Dissecting the tumor microenvironment of papillary thyroid carcinomas in primary tumor and metastatic lymph nodes at single-cell resolution.
Authors: Nguyen Anthony Tuan , Viramontes Jolene , Dar Tahir , Stevens Satchel , De Simone Marco , Jang Julie , Sacks Wendy L. , Sandler Howard M. , Zumsteg Zachary Stephan , Guarnerio Jlenia , Shon Wonwoo , Ho Allen S. , Shiao Stephen Lawrence 
Published: June 2023

e15182 Background: Although often well-differentiated and indolent, papillary thyroid carcinomas (PTCs) frequently metastasize to regional lymph nodes (LNs) especially in younger patients. It is unclear what factors of the local microenvironment promote LN metastases. To this end, we used single-cell RNA sequencing (scRNA-seq) to characterize the immune compartment of pair-matched primary tumor and metastatic LN samples from patients with PTC. Methods: In total, we processed 14 fresh surgical specimens from 7 patients undergoing primary total thyroidectomy and bilateral modified radical neck dissection between July 2018 and July 2019. CD45+ immune cells were isolated for library generation per 10X Genomics protocol for Single Cell 5’ v2 chemistry dual index. Data analysis was performed by Seurat and BBrowser. Differentially expressed genes were identified by Venice. Results: Pair-matched primary tumor and metastatic LN samples from 7 patients were included (mean [SD] age, 42.9 [17.4]). Histologic subtypes include classical PTC (n = 3), anaplastic (n = 2), tall cell variant (n = 1), and diffuse sclerosing variant (n = 1). The median number of positive metastatic LNs was 9 (range: 4-41) and the median number of examined LNs was 67 (range: 15-86). The majority of patients were pN1b (n = 6) or pN1a (n = 1) per AJCC 8th Edition. In total, we analyzed 26,659 individual cells by scRNA-seq, including 10,558 cells from the primary tumor and 16,101 cells from the metastatic LN. Unbiased clustering identified 6 major populations including B cells (CD79A/B, IGKC), T/natural killer (NK) cells (CD3E, CD247), myeloid cells (LYZ, FCER1G), thyrocytes (TG, EPCAM), endothelial cells (PECAM1, CD34), and fibroblasts (COL1A1/2, ACTA2). In a pooled analysis, we identified increased myeloid cells and thyrocytes within primary tumor samples. By contrast, there were increased B cells and T/NK cells within the metastatic LN samples. We found that IL7R is highly enriched by B and T/NK cells within metastatic LNs compared to primary tumor (B cells: log2 fold-change -1.26, P= 1.36x10-65; T/NK cells: log2 fold-change -1.23, P= 1.56x10-147). Further, CCL2 is significantly enriched by myeloid cells within the primary tumor compared to cells within the metastatic LN (log2 fold-change 2.16, P= 1.70x10-83). Lastly, TNFRSF12A (TWEAKR) is highly upregulated by thyrocytes within the metastatic LN as compared to cells from the primary tumor. Ongoing validation of these three targets (IL7R/FOXP3, CCL2/CD163, TNFRSF12A/PAX8) will be performed in an independent set of 25 patients with PTC with paired primary tumor-metastatic LNs. Conclusions: Collectively, our data indicate that there are increased immunosuppressive signals with the microenvironment that allow for PTC to colonize distant metastatic LNs. IL7R and TNFSRF12A may serve as a novel therapeutic target for patients with locally advanced PTC.