Cancer Associated Fibroblasts and Senescent Thyroid Cells in the Invasive Front of Thyroid Carcinoma

Abstract

Thyroid carcinoma (TC) comprises several histotypes with different aggressiveness, from well (papillary carcinoma, PTC) to less differentiated forms (poorly differentiated and anaplastic thyroid carcinoma, PDTC and ATC, respectively). Previous reports have suggested a functional role for cancer-associated fibroblasts (CAFs) or senescent TC cells in the progression of PTC. In this study, we investigated the presence of CAFs and senescent cells in proprietary human TCs including PTC, PDTC, and ATC. Screening for the driving lesions BRAFV600E and N/H/KRAS mutations, and gene fusions was also performed to correlate results with tumor genotype. In samples with unidentified drivers, transcriptomic profiles were used to establish a BRAF- or RAS-like molecular subtype based on a gene signature derived from The Cancer Genome Atlas. By using immunohistochemistry, we found co-occurrence of stromal CAFs and senescent TC cells at the tumor invasive front, where deposition of collagen (COL1A1) and expression of lysyl oxidase (LOX) enzyme were also detected, in association with features of local invasion. Concurrent high expression of CAFs and of the senescent TC cells markers, COL1A1 and LOX was confirmed in different TC histotypes in proprietary and public gene sets derived from Gene Expression Omnibus (GEO) repository, and especially in BRAF mutated or BRAF-like tumors. In this study, we show that CAFs and senescent TC cells co-occur in various histotypes of BRAF-driven thyroid tumors and localize at the tumor invasive front.

Keywords: BRAF- and RAS-like signaling; BRAFV600E; CAFs; Thyroid cancer; senescent cells.

Figure 1

Cancer-associated fibroblasts (CAFs) assessment by α-SMA immunohistochemical staining in human thyroid cancers. (A) A boxplot with a scatterplot showing IHC results expressed as α-SMA positive areas calculated by image digital quantification on the stained tissues section from non-neoplastic thyroids (NT) and thyroid tumors (scored separately for invasive front (IF) and tumor center (TC)). Each dot represents the mean of 2–3 fields scored for each sample. Below an example of α-SMA stained tissue section (scale bar: 5 mm) with representative scored regions (high magnification, scale bar: 500 μm) and the corresponding ImageJ threshold mask with the measured positive area (expressed as a percentage). (B) Tumor genotyping results. Pie charts represent the distribution of identified driving lesions or BRAF-/RAS-like signaling in tumor tissues; wt: samples with no alterations in the tested lesions. (C) Boxplot with scatterplot showing α-SMA IHC results in tumor samples stratified for genetic driving lesion or BRAF-/RAS-like signaling. *** p-value < 0.0001 and * p-value < 0.05 by Mann-Whitney test.

Figure 2

α-SMA, COL1A1, and LOX gene expression in human thyroid cancers. (A) Boxplot with a scatterplot showing mRNA expression of α-SMA (ACTA2 gene), COL1A1, and LOX genes using quantitative RT-PCR in paired non-neoplastic thyroids (NT) and thyroid tumors. (B) The same genes displayed in tumor samples stratified for driving lesion or BRAF-/RAS-like signaling. qRT-PCR data are shown as a relative quantity normalized to the HPRT gene used as endogenous control for RNA input normalization. Each dot represents the mean of three technical replicates. * p-value < 0.05 using a Wilcoxon. matched test for pairs NT/tumor or the Mann-Whitney test.

Figure 3

α-SMA, LOX, and COL1A1 immunostaining in human thyroid cancers. Representative thyroid tumors serial sections from two different patients stained by IHC for α-SMA, COL1A1, and LOX protein expression and localization. In the top panel, the tumor edge/invasive front is specifically shown (scale bar 500 µm), while lower panel has higher magnification (scale bar 200 µm). * clusters of tumor invading cells detaching from the principal tumor mass (T).

Figure 4

Senescent thyrocytes and CAFs assessment in human thyroid cancers. (A) Boxplot with a scatterplot of p16 mRNA expression by qRT-PCR in non-neoplastic thyroids (NT) and thyroid tumors and (B) in tumor samples stratified for driving lesion or BRAF-/RAS-like signaling. qRT-PCR data are shown as a relative quantity normalized to the HPRT gene used as an endogenous control. Each dot represents the mean of three technical replicates. (C) Representative serial sections of a BRAFV600E mutated thyroid tumor IHC stained with p16 and α-SMA (senescent cells and CAFs markers, respectively). Scale bar: 100 µm. (D) Correlation between α-SMA positive areas and p16 positive cells established by IHC. (E) Boxplot with a scatterplot of p16 IHC staining results in tumors stratified for driving lesions or signaling.

Figure 5

Unsupervised hierarchical clustering analysis of proprietary gene dataset on 5 tumoral and stromal genes. The color scale bar represents the relative gene expression levels normalized by the standard deviation. Color legend for tissue and BRAF-like/RAS-like signaling is reported. α-SMA and p16 are indicated by the corresponding gene symbol, ACTA2 and CDKN2A, respectively.

Figure 6

Assessment of tumoral and stromal derived genes in human thyroid cancers gene sets derived from Gene Expression Omnibus (GEO). (A) Gene sets description. (B) Unsupervised hierarchical clustering analysis of 407 samples derived from GEO datasets on five tumoral and stromal genes. The color scale bar represents the relative gene expression level normalized by the standard deviation. Color legend for the tissue, BRAF-like/RAS-like signaling, and CAF score is reported. α-SMA and p16 are indicated by the corresponding gene symbol, ACTA2 and CDKN2A, respectively.