Patient-Derived Papillary Thyroid Cancer Organoids for Radioactive Iodine Refractory Screening

Abstract

Patients with well-differentiated thyroid cancer, especially papillary thyroid cancer (PTC), are treated with surgical resection of the thyroid gland. This is followed by post-operative radioactive iodine (I¹³¹), resulting in total thyroid ablation. Unfortunately, about 15-33% of PTC patients are unable to take up I¹³¹, limiting further treatment options. The aim of our study was to develop a cancer organoid model with the potential for pre-treatment diagnosis of these I¹³¹-resistant patients. PTC tissue from thirteen patients was used to establish a long-term organoid model. These organoids showed a self-renewal potential for at least five passages, suggesting the presence of cancer stem cells. We demonstrated that thyroid specific markers, a PTC marker, and transporters/receptors necessary for iodine uptake and thyroid hormone production were expressed on a gene and protein level. Additionally, we cultured organoids from I¹³¹-resistant PTC material from three patients. When comparing PTC organoids to radioactive iodine (RAI)-refractory disease (RAIRD) organoids, a substantial discordance on both a protein and gene expression level was observed, indicating a treatment prediction potential. We showed that patient-derived PTC organoids recapitulate PTC tissue and a RAIRD phenotype. Patient-specific PTC organoids may enable the early identification of I¹³¹-resistant patients, in order to reduce RAI overtreatment and its many side effects for thyroid cancer patients.

Keywords: RAI therapy; cancer stem cells; papillary thyroid carcinoma; patient-derived tumor organoids; three-dimensional culture; treatment prediction; tumor organoids.

Figure 1

In vitro self-renewal of organoid-forming cells from human putative papillary thyroid cancer (PTC) cancer stem cells. (A) Schematic representation of PTC tissue isolation, primary tissue culture, and the self-renewal assay. (B) Primary PTC organoids 7 days in culture (p0), and organoids in passage 2 and 5. Scale bar = 250 µm. (C) Primary radioactive iodine (RAI)-refractory disease (RAIRD) organoids 7 days in culture (p0), and organoids in passage 1 and 2. Scale bar = 250 µm. (D) Self-renewal potential of PTC organoids during passaging (n = 13, error bars represent SEM). (E) Self-renewal potential of RAIRD organoids during passaging (n = 3, error bars represent SEM).

Figure 2

qPCR analysis of the organoid characterization marker panel mRNA level in patient material, organoids, cell lines, and 3D-spheroids (error bars represent the SEM of three biological replicates, normalized to thyroid tissue = dotted line. * p < 0.05).

Figure 3

Immunofluorescent characterization of PTC and RAIRD organoids. Representative images of organoids and tissue showing thyroid-specific markers, PTC markers, and markers for thyroid hormone synthesis. Scale bar tissue = 50 µm, and organoids = 10 µm. Markers indicated on the left side are shown as a red fluorescent signal. Nuclei are shown as a blue fluorescent signal.

Figure 4

Immunofluorescence quantification analysis of the organoid characterization marker panel (error bars represent the SEM of 10 quantified organoids). Significance is indicated with an asterisk: p < 0.001.