An integrated single-cell and spatial transcriptomic atlas of thyroid cancer progression identifies prognostic fibroblast subpopulations
Matthew A. Loberg, George J. Xu, Sheau-Chiann Chen, Hua-Chang Chen, Claudia C. Wahoski, Kailey P. Caroland, Megan L. Tigue, Heather A. Hartmann, Jean-Nicolas Gallant, Courtney J. Phifer, Andres A. Ocampo, Dayle K. Wang, Reilly G. Fankhauser, Kirti A. Karunakaran, Chia-Chin Wu, Maxime Tarabichi, Sophia M. Shaddy, James L. Netterville, Sarah L. Rohde, Carmen C. Solórzano, Lindsay A. Bischoff, Naira Baregamian, Barbara A. Murphy, Jennifer H. Choe, Jennifer R. Wang, Eric C. Huang, Quanhu Sheng, Luciane T. Kagohara, Elizabeth M. Jaffee, Ryan H. Belcher, Ken S. Lau, Fei Ye, Ethan Lee, Vivian L. Weiss
Matthew A. Loberg, George J. Xu, Sheau-Chiann Chen, Hua-Chang Chen, Claudia C. Wahoski, Kailey P. Caroland, Megan L. Tigue, Heather A. Hartmann, Jean-Nicolas Gallant, Courtney J. Phifer, Andres A. Ocampo, Dayle K. Wang, Reilly G. Fankhauser, Kirti A. Karunakaran, Chia-Chin Wu, Maxime Tarabichi, Sophia M. Shaddy, James L. Netterville, Sarah L. Rohde, Carmen C. Solórzano, Lindsay A. Bischoff, Naira Baregamian, Barbara A. Murphy, Jennifer H. Choe, Jennifer R. Wang, Eric C. Huang, Quanhu Sheng, Luciane T. Kagohara, Elizabeth M. Jaffee, Ryan H. Belcher, Ken S. Lau, Fei Ye, Ethan Lee, Vivian L. Weiss
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Research Article Genetics Oncology

An integrated single-cell and spatial transcriptomic atlas of thyroid cancer progression identifies prognostic fibroblast subpopulations

Abstract

Although well-differentiated thyroid carcinoma (WDTC) is characterized by a robust treatment response, aggressive subtypes, such as anaplastic thyroid carcinoma (ATC), remain highly lethal. To understand thyroid cancer evolution in both children and adults, we analyzed single-cell transcriptomes of 423,733 cells from 81 samples and spatially resolved key tumor and microenvironment populations across 28 tumors with spatial transcriptomics, including rare and unique composite WDTC/ATC tumors and pediatric diffuse sclerosing thyroid carcinomas. Additionally, we identified gene signatures of stromal cell populations in 5 large thyroid cancer bulk RNA-sequencing cohorts. Through this multi-institutional effort, we defined a population of POSTN+ myofibroblast cancer-associated fibroblasts (myCAFs) that are intimately associated with invasive tumor cells and correlate with poor prognosis, lymph node metastasis, and disease progression in thyroid carcinoma. We also revealed a population of inflammatory CAFs that are distant to tumor cells and are found in the inflammatory stromal microenvironment of autoimmune thyroiditis. Together, our study provides spatial profiling of thyroid cancer evolution in samples with mixed WDTC/ATC histopathology and identifies a prognostic myCAF subtype with potential clinical utility in predicting aggressive disease in both children and adults.

Authors

Matthew A. Loberg, George J. Xu, Sheau-Chiann Chen, Hua-Chang Chen, Claudia C. Wahoski, Kailey P. Caroland, Megan L. Tigue, Heather A. Hartmann, Jean-Nicolas Gallant, Courtney J. Phifer, Andres A. Ocampo, Dayle K. Wang, Reilly G. Fankhauser, Kirti A. Karunakaran, Chia-Chin Wu, Maxime Tarabichi, Sophia M. Shaddy, James L. Netterville, Sarah L. Rohde, Carmen C. Solórzano, Lindsay A. Bischoff, Naira Baregamian, Barbara A. Murphy, Jennifer H. Choe, Jennifer R. Wang, Eric C. Huang, Quanhu Sheng, Luciane T. Kagohara, Elizabeth M. Jaffee, Ryan H. Belcher, Ken S. Lau, Fei Ye, Ethan Lee, Vivian L. Weiss

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Figure 8

myCAFs are enriched in epithelial ATCs.

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myCAFs are enriched in epithelial ATCs. (A and B) UMAP showing subcluste...
(A and B) UMAP showing subclustering of ATC tumor cells colored by (A) sample (tumor label) or (B) BRAFV600E mutation status. (C) Milo differential abundance testing of CAF and perivascular stromal cell populations between BRAF WT and BRAFV600E ATC tumors. Individual dots depict neighborhoods calculated by Milo. Coloring of individual neighborhoods as dark blue (BRAFV600E) or light blue (BRAF WT) indicates differential abundance with a spatial FDR of less than 0.1. (D) Heatmap showing scaled expression of the top 10 differentially expressed genes with expression in at least 80% of cells in the population of interest between BRAF WT and BRAFV600E ATCs. (E) UMAP of ATC subclustering colored by expression of myCAF genes (top row) or epithelial keratins (bottom row). (F) Violin plots of expression of genes from E in ATC tumor cells split by BRAF status. (G) Representative multiplex immunofluorescence images from staining of 33 ATCs in the VUMC/UW cohort for pan-cytokeratin (PanCK, green) and FAP (red). Left: representative image of ATC with PanCK+ tumor cells and tumor-adjacent FAP+ fibroblasts. Right: representative image of ATC with no PanCK staining, FAP+ tumor cells, and minimal stromal FAP staining. White arrows point to malignant nuclei with membranous FAP staining. (H) Bar plots showing pathologist scoring of multiplex immunofluorescence. Tumors are split by BRAF status. P values calculated with Fisher’s exact test. (I) Corrplot showing Spearman’s rho correlations for ATC multiplex immunofluorescence staining samples comparing PanCK tumor cell intensity, FAP tumor cell intensity, FAP fibroblast intensity, ssGSVA scores for stromal subpopulations, ssGSVA MAP score, BRAF score, and RAS score in the VUMC/UW ATC cohort. Axes ordered by hierarchical clustering. Boxes indicate hierarchical clustering groups. Significance levels indicate *P < 0.05, **P < 0.01, or ***P < 0.001. (J) PFS (left) and OS (right) survival curves showing VUMC/UW ATCs split by 50th percentile myCAF score. P values calculated by log-rank test.