Anaplastic thyroid cancer cell-secreted TGFβ1 plays a key role in inducing macrophage polarization of human monocytes

Abstract

Anaplastic thyroid cancer (ATC) is a clinically aggressive form of undifferentiated thyroid cancer with limited treatment options. Tumor-associated macrophages (TAMs) constitute over 50% of ATC-infiltrating cells, and their presence is associated with a poor prognosis. We have previously shown that paracrine signals released by ATC cells induced pro-tumor M2-like polarization of human monocytes. However, which soluble factors derived from ATC cells drive monocyte activation, are largely unknown. In this study we investigated the participation of transforming growth factor β1 (TGFβ1) on the phenotype of macrophage activation induced by ATC cell-derived conditioned media (CM). THP-1 cells exposed to CM derived from ATC cells and recombinant human TGFβ1 induced M2-like macrophage polarization, showing high CD163 and Dectin1 expression. Moreover, we showed that TGFβ1 induced the messenger RNA (mRNA) and protein expression of the transcription factors SNAIL and SLUG. Accordingly, increased TGFβ1 secretion from ATC cells was confirmed by enzyme-linked immunosorbent assay (ELISA). Addition of SB431542, a TGFβ receptor inhibitor, significantly decreased the Dectin1, CD163, SNAIL and SLUG expression stimulated by ATC cell-derived CM. We validated the clinical significance of the expression of TGFβ ligands, their receptors, as well as SNAIL and SLUG in human ATC by analyzing public microarray datasets. We found that the expression of the main TGFβ ligands, TGFβ1 and TGFβ3, along with their receptors, TGFR1 and TGFR2, as well as SLUG, was significantly higher in human ATC tissue samples than in normal thyroid tissues. Our findings indicate that ATC cell-secreted TGFβ1 may play a key role in M2-like macrophage polarization of human monocytes and in the up-regulation of SNAIL and SLUG transcription factors. Thus, ours results uncovered a novel mechanism involved in the activation of TAMs by soluble factors released by ATC cells, which suggest potential therapeutic targets for ATC.

Keywords: Anaplastic thyroid cancer; M2-like macrophage polarization; TGFβ1; tumor-associated macrophages.

Figure 1

The effect of transforming growth factor β1 (TGFβ1) on the phenotype of THP-1 cells. (A-I-A-III) Soluble factors derived from anaplastic thyroid cancer (ATC) cells induced morphological changes in THP-1 cells. Monocytes were treated for 18 hours (h) with 8505C-derived conditioned media (CM) and TGFβ1 20 ng/ml. Representative bright-field imaging of THP-1 cells control (A-I) or treated with 8505C-derived CM (A-II) and TGFβ1 20 ng/ml (A-III). (B) Proliferation of THP-1 cells incubated with 8505C-derived CM and TGFβ1 12.5 ng/ml for 24 h and 48 h, estimated by cell counting. (C) Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay to detect human C-C motif chemokine ligand 13 (hCCL13), hDectin1 and CD163 in THP-1 cells grown under normal conditions (control) and treated with 8505C-derived CM and TGFβ1 20 ng/ml for 24 h. (D, E) Flow cytometry analysis showed significant induction of hDectin1 in THP-1 cells treated with 8505C-derived CM and TGFβ1 20 ng/ml for 24 h compared to THP-1 control (E). Representative histograms and quantification (D) are shown. (F) Concentrations of TGFβ1 in Dulbecco’s modified Eagle’s medium (DMEM) alone, DMEM FBS 5% (control) and in the CM of 8505C and C643 cells, determined by enzyme-linked immunosorbent assay (ELISA) as described in Methods and Materials. Data are expressed as mean ± SEM. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001.

Figure 2

SNAIL and SLUG are up-regulated in THP-1 cell activation induced by cancer cell-secreted TGFβ1. (A, B) Immunoblot analysis of SNAIL in THP-1 cells grown under normal conditions (control) or exposed to CM derived from 8505C cells and TGFβ1 20 ng/ml for 18 h and 24 h (A). The figure shows a representative Western blot of 4 independent experiments. Quantification of relative expression of SNAIL protein in human monocytes after using βActin as loading control (B). (C) Expression levels of SNAIL messenger RNA (mRNA) levels by RT-qPCR in control and THP-1 cells treated for 12 h and 24 h with 8505C-derived CM and TGFβ1 20 ng/ml. (D, E) Immunoblot for SLUG from THP-1 cells treated with CM derived from 8505C cells and TGFβ1 20 ng/ml for 18 h and 24 h. The same blot was stripped and re-blotted using anti-βActin antibody. The figure shows a representative Western blot of 4 independent experiments (D). Expression levels of SLUG mRNA levels by RT-qPCR in control and THP-1 cells treated for 18 h and 24 h with 8505C-derived CM and TGFβ1 20 ng/ml. Data are expressed as mean ± SEM. **P<0.005, ***P<0.0005, ****P<0.0001. ns, not significant.

Figure 3

TGFβ receptor inhibition affects 8505C-derived CM induced macrophage polarization as well as expression of SNAIL and SLUG. (A-C) THP-1 cells pre-incubated with SB431542 (20 µM) or dimethyl sulfoxide (DMSO) as control for 1 h, were treated with 8505C-derived CM for 18 h and 24 h. The expression of hCCL13 (A), hDectin1 (B) and hCD163 (C) were analyzed by RT-qPCR. (D, E) RT-qPCR of SNAIL (D) and SLUG (E) expression in THP-1 cells pre-treated with SB431542 (20 µM) before 8505C-derived CM treatment for 18 h and 24 h. Data are expressed as mean ± SEM. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. ns, not significant.

Figure 4

TGFβs, TGFβ receptors (TGFβRs), SNAIL and SLUG in ATC. (A-C) Boxplots showing TGFβ1-3 (A), TGFβ receptors, TGFβR1-3 (B), SNAIL and SLUG (C) gene expression in normal thyroids (NT) and ATC samples derived from Gene Expression Omnibus (GEO) datasets (GSE3467, GSE3678, GSE6004, GSE29265, GSE33630, GSE53157, GSE35570, GSE60542, GSE29265, GSE33630, GSE76039 and GSE65144). Statistical significance by Mann Whitney test; ****P<0.0001. ns, not significant. (D-G) Correlation between expression of TGFβ1 and SNAIL (D) and SLUG (E). Correlation between expression of SLUG and macrophages markers (CD68 and CD163).

Figure 5

Schematic model showing the contribution of ATC cell-secreted TGFβ1 on macrophage activation. ATC cells, among other soluble factors, produce and secrete high levels of TGFβ1, which activates its signaling pathway including up-regulation of SMAD2/SMAD3 transcription factors. Activation of the TGFβ1 signaling pathway in turn increases the expression of the transcription factors SNAIL and SLUG in THP-1 cells. The up-regulation of SNAIL and SLUG would induce M2-like macrophage polarization of human monocytes triggered by TGFβ1, thereby contributing to the progression of thyroid cancer. The activation of macrophages induced by ATC cell-secreted TGFβ1 would be independent of the IL-6/STAT3 signaling pathway activation induced by ATC cell-derived CM. Created with BioRender.