Date of Award

6-30-2016

Document Type

Open Access Dissertation

Department

Biological Sciences

First Advisor

Maria Marjorette O Pena

Abstract

The impact of tumor associated stromal cells in tumor formation, progression, and response to therapy has led to a paradigm shift in cancer therapy. For many years, tumors were considered as a mass consisting only of actively proliferating cancer cells and therapies were design to target these cells. However, research in the last two decades has shown that tumors are not only comprised of a heterogeneous population of neoplastic cells, but they are also comprised of and infiltrated by a heterogeneous population of non-tumor cells that contribute to tumor progression and potentially affect the efficacy of tumor-directed chemotherapies. Tumors are now considered as complex organ consisting of both cancerous and noncancerous cells interacting with each other within the tumor microenvironment to promote malignancy.

For decades, 5- fluorouracil (5-FU), an inhibitor of the enzyme thymidylate synthase (TS), has been used in the clinical management of colorectal cancer. Although it has been beneficial to some patients, its use has been severely limited by cytotoxic side effects due to its lack of specificity, affecting both rapidly proliferating cancer and healthy normal cells, and acquired resistance by cancer cells over time. To circumvent these limitations, we propose that genetic modulation of non-tumor or stromal cells in the microenvironment might enhance drug efficacy and reduce drug-induced cytotoxicity.

Genetic modulation of stromal cells may be accomplished by utilizing ribonucleic acid interference (RNAi) technologies. With RNAi, we were able to suppress intracellular protein levels of TS prior to therapy and chemo-sensitized cells to TS inhibitors. In vivo, we found that the use of constitutive H1 promoter RNAi vector systems to sensitize hematopoietic stem cells in ApcMin/+ mice, was problematic and toxic. However, using a mCMV constitutive RNAi vector which was not toxic in an in vivo model system, we found that we can sensitize stromal cells to the cytotoxic effects of 5-FU. To specifically direct chemo-sensitivity to 5-FU to cells in the tumor microenvironment we utilized the promoter of osteopontin, a gene that was only upregulated in hematopoietic cells in the tumor microenvironment, to drive the expression of a TS silencing shRNA. This allowed us to enhance chemo-sensitivity to 5-FU specifically within the tumor stroma.

In addition to enhancing the efficacy of TS inhibitors by modulating bone marrow derived cells, we also targeted mast cells by incorporating a pharmacological drug, Cromolyn that inhibits mast cells function, and combined this with 5-FU therapy. We had previously found that mast cells are resistant to 5-FU and are recruited into the tumor microenvironment upon systemic treatment of ApcMin/+ mice with 5-FU. We hypothesized that inhibition of mast cells in combination with 5-FU treatment might enhance anti-tumor efficacy of 5-FU or block the recurrence of tumors post-therapy. The results showed that administration of Cromolyn after systemic treatment with 5-FU decreased recurrence of tumor post therapy. These findings suggest that modulation of the tumor stromal cells by genetic modification or by pharmacological methods have the potential to enhance the anti-tumor efficacy of TS inhibitors. It will be necessary to assess the long-term effects and the mechanistic underpinnings of these studies to realize their clinical utility.

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Biology Commons

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