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Development of an extracellular vesicle-associated transcriptomic biomarker signature for vandetanib treatment response of anaplastic thyroid cancer cells
Abstract
Anaplastic thyroid cancer (ATC) is a relatively rare but highly aggressive form of thyroid carcinoma (TC), with a median overall survival between only 3 and 6 months due to its metastatic tendency and a lack of targeted therapy options. To increase the range of therapeutic options, ATC is one of the malignancies included in the REPO4EU drug repurposing project. In the scope of REPO4EU, we found sensitivity of the ATC cell line Cal62 to the receptor tyrosine kinase (RTK) inhibitor vandetanib, making this drug a repurposing candidate to be investigated for treatment of ATC. However, to assess response to a drug in clinical practice, imaging techniques are usually employed, meaning that it takes time before non-responders are identified, in which the tumor can grow and metastasize further. Since tumors are known to release a high amount of cell-free RNA (cfRNA), which can be found in patient blood samples, protected from degradation by association to extracellular vesicles (EVs), a liquid biopsy-based approach might provide information about the therapeutic success earlier. As a first step towards this goal, we aimed to identify an in vitro EV-associated cfRNA biomarker signature for the response of Cal62 cells to vandetanib.First, we assessed the EC50 of vandetanib for Cal62 cells in cell culture medium depleted of fetal calf serum EVs (8.8 μM). We then treated Cal62 cells with two doses of vandetanib (8.8 and 20 μM) in triplicates and isolated both the cellular and EV-associated cfRNA after 48 hours. EV concentration and size distribution was assessed by nanoparticle tracking analysis and flow cytometry. Using RT-qPCR, we observed significant transcriptional regulation of multiple transcription factors of the MAPK-pathway as well as their target genes, involved in cell-cycle control, RTK signaling feedback, and inflammation. Most notably, we measured 3.4-fold / 11.2-fold upregulation of cyclin dependent kinase inhibitor 1A (CDKN1A; p-values 0.034 and 0.007), 5.2-fold / 6.9-fold downregulation of cell division cycle 25A (CDC25A; p-values 0.018 and 0.001) and 3.1-fold / 3.6-fold downregulation of the ERBB receptor feedback inhibitor 1 (ERFFI1; p-values 0.003 and 0.003) compared to the DMSO control on cellular RNA level after treatment with 8.8 / 20 μM vandetanib treatment for 48 h, respectively. These results were confirmed in the cell-free RNA compartment.As a next step, we will assess how the transcription of these genes is regulated after different treatment times with vandetanib. Finally, we will investigate the whole transcriptomic change of Cal62 cells in the cellular and cell-free RNA by next-generation sequencing to identify an EV-associated biomarker signature that significantly predicts the response to vandetanib in vitro. These results may serve as a basis for further clinical studies, making this project an important basis towards assessing therapeutic success of vandetanib treatment for ATC patients in liquid biopsies.
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