BRAFV600E Induction in Thyrocytes Triggers Important Changes in the miRNAs Content and the Populations of Extracellular Vesicles Released in Thyroid Tumor Microenvironment

Abstract

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 BRAFV600E, 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 BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment.

BRAFV600E expression in thyrocytes causes recruitment of immunosuppressive STABILIN-1 macrophages

“…Expression of the BRAF V600E oncogene was obtained by intraperitoneal doxycycline injection in the Tg-rtTA/tetO-BRAF V600E mouse model and led to tissue changes similar to those found in human PTC [22]. In our previous work, we observed (i) a two-fold increase in thyroid lobe size, (ii) the appearance of papillae in colloidal spaces, (iii) a progressive decrease in the protein reserve contained in the colloid, as well as (iv) a decrease in the expression of thyrocyte molecular markers, as from Day 1 to Day 4 (REF22).…”

“…also highlighted the necessity of the CCR2 receptor for macrophage attraction and tumor development using this PTC model [19]. This PTC mouse model indeed presents several advantages: (i) ease of use as it only depends on the presence of two transgenes and the administration of doxycycline [41]; (ii) flexibility in the timing of oncogene induction and in the dose of doxycycline administered (either via food or via intra-peritoneal injections) [21,22]; (iii) short latency and high reproducibility as compared to sporadic thyroid cancer models [42-44]; and finally (iv), possibility to study early changes occurring in the thyroid which allows to investigate in situ PTC development as well as the thyroid immune microenvironment, impossible to study in xenograft models in immunosuppressed mice. Indeed, recruitment of TAMs described by Ryder et al .…”

“…A. Fagin [21]. Mice were intraperitoneally injected with a saline solution (CTL) or with doxycycline (1µg/g body weight) every 24h during maximum 4 days (Day 1 à Day 4) [22]. The Stabilin-1 knockout mice were generated at the UCLouvain Transgenesis platform as described [23].…”

“…After paraffin removal and rehydration, sections were either stained with hematoxylin and eosin for histological analysis or treated with 0.3% H2O2 for immunohistochemistry. Slides were processed as described [22] for antigen retrieval, permeabilization, blocking and antibody staining (listed in Supplementary Table S1). For both, histology and immunohistochemistry, slides were mounted in Dako aqueous Medium (Agilent Technologies) and scanned with the panoramic P250 digital slide scanner (3DHistech).…”

Role of Extracellular Vesicles in Thyroid Physiology and Diseases: Implications for Diagnosis and Treatment

“…To what extent EVs could mediate those communications in a healthy thyroid is unknown. Our group demonstrated abundant endothelial and immune cell-derived EV populations in EV isolated from adult mouse thyroid lobes [ 22 ]. During thyroid development, endothelial progenitor cell (EPC)-derived EVs were shown to stimulate folliculogenesis of embryonic thyroid lobes cultured on filters by promoting thyrocytes organization and lumen expansion [ 23 ], which could be one way for endothelial cells to promote thyroid embryogenesis [ 24 ].…”