CANCER-ASSOCIATED FIBROBLASTS (CAFs) MODULATE CALCIFICATION IN PAPILLARY THYROID CARCINOMA CELLS

INTRODUCTION AND OBJECTIVES: Papillary thyroid carcinoma (PTC) accounts for around 85% of carcinomas of the thyroid gland, and although it presents a good prognosis, recurrences can occur in about 10% to 30% of the cases. Calcifications are commonly found in PTCs and are related to the malignancy of the tumors, although the mechanisms related to their formation are still unknown. Previous studies by our group have shown that calcifications in PTCs are associated with the occurrence of lymph node metastases and that the osteopontin protein (OPN) is related to the presence of calcifications. Cancer-associated fibroblasts (CAFs) are cells activated by tumor molecules that have increased synthesis of growth factors and extracellular matrix (ECM) components, and are recruited and infiltrated into tumors, promoting their growth and progression. CAFs are known to secrete proteins related to calcification, but their participation in this process in thyroid cancer is still unknown. Therefore, this project aims to investigate whether CAFs modulate calcification in a PTC cell line. MATERIAL AND METHODS: The gingival fibroblast cell line (FGH) was cultured with 10 ng/mL TGF-β for 24 hours and the protein levels of fibroblast activation protein (FAP) was analyzed by immunoblotting. Conditioned media (CM) from FGH and CAFs cells were collected (named CM-FGH and CM-CAF, respectively), and added to the TPC1 (PTC derived cell line), in order to observe the response of secreted factors into the medium in the TPC1 basal calcification. TPC1 cells were then treated with RPMI or CM-FGH or CM-CAF for periods of 3 and 7 days. Calcifications were marked with the calcium Alizarin dye, to visualize these structures. The expression of genes related to calcification - SPP1, SPARK, BGLAP, COL1A1 - was also evaluated in TPC1 and FGH cells by real-time PCR. The number of TPC1 viable cells were also quantified in the three conditions (TPC1 cultured with RPMI or with CM-FGH or with CM-CAF). RESULTS AND CONCLUSION: Our results showed an increase in FAP protein levels in CAFs, confirming FGH fibroblast activation in CAF within 24 hours. Additionally, we found that calcification is intensified in the extracellular matrix in TPC1 cells treated with CM-CAFs. Genes related to calcification - SPP1, SPARK, BGLAP, COL1A1 - are expressed by both the tumor and FGH cell lines. We observed that TPC1 cells treated with CM-CAF for three days showed an increase in the number of viable cells, however, no difference was observed in the number of TPC1 cells treated with CM-CAF in the period of 7 days. Together, these results may contribute to a better understanding of the biology and etiopathogenesis of the calcification process in papillary thyroid carcinoma cells.