Access Type

Open Access Dissertation

Date of Award

January 2021

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Pharmaceutical Sciences

First Advisor

Arun K. Iyer

Abstract

TARGETED COMBINATION THERAPY OF TRIPLE-NEGATIVE BREAST CANCERby KETKI BHISE May 2021 Advisor: Dr. Arun Iyer Major: Pharmaceutical Sciences Degree: Doctor of Philosophy Triple Negative Breast Cancer (TNBC) accounts for 10-20% of the total breast carcinoma cases. There is no FDA-approved targeted therapy for TNBC due to lack estrogen, progesterone and HER-2. We have identified the role of a hypoxia biomarker, carbonic anhydrase IX (CAIX) in proliferation and metastasis of TNBC. Based on this observation, we have developed CAIX-targeted Doxorubicin (Dox) prodrug, abbreviated as DoxAtz. To improve the delivery of DoxAtz to the tumor, we developed long circulating liposomes (LipoDoxAtz) and extensively characterized the liposomes before testing for their anti-cancer activity. We studied the effect of the DoxAtz drug and its liposomal formulation, LipoDoxAtz in arresting tumor growth in Doxorubicin-resistant cellular and animal model. We screened wild type and Doxorubicin-resistant phenotypes of two TNBC cell lines for studying the activity of these treatments in suppression of HIF-1a in vitro. We observed that compared to culturing the cells under physiological oxygen concentration, hypoxic culture conditions were best suited for studying the anti-cancer efficacy of individual therapies. LipoDoxAtz had the advantage of sustained release of the drug, which was reflected in its enhanced cytotoxicity and cellular uptake. Lastly, we tested the anti-tumor efficacy of different monotherapies in DoxR-MDA-MB-231 in athymic nu/nu mice. Although there was 2.5-fold improvement in tumor suppression observed with LipoDoxAtz treatment compared to the untreated control group at the half-time point of the experiment, the tumors were in the log phase of rapid cell division, and hence, the tumors outgrew the treatments in the due course of time. Finally, we studied the effect of combination therapies in tumor suppression in 4T1 syngeneic Balb/c tumors. We tested the efficacies of three immune checkpoint inhibitors, namely blockades for CD73, CTLA4 and PD-L1 in combination with LipoDoxAtz to assess reduction in tumor growth. Owing to the possibility that antiPD-L1 and antiCTLA4 act at different stages of the cancer immunity cycle, with CTLA4 blockade at the earlier stage than PD-L1 blockage, the tumors treated with LipoDoxAtz and CTLA4 combination showed a significant reduction in growth compared to the combination with PD-L1. CD73 blockade had no effect in arresting tumor growth. Our data with combination of LipoDoxAtz + CTLA4 showed a significant tumor regression, increased survival and no toxicity to the overall health of mice. Moreover, mechanistic studies indicated upregulation of proteins promoting immunogenic cell death, namely HMGB1 and calreticulin with the combination group. Higher infiltration of CD8+ Tc cells and secretion of IFN-g confirmed the involvement of immunogenic factors to arrest tumor growth in highly aggressive 4T1 tumors. These findings indicate promising therapeutic potentials for our newly developed hypoxia-targeted DoxAtz in combination with antiCTLA4 for effective TNBC therapy in clinic.

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