Nanomedicines and Combination Therapy of Doxorubicin and Olaparib for Treatment of Ovarian Cancer

Ovarian cancer is the fourth leading cause of death in women of developed countries, with dismal survival improvements achieved in the past three decades. Specifically, current chemotherapy strategies for second-line treatment of relapsed ovarian cancer are unable to effectively treat recurrent disease. This thesis aims to improve the therapeutic outcome associated with recurrent ovarian cancer by (1) creating a 3D cell screening method as an in vitro model of the disease (2) developing a nanomedicine of doxorubicin (DOX) that is more efficacious than PEGylated liposomal doxorubicin (PLD / Doxil ® ) and (3) evaluating additional strategies to enhance treatment efficacy such as mild hyperthermia (MHT) and combination therapy with inhibitors of the poly(ADP-ribose) polymerase enzyme family (PARP). Overall, this work demonstrates the use of 3D multicellular tumor spheroids (MCTS) as an in vitro drug testing platform which more closely reflects the clinical presentation of recurrent ovarian cancer relative to traditional monolayer cultures. With the use of this technology, it was found that tissue penetration of drug is not only an issue for large tumors, but also for invisible, microscopic lesions that result from metastasis or remain following cytoreductive surgery. A novel block-copolymer micelle formulation for DOX was developed and fulfilled the goal of iicontrolling drug release while enhancing intratumoral distribution and MCTS bioavailability of DOX, which resulted in a significant improvement in growth inhibition, relative to PLD. MHT appeared to enhance drug accumulation in MCTS in the short term, but not after 48 h of drug treatment. Drug combination studies of DOX together with the PARP inhibitor, olaparib (OLP, Lynparza ® ) were conducted in 2D monolayers and 3D MCTS. In these studies, the effectiveness of the DOX:OLP combination therapy in monolayers and MCTS was found to be ratio dependent such that equimolar ratios resulted in an additive effect, while a greater level of synergy was observed with more extreme ratios. The synergistic effect observed bears promise for future evaluation in vivo which warrants an appropriate delivery method to ensure that the determined molar ratios of both drugs accumulate at the tumor as such, despite differences in the pharmacokinetic profile of each drug, respectively.Ph.D

Characterization of micelle morphology and size.

<p>a) Transmission electron micrograph (Scale bar in represents 100 nm) and b) size distribution of BCM+DTX as determined by dynamic light scattering at 37°C.</p

3-D cultures as intermediary between 2-D cultures and animal models.

<p>Intermediate in complexity, 3-D cultures permit the systematic, high-throughput assessment of formulation properties in a controlled environment that approximates important properties of <i>in vivo</i> tumors in the absence of complex parameters which may confound data interpretation.</p

Spatial distribution of proliferating cells in spheroids.

<p>Ki67 positive signal distribution relative to radial position in a) HeLa and b) HT29 MCTS as a percent of total positive stain, n = 6.</p

Inhibition of spheroid growth.

<p>a) Sequential images of the same HeLa and HT29 spheroids following treatment with BCM+DTX at a concentration of 20 ng/mL. Bars represent 100 µm. Growth inhibition of HeLa (b,c) and HT29 (d,e) MCTS by BCM+DTX and <b>Taxotere</b>® at concentrations of 2, 20 and 200 ng/mL. Cells were re-treated after two weeks (arrow). Box represents expanded region of plots b) and d). Data is expressed as the mean volume of six spheroids (n = 6) ± SD. “*” represents a significant difference between BCM 20 and TAX 20, p<0.05.</p

Clonogenic potential of cells following treatment.

<p>Clonogenic survival of HeLa and HT29 cells following 24 h treatment with 20 ng/mL of BCM+DTX or Taxotere® as a) monolayers, b) disaggregated spheroids and c) intact spheroids.</p

Histological assessment of spheroid microenvironment.

<p>HeLa (a–c) and HT29 (d–f) MCTS cross-sections stained with H&E (a, d), Ki67 proliferation marker (b, e) and EF5 (c, f), a marker of hypoxia. Scale bars represent 100 µm. g) Properties of the spheroid microenvironment and their spatial distribution. “++”, “+”, and “–”, indicate high, intermediate and low levels of the corresponding feature, respectively.</p

Multicellular Tumor Spheroids for Evaluation of Cytotoxicity and Tumor Growth Inhibitory Effects of Nanomedicines <i>In Vitro</i>: A Comparison of Docetaxel-Loaded Block Copolymer Micelles and Taxotere®

<div><p>While 3-D tissue models have received increasing attention over the past several decades in the development of traditional anti-cancer therapies, their potential application for the evaluation of advanced drug delivery systems such as nanomedicines has been largely overlooked. In particular, new insight into drug resistance associated with the 3-D tumor microenvironment has called into question the validity of 2-D models for prediction of <i>in vivo</i> anti-tumor activity. In this work, a series of complementary assays was established for evaluating the <i>in vitro</i> efficacy of docetaxel (DTX) -loaded block copolymer micelles (BCM+DTX) and Taxotere® in 3-D multicellular tumor spheroid (MCTS) cultures. Spheroids were found to be significantly more resistant to treatment than monolayer cultures in a cell line dependent manner. Limitations in treatment efficacy were attributed to mechanisms of resistance associated with properties of the spheroid microenvironment. DTX-loaded micelles demonstrated greater therapeutic effect in both monolayer and spheroid cultures in comparison to Taxotere®. Overall, this work demonstrates the use of spheroids as a viable platform for the evaluation of nanomedicines in conditions which more closely reflect the <i>in vivo</i> tumor microenvironment relative to traditional monolayer cultures. By adaptation of traditional cell-based assays, spheroids have the potential to serve as intermediaries between traditional <i>in vitro</i> and <i>in vivo</i> models for high-throughput assessment of therapeutic candidates.</p></div

Spheroid packing density and growth.

<p>a) Cells per HeLa and HT29 spheroid of given volume, n = 12. b) Growth of HeLa and HT29 spheroids, n = 6. Data was fit using the Gompertz equation for tumor growth. The dashed lines indicate spheroid properties used in the studies.</p