerjee, S.; Kaye, S. B. Advanced Epithelial Ovarian Cancer: From Standard Chemotherapy to Promising Molecular Pathway Targets - Where Are we Now? Anticancer Res. 2014, 34 (5), 2069−2077. (7) Gonzalez-Martin, A.; Sanchez-Lorenzo, L.; Bratos, R.; Marquez, R.; Chiva, L. First-line and maintenance therapy for ovarian cancer: current status and future directions. Drugs 2014, 74 (8), 879−89. (8) Schmid, B. C.; Oehler, M. K. New perspectives in ovarian cancer treatment. Maturitas 2014, 77 (2), 128−36. (9) Staropoli, N.; Ciliberto, D.; Botta, C.; Fiorillo, L.; Grimaldi, A.; Lama, S.; Caraglia, M.; Salvino, A.; Tassone, P.; Tagliaferri, P. Pegylated liposomal doxorubicin in the management of ovarian cancer: a systematic review and metaanalysis of randomized trials. Cancer Biol. Ther. 2014, 15 (6), 707−20. (10) Safra, T.; Borgato, L.; Nicoletto, M. O.; Rolnitzky, L.; PellesAvraham, S.; Geva, R.; Donach, M. E.; Curtin, J.; Novetsky, A.; Grenader, T.; Lai, W. C. V.; Gabizon, A.; Boyd, L.; Muggia, F. BRCA Mutation Status and Determinant of Outcome in Women with Recurrent Epithelial Ovarian Cancer Treated with Pegylated Liposomal Doxorubicin. Mol. Cancer Ther. 2011, 10 (10), 2000−2007. (11) Adams, S. F.; Marsh, E. B.; Elmasri, W.; Halberstadt, S.; VanDecker, S.; Sammel, M. D.; Bradbury, A. R.; Daly, M.; Karlan, B.; Rubin, S. C. A high response rate to liposomal doxorubicin is seen among women with BRCA mutations treated for recurrent epithelial ovarian cancer. Gynecol. Oncol. 2011, 123 (3), 486−491. (12) Deeks, E. D. Olaparib: first global approval. Drugs 2015, 75 (2), 231−40. (13) AstraZeneca LYNPARZA® (olaparib) receives additional FDA approval in the US for ovarian cancer. https://www.astrazeneca-us. com/media/press-releases/2017/lynparza-olaparib-receivesadditional-fda-approval-in-the-us-for-ovarian-cancer-08172017.html (08/17/2017). (14) Scott, L. J. Niraparib: First Global Approval. Drugs 2017, 77 (9), 1029−1034.
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CONCLUSIONS In summary, this report highlights the challenges that tumor heterogeneity poses to the effective treatment of recurrent HGSOC. Cell line characteristics such as BRCA status and single-drug sensitivity were not predictive of response to the DOX−OLP combination. Overall, the effectiveness of the DOX:OLP combination therapy in monolayers and MCTS was found to be ratio-dependent, such that more equimolar ratios (1:2 and 2:1 DOX:OLP) resulted in additive (or antagonistic) effects, while a greater level of synergy was observed with more extreme ratios (1:100 and 100:1 DOX:OLP), especially at the higher effect level of 0.75. Future studies by our group will examine the impact of treatment with the synergistic molar ratios identified in this study, with or without OLP maintenance therapy, as well as dosing regimens, on efficacy of DOX and OLP in relevant animal models of HGSOC.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b00843. STR profiles, primer sequences, HGSOC characteristics, BRCA1 or BRCA2 status, Gompertz growth equation parameters, MCTS images, OLP treatment effects, and MCTS combination treatment (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Christine Allen: 0000-0002-4916-3965 Author Contributions †
S.E. and J.C.E. made equal contribution. 482
DOI: 10.1021/acs.molpharmaceut.7b00843 Mol. Pharmaceutics 2018, 15, 472−485
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