Single-cell transcriptomic analysis identifies decreased CEBPB and PAX8 expression associated with immune landscape differences between medullary and papillary thyroid carcinoma.

BACKGROUND: Medullary thyroid carcinoma (MTC) is an aggressive neuroendocrine malignancy originating from thyroid C cells, characterized by higher metastatic potential and poorer prognosis compared with papillary thyroid carcinoma (PTC), the most common differentiated thyroid cancer subtype. However, the molecular mechanisms underlying the distinct malignant phenotypes between MTC and PTC remain incompletely elucidated. This study aimed to exploratorily characterize potential transcriptional and immunological differences between PTC and MTC at single-cell resolution. METHODS: We performed single-cell RNA-sequencing (scRNA-seq) analysis on tissues from one PTC and one MTC. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to detect the expression of CEBPB and PAX8 in TPC-1 and TT cells; immunohistochemistry (IHC) was performed to detect the expression of CEBPB and PAX8 in PTC and MTC tissues. RESULTS: The scRNA-seq analysis generated data on 19,334 genes and 0.11 billion unique molecular identifiers (UMIs), derived from 10,039 cells in MTC samples and 9,295 cells in PTC samples, classified into 22 clusters, each annotated to represent 10 cellular lineages. We observed an elevated quantity of epithelial and fibroblastic compartments in MTC, and a predominant population of T cells and natural killer (NK) cells were derived from PTC tissues. Our analysis integrated gene set variation analysis (GSVA), Single-Cell rEgulatory Network Inference and Clustering (SCENIC) analysis, and pseudotime trajectory analysis to present the upmost transcription factors and pathways in different cell clusters of MTC and PTC. We then proceeded to analyze subcluster 0 of epithelial cells, where MTC accounted for the major portion with a low degree of differentiation. Meanwhile, CEBPB, CEBPD and PAX8 exhibited the highest activity in cluster 0, which was further shown to display a downward trend through pseudotime trajectory analysis. RT-qPCR and IHC results revealed that the relative expression of CEBPB and PAX8 were downregulated in MTC compared with PTC. CONCLUSIONS: Collectively, our preliminary findings suggested that the downregulation of CEBPB with PAX8 may be associated with impaired immune surveillance in MTC, potentially contributing to its more aggressive clinical behavior compared with PTC. These exploratory observations provided a hypothesis-generating framework and candidate molecular targets for enhancing anti-tumor immunity in MTC.