Date of Award

4-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Samuel Z. Soffer, MD

Second Advisor

Marc Symons, PhD

Third Advisor

Bettie M. Steinberg, PhD

Abstract

Osteosarcoma is the most common malignant bone tumor in children and is the third most common cancer of adolescence. Osteosarcoma is highly metastatic and up to 40% of newly diagnosed patients who initially present with localized disease will go on to develop pulmonary metastases. This high metastatic rate is thought to result from subclinical micrometastatic disease present in the lungs even before diagnosis. The 5-year survival for children with metastatic osteosarcoma is extremely low and the long- term outcomes for these patients have not improved over the last 30 years. Novel therapies capable of suppressing these microscopic deposits of tumor from blossoming into overt metastatic disease are urgently needed to improve survival. Tumor-associated macrophages (TAMs) have been shown to contribute to the malignant progression of a variety of cancers. TAMS have been demonstrated to produce epidermal growth factor receptor (EGFR) ligands that bind to and stimulate tumor cells to become more invasive. Previous studies in carcinomas have described a paracrine loop whereby tumor cells secrete colony stimulating factor 1 (CSF-1) that then induces macrophages to make EGF, and inhibition of EGFR signaling has been demonstrated to block macrophage-promoted tumor invasion and metastasis. At present the role of TAMs and EGFR signaling in the progression of pulmonary metastasis has not been evaluated in osteosarcoma.

In this thesis, I hypothesized that 1) macrophages induce osteosarcoma tumor cell invasion and metastasis 2) inhibiting tumor-macrophage crosstalk will decrease tumor cell invasion, and 3) inhibiting tumor cell crosstalk will lead to inhibition of pulmonary metastasis. I utilized a series of in vitro experiments to explore the impact of macrophages and of a CSF-1R and EGFR inhibition on osteosarcoma tumor cell activity, including proliferation, migration and invasion. Additionally, I characterized a syngeneic murine model of metastatic osteosarcoma to explore the efficacy of inhibiting crosstalk as a method to prevent pulmonary metastasis.

I demonstrated that macrophages promote osteosarcoma invasion and contribute to the development of pulmonary metastasis, which is significantly inhibited by treatment with the FDA-approved EGFR inhibitor gefitinib. I demonstrated that while both gefitinib and CSF-1R inhibition impacts macrophage function in vitro, the CSF-1R/EGFR paracrine loop described in carcinoma is not at play. Additionally, I show that gefitinib acts on macrophages in vitro and alters macrophage phenotype in the lungs of tumor-bearing mice. Although gefitinib is clinically approved as an EGFR inhibitor, I demonstrate for the first time that gefitinib inhibition of macrophage-promoted invasion is mediated through inhibition of an alternative target, receptor interacting protein kinase 2 (RIPK2) in macrophages. These data indicate that upfront treatment with gefitinib may limit metastatic progression of osteosarcoma by modulating macrophages via RIP2K inhibition.

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