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. 2016 Oct;7(5):451.
doi: 10.4172/2155-9899.1000451. Epub 2016 Sep 16.

Immune Suppression Mediated by Myeloid and Lymphoid Derived Immune Cells in the Tumor Microenvironment Facilitates Progression of Thyroid Cancers Driven by Hras G12V and Pten Loss

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Immune Suppression Mediated by Myeloid and Lymphoid Derived Immune Cells in the Tumor Microenvironment Facilitates Progression of Thyroid Cancers Driven by Hras G12V and Pten Loss

Lee Ann Jolly et al. J Clin Cell Immunol. .
Free PMC article

Abstract

Thyroid cancer is the most common endocrine malignancy and is predicted to be the 4th 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 HrasG12V at endogenous levels and Pten inactivation (HrasG12V/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 HrasG12V/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. HrasG12V/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 HrasG12V/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, HrasG12V/Pten-/-/TPO-cre tumors contained Arginase-1 positive cells as determined by immunohistochemical analysis, supporting an immunosuppressive TME in HrasG12V/Pten-/-/TPO-Cre thyroid tumors. We next evaluated whether or not cytotoxic (CD8+) or helper T cells (CD4+) were recruited to HrasG12V/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. HrasG12V/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 HrasG12V/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.

Keywords: Hras; Immune cells; Immunosuppression; Thyroid cancer; Tumor microenvironment.

