Citrullination-associated transcriptional states identify a non-canonical invasive program in malignant epithelial cells and correlate with SPP1-CD44 immune microenvironment niches in thyroid cancer

Abstract

Background Citrullination, a post-translational modification catalyzed by protein arginine deiminases (PADIs), has been implicated in malignant progression across multiple cancer types. However, its cellular distribution, functional significance, and role in microenvironment remodeling in thyroid cancer remain largely unknown. Methods We integrated single-cell transcriptomics (GSE232237, 92,519 cells), spatial transcriptomics (GSE250521), and TCGA-THCA bulk transcriptomic data. Malignant epithelial cells were identified using CopyKAT. A PADI-family-based citrullination score (CitrScore) was calculated per cell. Functional states, regulon networks, co-expression modules, drug sensitivity, cell-cell communication, and in silico CD44 knockout were systematically analyzed. CD44 knockdown and overexpression experiments were performed in TPC-1 and 8505C cells. Results CitrScore was significantly enriched in malignant epithelial cells (P < 0.001). High-CitrScore malignant cells exhibited a non-canonical "high-invasion/high-apoptosis" program without concordant EMT elevation, accompanied by Ras-MAPK and PI3K-AKT-mTOR pathway activation. The C_core-up signature mapped to a pharmacologic vulnerability centered on the CDC37-HSP90-PI3K/MAPK axis. In the myeloid compartment, conventional dendritic cells (mDCs) represented the predominant Citr-high subset, with higher enrichment in PTC than ATC. The SPP1-CD44 axis dominated macrophage-malignant communication and marked a niche associated with Citr-low malignant cells, supported by spatial transcriptomics. In silico CD44 knockout preferentially perturbed the C_core program (P < 0.001). Experimentally, CD44 knockdown suppressed proliferation, migration, invasion, and EMT while promoting apoptosis; CD44 overexpression produced opposite effects. Conclusion This study delineates a citrullination-associated malignant state in thyroid cancer and highlights the SPP1-CD44 axis as a functionally relevant macrophage-malignant communication hub, nominating CD44 as a pro-tumorigenic molecule and candidate therapeutic target.