The Creation of 3D Thyroid Models to Investigate the Effects of Sorafenib on Thyroid Tumour Tissue

Thyroid cancer (TC) is the most common endocrine malignancy globally. While surgery and radioactive iodine (RAI) therapy generally yield favourable outcomes, prognosis worsens in RAI-refractory or resistant cases, highlighting the need for alternative therapies such as tyrosine kinase inhibitors (TKI). High attrition rates in anti-cancer drug discovery often result from inadequate representation of the tumour microenvironment (TME) in traditional 2D culture models. Spheroids and organoids (3D models), which better replicate in vivo tumour conditions, have demonstrated superior predictive capabilities by exhibiting dose-dependent drug responses and IC50 values comparable to 2D studies, despite greater drug diffusion distances.The current study established, optimised and demonstrated the effectiveness of 3D cultures from two TC cell lines, papillary (K1) and anaplastic (8305c), as reliable drug screening platforms. The aim was to evaluate the efficacy of sorafenib, a commonly-used TKI, in TC spheroids and organoids, under both static and dynamic conditions. Consistently well-defined spheroids were created, displaying reproducible sizes, shape and viability. Spheroids were subjected to sorafenib treatment for a duration of 48 h, with the drug medium replenished at the 24 h mark to simulate the pharmacokinetic profile of clinical patient dosing regimens. Post-treatment, spheroids were evaluated using viability assays, microscopy analysis and diameter measurements. Both papillary and anaplastic spheroids, with and without necrotic cores, were successfully developed. The addition of the 3D matrix, Matrigel® to the spheroids, resulted in a doubling in overall size and greater viability compared to those without the additive. The 8305c spheroids displayed greater sensitivity to sorafenib compared to K1 spheroids.Preliminary trials involving spheroids maintained on a microfluidic chip, with a constant flow of nutrients and removal of waste, validated their potential by demonstrating dose-dependent drug responses resembling those observed in static 3D models within this study. Furthermore, the spheroids within the microfluidic devices displayed heightened sensitivity to sorafenib, aligning with findings in the literature that dynamic culture systems often exhibit greater drug sensitivity compared to static models.Patient-derived thyroid tissues, both malignant (n = 7) and benign (n = 4), were dissociated into primary cells and plated in either Matrigel® or VitroGel® matrices for generation of organoids or primary spheroids. Of the 11 primary cultures, 6 formed spheroids or spheroid-like structures. Notably, co-cultures with K1 cells resulted in more defined and stable spheroidal formations, suggesting a supportive role of immortalised cell lines in enhancing primary spheroid development. However, the success of organoid cultivation was limited, with issues such as inadequate aggregation and variability across different matrices. This study highlights the potential use of spheroids for high throughput testing of both mono and combination therapies. It is clear that the consistent generation of organoids and spheroids from patient-derived tissue will require additional work, however the promise that these cultures offer, being more closely representative of the in vivo situation, for personalised medicine makes this work a priority.