Spatial immune profiling reveals varying tumor microenvironments in papillary thyroid microcarcinoma

Introduction: Papillary thyroid microcarcinomas (PTMC) are usually indolent, but up to 10% present with clinically significant lymph node metastases or very rarely with distant metastases (M1). This study analyzes transcriptomic signatures and spatially resolved tumor microenvironment profiles in metastatic versus non-metastatic PTMC to refine risk stratification. Methods: Patients diagnosed with indolent non-metastatic (N0/Nx) PTMCs and with primary tumors (PT) metastatic to the lateral neck lymph nodes or distant sites (N1b/M1) were selected, and RNA sequencing was done on PT and adjacent normal tissues. Multiplex immunofluorescence was performed, staining using CD8a and CD20 antibodies. Images were analyzed in a single-blinded manner using HALO (Indica Labs), and differential spatial expression was tested using a t-test or a Mann-Whitney test, depending on data distribution. Spatial proteomic profiling was performed using the NanoString (Bruker) GeoMX® platform, including Human Immune Cell Profiling, Typing and Activation Status, and MAPK Signaling Protein Modules for the nCounter analysis. Protein expression data were log2-transformed, and between-groups differential expression was assessed using Welch's two-sample t-tests with Benjamini-Hochberg adjustment. Proteins with adjusted p<0.05 and absolute log2 fold-change >0.5 were considered significant. Results: The N0/Nx and N1b/M1 study cohorts consisted of 17 and 19 patients, respectively. No difference in the clinical characteristics, viz., age (p=0.64), sex (p=0.24), and median PT size (p=0.27), was found between the groups. Transcriptome analysis indicated significant downregulation of pathways involved in the B cell activation without a significant difference in B cell (CD20 + /CD8a ‑ ) infiltration in the PT (p=0.43) nor in the PT rims (p=0.92) from the N1b/M1 compared to N0/Nx group. However, cytotoxic T cells (CD8a + /CD20 - ) significantly populated the rims of the PT from N1b/M1 group compared to N0/Nx (p=0.007). Spatial immune profiling revealed that N1b/M1 PT had significantly higher T-cell infiltration (CD3; padj=0.02) than N0/Nx tumors and showed pronounced expression of the immune evasion molecule PD-L2 (padj=0.02). N1b PT also showed high stemness (CD44, padj=0.001). Compared to N1b PT, M1 showed higher immune evasion (PD-L1, padj=0.01; PD-L2, padj=0.049). Conclusion: Metastasis-prone PTMC is characterized by enhanced stemness, and increased T cell infiltration, accompanied by upregulation of immune evasion molecules, suggesting that immune evasion may play a critical role in pathogenesis of metastases.