Abstract 4071: Investigation of thyroid tumor microenvironment by immunohistochemical and bioinformatic analyses
Authors: Minna Emanuela , Brich Silvia , Todoerti Katia , Pilotti Silvana , Perrone Federica , Paielli Nicholas , Agnelli Luca , Bersani Ilaria , Mortarini Roberta , Anichini Andrea , Collini Paola , Neri Antonino , Greco Angela , Borrello Maria Grazia 
Published: August 2018

Abstract
Introduction
Tumor microenvironment plays an essential role in cancer development and progression. In the context of thyroid cancer (TC) it still represents a poorly investigated field. Recent data from in vitro and in vivo studies proposed a model of TC progression promoted by the converging action of thyroid tumor cells and cancer-associated fibroblasts (CAFs). According to this mouse model CAFs are recruited in tumor stroma where synthesize and deposit collagen (COL1A1) that is, in turn, cross-linked by the enzyme LOX, produced by the thyroid tumor cells. Collectively this coordinated action lead to matrix stiffness and TC progression. Interestingly, this process is reported to be specifically observed in murine thyroid tumors expressing BRAFV600E mutation, but not in those harboring RAS mutation. In this study we tested this cross-talk in human thyroid cancer tissues.
Experimental procedures
A series of thyroid specimens, comprising different tumor subtypes (PTC and PDTC) and histologic variants, was collected at our institution and investigated by immunohistochemical (IHC) analyses for the expression of α-SMA (marker for activated fibroblasts), collagen COL1A1 and LOX. Tumor samples were also screened for the most common genetic lesions reported in TC including BRAFV600E and N/H/KRAS mutations. Publicly available datasets of thyroid tumors and non-neoplastic thyroid controls, analyzed on Affymetrix HG-U133 Plus 2.0 array, were selected to investigate the expression profiles of α-SMA, COL1A1 and LOX genes. Robust Multi Array Average (RMA) normalization, Brain Array annotation and batch correction procedures were applied to obtain gene expression profiles across all samples.
Results
In IHC analyses α-SMA decoration was observed in the stroma of thyroid tumor tissues while was restricted to blood vessels in the adjacent non-neoplastic thyroid. Stromal α-SMA staining was not evenly distributed in the tumors but localized preferentially at the tumor invasive border, organized in peripheral structures as fibrous septa or capsule. In agreement with the proposed model, higher level of α-SMA staining were specifically observed in PTC tumors harboring BRAFV600E mutation compared with tumors harboring RAS mutation. IHC analyses on tissue serial sections confirmed the coordinated expression of α-SMA, COL1A1 and LOX; COL1A1 localized in tumor stroma and was closely correlated with α-SMA positive areas while LOX was expressed by thyroid tumor cells. The coordinated expression of the three genes was confirmed by meta-analysis of public datasets.
Conclusions
In this study we provide evidence of the coordinated presence of thyroid tumor cells, activated fibroblasts and proteins of extracellular matrix, such as COL1A1 and LOX, in the contest of human thyroid carcinoma.
Citation Format: Emanuela Minna, Silvia Brich, Katia Todoerti, Silvana Pilotti, Federica Perrone, Nicholas Paielli, Luca Agnelli, Ilaria Bersani, Roberta Mortarini, Andrea Anichini, Paola Collini, Antonino Neri, Angela Greco, Maria Grazia Borrello. Investigation of thyroid tumor microenvironment by immunohistochemical and bioinformatic analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4071.