Transcriptomic Biomarker Discovery from Extracellular Vesicles: A Workflow in Action Using Off-Label Treatment of Anaplastic Thyroid Cancer with Vandetanib as an in Vitro Model

<p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="first" dir="auto" id="d26944192e240">Anaplastic thyroid cancer (ATC) is a rare disease with poor prognosis and limited treatment options. Drug repurposing - such as off-label use of vandetanib for tumors overexpressing EGFR – holds great therapeutic potential. Timely assessment of treatment efficacy is critical in ATC, given its rapid progression and tendency for early metastasis. Extracellular vesicle (EV)-associated cell-free RNA from liquid biopsies shows promise as a non-invasive biomarker source for monitoring therapeutic response. We present a pipeline for the development and analysis of EV-associated transcriptomic biomarkers, applied here to evaluate vandetanib treatment efficacy in ATC. We highlight key considerations, pitfalls, and bioinformatic tools in biomarker validation by RT-qPCR. <p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" dir="auto" id="d26944192e242">EVs were precipitated from Cal62 cell culture supernatants after treatment with three doses of vandetanib. Characterization was performed via NTA and flow cytometry. Total cellular RNA-Seq and label-free proteomics of cells and EVs, were conducted to identify candidate biomarkers. Selected transcripts were validated using digital and conventional RT-qPCR. <p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" dir="auto" id="d26944192e244">Vandetanib induced dose-dependent transcriptomic and proteomic changes in Cal62 cells. Dimensional reduction and pathway analysis identified a signature of eight transcripts (two upregulated, six downregulated). This signature was validated across cellular and cell-free RNA samples. Biological relevance and discriminatory potential were confirmed through pathway over-representation analysis and partial least-squares regression. <p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" dir="auto" id="d26944192e246">The identified EV-RNA biomarker signature reliably differentiates vandetanib-treated from untreated Cal62 cells and can be quantified robustly via RT-qPCR. These findings demonstrate the utility of our biomarker development pipeline and provide a foundation for future clinical validation with liquid biopsy samples from ATC patients undergoing off-label vandetanib therapy.