Differential diagnosis of thyroid cancer and benign nodules is still one of the most challenging issues in the field of endocrinology. To overcome overdiagnosis of papillary thyroid carcinomas (PTC) and the consecutive overtreatment of multinodular diseases, the search for easily accessible, sensitive and accurate biomarkers is critical. Several micro-RNAs (miRNAs) freely circulating in peripheral blood or enclosed in extracellular vesicles (EVs) have been proposed as potential biomarkers from non-invasive liquid biopsies. However, protocols are rarely comparable and conflicting data exist in the literature. In this work, we aimed to assess the diagnostic value of six micro-RNAs by comparing their expression in thyroid tissue to their abundance in bulk plasma and in plasma-EVs, before and after thyroid surgery. Plasma-EVs were isolated using a sequential density- and size-based fractionation, followed by in-depth characterization, confirming EV purity. Micro-RNA levels were measured by RT-qPCR in thyroid tissue, plasma and plasma-EVs. Among the six candidates, only miR-146b-5p and miR-21a-5p displayed a significant differential abundance in purified plasma-derived EVs from patients with PTC and benign disease. However, no difference could be demonstrated in bulk plasma through our cohort of patients. Overall, our work supports the use of a well-defined protocol of plasma-EV miRNAs purification for biomarker discovery, rather than the use of freely circulating miRNAs in bulk plasma. Our work also demonstrates that standardized pre-analytical and analytical procedures as well as optimized EV-miRNAs detection methods are essential.

Extracellular vesicles are spherical subcellular structures delimited by a lipid bilayer and released by most cells in the human body. They are loaded with a myriad of molecules (i.e., nucleic acids and proteins) depending on their cell of origin and provide the ability to transmit a message to surrounding or distant target cells. In several organs, including the thyroid, abundant recent literature reports that extracellular vesicles are responsible for intercellular communication in physiological and pathological processes, and that their utilization as a potential biomarker of pathological states (i.e., cancer, autoimmune diseases) or as therapeutic delivery vehicles promise clinical options. In this review, we present the current knowledge and understanding regarding the role of extracellular vesicles in developing thyroid diseases and diagnosis.

Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAF^V600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAF^V600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment.

Papillary thyroid carcinoma (PTC) is the most frequent histological subtype of thyroid cancers (TC), and BRAF^V600E genetic alteration is found in 60% of this endocrine cancer. This oncogene is associated with poor prognosis, resistance to radioiodine therapy, and tumor progression. Histological follow-up by anatomo-pathologists revealed that two-thirds of surgically-removed thyroids do not present malignant lesions. Thus, continued fundamental research into the molecular mechanisms of TC downstream of BRAF^V600E remains central to better understanding the clinical behavior of these tumors. To study PTC, we used a mouse model in which expression of BRAF^V600E was specifically switched on in thyrocytes by doxycycline administration. Upon daily intraperitoneal doxycycline injection, thyroid tissue rapidly acquired histological features mimicking human PTC. Transcriptomic analysis revealed major changes in immune signaling pathways upon BRAF^V600E induction. Multiplex immunofluorescence confirmed the abundant recruitment of macrophages, among which a population of LYVE-1+/CD206+/STABILIN-1+ was dramatically increased. By genetically inactivating the gene coding for the scavenger receptor STABILIN-1, we showed an increase of CD8+ T cells in this in situ BRAF^V600E-dependent TC. Lastly, we demonstrated the presence of CD206+/STABILIN-1+ macrophages in human thyroid pathologies. Altogether, we revealed the recruitment of immunosuppressive STABILIN-1 macrophages in a PTC mouse model and the interest to further study this macrophage subpopulation in human thyroid tissues.

Papillary thyroid cancer (PTC) is the most common endocrine malignancy which diagnosis and recurrences still challenge clinicians. New perspectives to overcome those issues could come from the study of extracellular vesicles (EVs) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAF^V600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. Cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to healthy thyroid, and that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Pathway analysis showed that the more abundant EV-microRNAs could impact on immune processes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAF^V600E induction, and their cellular origin. Thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment.

Papillary thyroid carcinoma (PTC) is the most frequent histological subtype of thyroid cancers (TC), and BRAF ^V600E genetic alteration is found in 60% of this endocrine cancer. This oncogene is associated with poor prognosis, resistance to radioiodine therapy and tumor progression. Histological follow-up by anatomo-pathologists reveals that 2/3 of surgically-removed thyroids do not present malignant lesions. Continued fundamental research into the molecular mechanisms of TC downstream of BRAF ^V600E remains thus central to better understand the clinical behavior of these tumors. To study PTC, we used a mouse model in which expression of BRAF ^V600E is specifically switched on in thyrocytes by doxycycline administration. Upon daily intraperitoneal doxycycline injection, thyroid tissue rapidly acquired histological features mimicking human PTC. Transcriptomic analysis revealed major changes in immune signaling pathways upon BRAF ^V600E induction. Multiplex immunofluorescence confirmed the abundant recruitment of macrophages, among which a population of LYVE-1+/CD206+/STABILIN-1+ was dramatically increased. By genetically inactivating the gene coding for the scavenger receptor STABILIN-1, we showed an increase of CD8+ T cells in this in situ BRAF ^V600E dependent TC. Finally, we demonstrated the presence of CD206+/STABILIN-1+ macrophages in human thyroid pathologies. Altogether, we revealed the recruitment of immunosuppressive STABILIN-1 macrophages a PTC mouse model and the relevance of these observations in human thyroid tissues.