Thyroid-cell derived EVs harbour surface thyrotropin-receptor and intravesicular thyroglobulin offering a novel approach for detecting thyroid cancer recurrence http://creativecommons.org/licenses/by/4.0/legalcode application/pdf 26172591 bytes eng Cell line, Extracellular vesicles, Tetraspanin, Thyroid cancer, Thyroglobulin, Thyrotropin receptor Srpsko društvo istraživača raka Bobar, Nevena Živaljević, Vladan Mitić, Ninoslav Kosanović, Maja Šelemetjev, Sonja Išić Denčić, Tijana Žarković, Miloš Janković Miljuš, Jelena Background: Thyroid cancer (TC) poses diagnostic challenges in differentiating benign from malignant tumors and detecting recurrence, which occurs in 21–27% of patients. Analysis of serum thyroglobulin (Tg) can detect recurrence early, but the marker is not applicable in patients harboring Tg antibodies (TgAt) due to TgAb interference in standard Tg detection tests. Extracellular vesicles (EVs) present a promising target for non-invasive diagnostics as they are enriched in transmembrane proteins such as tetraspanins and tissue-specific markers. We investigated whether thyroid-cell derived EVs retain tissue specific surface characteristics (thyrotropin receptor – TSH-R) and intravesicular cargo (Tg) that can enable their use in recurrent TC detection. Materials and Methods: EVs were extracted via differential ultracentrifugation (dUC) from FBS-free cell culture media of normal thyroid (Nthy-Ori 3-1) and cancer cell lines (TPC-1 and OCUT2) and patient plasma (benign, malignant, recurrent). EVs number, diameter and zeta potential were assessed using Nanotracking Analysis (NTA), and their presence was confirmed by Transmsion electron microscopy (TEM). Tetraspanin CD63 – an EV marker, TSH-R, and Tg - thyroid specific protein markers were detected via Dot Blot and Western Blot. Results: NTA detected 130–160 nm particles, in all samples implying that EV pellets were enriched with small EVs with TEM analysis confirming that conclusion. Zeta potential differed between EVs from Nthy-Ori 3-1 cell line and TPC-1 derived EVs. CD63 was present in EVs from all cell lines and patient plasma. The expression of TSH-R was higher in Nthy-Ori 3-1 cell lysates, compared to TPC-1 and OCUT2, corresponding to their differentiation state. Consistent with this, TPC-1 EVs were positive while OCUT2 EVs were negative of TSH-R, however Nthy-Ori 3-1 EVs were deprived of TSH-R. Tg was confirmed in cell lysates and EVs from Nthy-Ori 3-1 and TPC-1 EVs but absent in OCUT2 cells and EVs. EVs from plasma of patients with thyroid tumors as well as recurrence were positive for both TSH-R and Tg. The highest Tg signal was observed in a malignant sample with larger tumor size. Conclusion: This pilot study highlights the potential of using CD63 and TSH-R proteins for isolating thyroid-specific EVs. The presence of thyroid-specific TSH-R and intravesicular Tg in all patient samples paves the way for studies of larger sample cohorts to address early recurrence detection. Thyroid-cell derived EVs harbour surface thyrotropin-receptor and intravesicular thyroglobulin offering a novel approach for detecting thyroid cancer recurrence https://phaidrabg.bg.ac.rs/o:36817 ISSN: 3009-3848 2025 info:eu-repo/semantics/conferenceProceedings