Immune Suppression Mediated by Myeloid and Lymphoid Derived Immune Cells in the Tumor Microenvironment Facilitates Progression of Thyroid Cancers Driven by HrasG12V and Pten Loss

摘要

Thyroid cancer is the most common endocrine malignancy and is predicted to be the 4^th most commonly diagnosed cancer by 2030. Approximately one-half of follicular thyroid carcinomas (FTC) contain genetic alterations in RAS family members. Furthermore, Cowden's disease, which is characterized by loss of PTEN, predisposes for the development of FTC in humans. We have shown that thyroid specific expression of Hras^G12V at endogenous levels and Pten inactivation (Hras^G12V/Pten^−/−/TPO-cre mice) leads to the development of FTCs that closely recapitulate human disease, with complete penetrance at one year. In patients, FTCs metastasize via the bloodstream to distant sites, frequently the lungs, bones and brain. The first objective of the study was to determine if these mice developed de novo metastasis to relevant sites. Indeed, spontaneous metastasis to the lungs was observed in 56% of Hras^G12V/Pten^−/−/TPO-cre mice. We next sought to identify the cellular components within the tumor microenvironment (TME) of FTC that contribute to tumor progression and metastasis via FACS analysis. Surprisingly, a large amount of immune infiltrate was observed. Hras^G12V/Pten^−/−/TPO-Cre thyroid tumors were comprised of 68.5 ± 11.79% CD45⁺ cells, in stark contrast to wild-type (WT) thyroids which were comprised of 17.6% CD45⁺ cells. Further, 53.1 ± 10.9% of the CD45⁺ cells from Hras^G12V/Pten^−/−/TPO-Cre thyroid tumors were of myeloid-lineage (CD11b⁺), consisting of macrophages (F4/80⁺Gr-1⁻) and myeloid-derived suppressor cells (F4/80⁻Gr-1⁺). Further, Hras^G12V/Pten^−/−/TPO-cre tumors contained Arginase-1 positive cells as determined by immunohistochemical analysis, supporting an immunosuppressive TME in Hras^G12V/Pten^−/−/TPO-Cre thyroid tumors. We next evaluated whether or not cytotoxic (CD8⁺) or helper T cells (CD4⁺) were recruited to Hras^G12V/Pten^−/−/TPO-Cre tumors. The majority of T cells in these tumors were double positive for CD4 and CD25, markers of immune suppressive regulatory T cells (Treg). Additionally, we identified Foxp3 positive cells by immunohistochemical analysis of tumor sections, indicating a functional suppressive Treg phenotype in vivo. Hras^G12V/Pten^−/−/TPO-Cre tumor cell lines displayed increased secretion of SDF-1, I-TAC, CCL9/10, and MCP5, cytokines that have been reported to play a direct role in the chemotaxis of immune cells and thus could contribute to the increased recruitment of myeloid and lymphoid derived cells in Hras^G12V/Pten^−/−/TPO-Cre tumors. These studies are the first to identify and implicate the interaction between tumor cells and immune cells in Ras-driven thyroid cancer progression, which we hope will lead to the development of more effective therapeutic approaches for aggressive forms of thyroid cancer that target the TME.