Figures

Figure 1
Figure 1
HrasG12V and Pten loss leads to FTCs that progress to PDTC in HrasG12V/Pten−/−/TPO-Cre mice. (A) Top panel: Gross histology of wild-type (WT, left) and HrasG12V/Pten−/− (right) thyroid at 50 weeks of age. Tr=Trachea. HrasG12V/Pten−/− thyroid is significantly enlarged. (A) Bottom panel: H and E staining of 5 μm thick tissue sections from WT (left) and HrasG12V/Pten−/− (right) thyroid tissue. Th=Thyroid tissue, Tr=Trachea. WT thyroid contains organized follicles filled with colloid, in contrast to HrasG12V/Pten−/− thyroids, in which the normal follicular architecture is disrupted. 20X magnification, scale bar is 100 μM. (B) Survival of mice with heterozygous activation of HrasG12V (black line) is 100% at one year of age, while homozygous activation of HrasG12V (red line) is lethal by 40 weeks of age, with no mice surviving past this time point.
Figure 2
Figure 2
HrasG12V/Pten−/−/TPO-Cre mice develop tumors with features of classical and aggressive FTC that metastasize to the lungs. H and E stained 5 μm thick tumor sections from wild type and HrasG12V/Pten−/− thyroid sections. (A) 10X image of wild type thyroid. Scale bar=200 μM. (B) 10X image of HrasG12V/Pten−/− tumor tissue with multiple foci (arrows). (C) 10X image of tumor tissue invading into tracheal cartilage (arrow). (D). 10X image of tumor displaying extrathyroidal extension into surrounding soft tissue (arrow). (E) 40X image showing invasion of tumor tissue into lymph vessel (arrow). Scale bar=50 μM. (F) Gross histological image of lungs from a HrasG12V/Pten−/−/TPO-Cre mouse, dashed line highlights the perimeter of metastatic nodule. By one year of age, 56% of HrasG12V/Pten−/−/TPO-Cre mice (n=16) with FTC develop metastasis to the lungs.
Figure 3
Figure 3
Robust recruitment of immune cells to HrasG12V/Pten−/−/TPO-Cre tumors. (A) Representative FACS analysis of CD45+ (immune) cells in pooled wild type thyroids (left) and a HrasG12V/Pten−/−/TPO-Cre tumor (right). (B) Quantification of the percentage of CD45+ cells detected out of all live, single cells analyzed from 2 separate pools of 10 wild-type (WT) thyroids and 8 HrasG12V/Pten−/−/TPO-Cre tumors. (C) 20X images of H and E stained sections from WT (left) or HrasG12V/Pten−/−/TPO-Cre tumor tissue (right). Arrows point to immune infiltration. Scale bar=100 μm. (D) 20X image of HrasG12V/Pten−/−/TPO-Cre tumor section immunostained with CD45-FITC antibody (green). Arrows point to clusters of CD45+ cells. Nuclei are counterstained with DAPI. Scale bar=100 μm.
Figure 4
Figure 4
Macrophages and MDSCs are recruited to HrasG12V/Pten−/− tumors. (A) Top: Representative FACS analysis of myeloid (CD11b+) cells within a HrasG12V/Pten−/− thyroid tumor. Bottom: Representative FACS analysis of macrophage (F4/80+Gr-1-) and MDSC (F4/80Gr-1+) populations within a HrasG12V/Pten−/− thyroid tumor. (B) Average percentages of MDSCs and Macrophages out of all myeloid derived cells (CD11b+) in HrasG12V/Pten−/− thyroid tumors (N=8). (C) 20X image of 5 μm thick HrasG12V/Pten−/− tumor section immunostained with F4/80 to confirm presence of macrophages (arrow) Scale bar=100 μm.
Figure 5
Figure 5
HrasG12V/Pten−/− tumors are positive for Arginase-1 and express cytokines that induce M2 polarization. (A) 20X (top) and 40X (bottom) images of 5 μm thick HrasG12V/Pten−/− tumor sections immunostained with Arginase-1, a marker of M2 polarization. Scale bar=100 μm for 20X image, 200 μm for 40X. (B) Elisa analysis of TGFβ1 secretion from primary wild-type (WT) thyrocytes and tumor cells isolated from a HrasG12V/Pten−/− tumor (H340T). (C) RT-PCR analysis of mcsf expression in primary WT thyrocytes and three independent tumor cell lines isolated from HrasG12V/Pten−/− tumors (Hras1, H245T, and H340T).
Figure 6
Figure 6
Recruitment of immunosuppressive Tregs to HrasG12V/Pten−/− thyroid tumors. (A) Representative image of FACS analysis showing gating used to identify T cell populations. CD3+ cells (T-cells) were gated out of all CD45+CD11b immune cells and further classified according to CD8 expression (cytotoxic T cells) and CD4 expression (T helper cells). CD4 T cells were classified as regulatory T cells (Treg) based on expression of CD25. (B) 20X images of 5 μm thick WT or HrasG12V/Pten−/− tumor tissue sections immunostained with Foxp3, a transcription factor exclusively expressed by immunosuppressive regulatory T cells. In WT thyroid, non-specific DAB staining occurs in thyroid follicles due to presence of colloid, however there no observed Foxp3 nuclear staining in thyrocytes or T-cell populations. Scale bar=100 μm. (C) Cytokine densitometry analysis from conditioned medium isolated from independent tumor cell lines derived from BrafV600E/Pten−/− (Braf1, B297T) and HrasG12V/Pten−/− (Hras1, H340T) thyroid tumors. *, P<0.01, significant difference observed between genotypes (Hras1 versus Braf 1, H340T versus B297T); a, P<0.05, significant difference between values obtained from cell lines of the same genotype (Hras1 versus H340T, Braf1 versus B297T) based on two-way ANOVA with post-hoc analysis (Tukey).
Figure 7
Figure 7
Proposed model of HrasG12V driven remodeling of the TME that contributes to progression of thyroid cancer. Activation of HrasG12V and loss of Pten in thyroid cells leads to transformation of thyroid epithelial cells and development of FTCs that progress to PDTC. HrasG12V/Pten−/− tumor cells stimulate the recruitment of myeloid and lymphoid derived immune cells, including macrophages, CD4+ Tregs, and MDSCs, to the TME via secretion of key cytokines that are chemotactic for these cell types. HrasG12V/Pten−/− tumor cell derived TGFβ1 and MCSF induces macrophages, CD4+ T cells, and MDSCs to upregulate immune suppressive molecules including IL-10, TGFβ, and CTLA4, as well as pro-angiogenic factors such as VEGFA. This results in further immune suppression and angiogenesis in the TME, promoting thyroid cancer progression.

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