Abstract A77: Differential recruitment of myeloid derived immune cells and fibroblasts to the thyroid tumor microenvironment in mouse models of papillary and follicular thyroid cancer
Authors: King Lee Ann , Novitsky Sergey , Ricarte-Filho Julio , Owens Philip , Franco Aime T. 
Published: October 2015

Thyroid cancer is the most common endocrine malignancy, and its incidence has steadily increased worldwide over the last 30 years. Genetic alterations leading to the activation of the MAPK signaling pathway are crucial for the initiation and progression of thyroid cancer, as evidenced by the high frequency of activating mutations in BRAF, HRAS, NRAS, RET and TRK. Mutations in BRAF are observed in ~45% of papillary thyroid cancers (PTCs), while follicular thyroid cancer (FTC) is often associated with mutations in RAS. We have shown that thyroid specific expression of HrasG12V and Pten inactivation leads to the development of FTCs and poorly differentiated thyroid cancer (PDTC) in HrasG12V/Ptenhom/TPO-cre mice while thyroid specific expression of BrafV600E and loss of Pten leads to the rapid development of PTC and PDTC in BrafV600E/Ptenhom/TPO-cre mice with complete lethality by weaning. We sought to determine whether activation of Braf or Hras resulted in the differential recruitment of stromal cells to the thyroid tumor microenvironment in HrasG12V/Ptenhom/TPO-cre and BrafV600E/Ptenhom/TPO-cre mice. FACS analysis revealed a significant increase in the overall amount of CD45+ cells in Hras (60.95±5.28%) versus Braf (18.35±1.3%) driven murine thyroid tumors. However, we found that the population of CD45+ cells in Braf driven tumors was comprised of a higher percentage of CD11b+ myeloid cells (62.1±1.3%) in comparison to Hras (37.0±5.5%) driven tumors. We next measured levels of cytokine expression in tumor cell lines derived from BrafV600E/Ptenhom/TPO-cre and HrasG12V/Ptenhom/TPO-cre mice. Braf driven tumor cells expressed higher levels of GM-CSF in comparison to Hras driven tumor cells, which we hypothesize contributes to the larger myeloid compartment observed in BrafV600E/Ptenhom/TPO-cre thyroid tumors. Studies are ongoing to further characterize the immune cell populations in these tumors. FACS analysis also revealed an increase in CD45-CD326-CD140α+ fibroblast infiltrate in Braf versus Hras driven thyroid tumors (68.4±0.424% versus 9.8±4.27%, p&lt;.0001). Fibroblasts are a major source of type I collagen (Col1a1), which has been linked to the immunosuppression of tumor infiltrating leukocytes via inhibition of Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1). Therefore, we hypothesized that the increase in fibroblast infiltrate observed in Braf driven thyroid tumors would result in a subsequent increase in Col1a1 production, thus potentially leading to a more immunosuppressive thyroid tumor microenvironment in PTC. Indeed, levels of Col1a1 gene expression were significantly upregulated an average of 80 fold (p&lt;.0001) in Braf versus Hras driven thyroid tumors, with no significant difference in Col1a1 expression between Hras driven tumors and WT thyroid controls. Histopathological analysis via Picrosirius red staining confirmed higher levels of total and fibrillar collagen in Braf versus Hras driven tumors. In addition, microarray analysis of human thyroid tumors revealed that Col1a1 expression levels are highest in PTCs, particularly in those with mutations in BRAF, and lowest in FTCs and follicular variant PTC, which are both associated with a high RAS mutation rate. Because FTCs and PTCs have distinct pathological features and differ in prognosis and responses to therapy, we aim to translate these findings to the discovery of more personalized diagnostic and treatment strategies that are targeted toward the thyroid tumor microenvironment.

Citation Format: Lee Ann King, Sergey Novitsky, Julio Ricarte-Filho, Philip Owens, Aime T. Franco. Differential recruitment of myeloid derived immune cells and fibroblasts to the thyroid tumor microenvironment in mouse models of papillary and follicular thyroid cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A77.