Visualizing malignant progression: in situ CD109-based spatial immunofluorescence assay delineates papillary to...

Anaplastic thyroid carcinoma (ATC) is the rarest and most aggressive subtype of thyroid cancer, and considered to arise from differentiated thyroid carcinoma, however, the underlying molecular processes remain largely unknown. Using CD109 as a malignant marker, we analyzed an ATC case containing a papillary thyroid carcinoma (PTC) component. Our newly developed spatial immunofluorescence (SPI) assay, which enabled the differential expression of CD109 and CK8/18, a PTC marker, demonstrated gradual and completely opposed changes at the boundary between ATC and PTC components. Similar specific expression patterns were observed in E-cadherin, vimentin, PCNA, αSMA, Iba-1, collagen (COL) III/VI, TGFβ1-induced (TGFBI), active Yes-associated protein, periostin, and S100. The zonal and reciprocal transitions between PTC and ATC markers suggested that anaplastic transformation was not merely the result of clonal expansion, but rather it was executed by ATC-specific tumor microenvironment (ATC-TME) that recruited more Iba-1- and S100-positive macrophages, along with unique ATC-cancer-associated fibroblasts (CAFs), which deposited more COL III/VI and TGFBI. We proposed that expansion of ATC-CAFs caused extracellular matrix stiffening and compromised PTC cells, thereby inducing necroptosis and S100 release. This process simultaneously promoted the epithelial-mesenchymal transition in PTC cells and selected pre-existing PTC cells harboring additional gene mutations sufficient for anaplastic transformation.