Journal: PLoS ONE

Article Title: Flavonoid Rutin Increases Thyroid Iodide Uptake in Rats

doi: 10.1371/journal.pone.0073908

Figure Lengend Snippet: (A) Thyroid TSH receptor (TSHR) protein (n = 12 per group) (B) and mRNA (fold increase=1.93; n = 10 per group) levels in rats treated with rutin (20mg/Kg BW, daily injections, sc.) for five days. GAPDH was used as internal control. *p < 0.05 vs. control.

Article Snippet: NIS antibody (kindly provided by Dr. Nancy Carrasco) was diluted 1:2000, while the TSH receptor antibody (purchased from Santa Cruz Biotechnology - California, USA) was diluted at 1:500.

Techniques: Control

Journal: Journal for Immunotherapy of Cancer

Article Title: TSH-TSHR axis promotes tumor immune evasion

doi: 10.1136/jitc-2021-004049

Figure Lengend Snippet: TSH released by moDCs promotes tumor proliferation (A) Expression of TSHR in different cancer types of TCGA cohorts. Tumors (red) and paired normal samples (green) are shown for each type. GBM, glioblastoma multiforme; LGG, lower grade glioma; THCA, thyroid carcinoma; THYM, thymoma. (B) Thyroid cancers (KTC1, BCPAP), glioma (U87, U251), and breast cancer (MCF7; wild type (WT) and TSHR-overexpressed (TSHR-OE)) were subjected for immunoblot analysis of TSHR and β-actin. (C–G) Evaluation of tumor proliferation through CCK-8 assay. KTC1, BCPAP, U87, U251, and MCF7 were treated with different concentrations of TSH (C) or culture supernatant of moDCs from patients with DTC and healthy donors (D). (E) BCPAP were treated with culture supernatant of moDCs from several patients with DTC. The correlation of BCPAP proliferation and TSHA(left)/TSHβ2(right) expression in moDCs. (F) KTC1 and U87 were treated with a culture supernatant of moDCs from patients with DTC and/or TSHR inhibitor (ML224). (G) WT and TSHR-OE MCF7 were treated with a culture supernatant of moDCs from patients with DTC. DTC, differentiated thyroid cancers; moDC, monocyte-derived dendritic cells; TCGA, the cancer genome atlas; TSH, thyroid-stimulating hormone; TSHR, TSH receptor.

Article Snippet: TSH (T9265, Sigma-Aldrich) and TSHR inhibitor (TSHRi) (ML224, MedChemExpress) were supplemented into the culture medium in the following experiments.

Techniques: Expressing, Western Blot, CCK-8 Assay, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: TSH-TSHR axis promotes tumor immune evasion

doi: 10.1136/jitc-2021-004049

Figure Lengend Snippet: TSH released by moDCs promotes tumor migration and invasion. (A) Effects of culture supernatant of moDCs and TSHRi on cell migratory abilities by wound healing assays in KTC1 and U87 cells. (B) Effects of culture supernatant of moDCs and TSHRi on cell invasive capacities by transwell assays in KTC1 and U87 cells. (C) Effects of culture supernatant of moDCs and TSHR-OE on cell migratory abilities by wound healing assays in MCF7 cells. (D) Effects of culture supernatant of moDCs and TSHR-OE on invasive capacities by transwell assays in MCF7 cells. Representative images of three independent experiments with similar results are shown. *P<0.05, **p<0.01, ***p<0.001. moDCs, monocyte-derived dendritic cells; TSHR, thyroid-stimulating hormone receptor; TSHRi, TSHR inhibitor; TSHR-OE, TSHR-overexpressed; WT, wild type.

Article Snippet: TSH (T9265, Sigma-Aldrich) and TSHR inhibitor (TSHRi) (ML224, MedChemExpress) were supplemented into the culture medium in the following experiments.

Techniques: Migration, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: TSH-TSHR axis promotes tumor immune evasion

doi: 10.1136/jitc-2021-004049

Figure Lengend Snippet: TSH released by moDCs promotes tumor immunosuppression (A) Volcano plot showing genes changed in KTC1 treated with culture supernatant of moDCs compared with control, as determined by RNA sequencing n=3 per group. Genes were upregulated (red; p<0.05, FC >2) or downregulated (green; p<0.05, FC<-2). (B) Heatmaps of the RNA sequencing data of KTC1 treated with the culture supernatant of moDCs. Representative genes from each category are shown. (C) KTC1, U87, and MCF7 were treated with the culture supernatant of moDCs (blue) or control medium (red) for 24 hours. Expression of CD31, PD-L1, PD-L2, and VISTA was assessed by flow cytometry. (D) Correlation of gene expression (PD-1, PD-L1, PD-L2, VISTA, IDO1) with infiltration level of dendritic cells by ‘TIMER’ tool in TCGA THCA, GBM, and LGG. (E, F) Tumor cells were co-cultured with moDCs through transwell with 0.4 µm pore size, which permitted molecules diffusion but not cell migration. KTC1 and U87 were treated with TSHR inhibitor (F). PD-L1 expression was assessed by flow cytometry. Representative histograms (left), PD-L1 MFI (median fluorescence intensity) (middle), and experiment schematics (right) were shown. (G) Immunofluorescence staining of differentiated thyroid cancers for PD-L1 (red) and TSHR (green). PD-L1 + TSHR + tumor cells were indicated in white rectangles. (H) Control and TSHR-overexpressed MCF7 were co-cultured with moDCs and T cells at a 2:1:1 ratio for 3 days and stained with PI and anti-CD45 antibodies. Representative PI +CD45- tumor cells (left), tumor death ratios calculated as PI + /CD45 - (right, up), and experiment schematics (right, down) were shown. FSC, forward scatter; GBM, glioblastoma multiforme; LGG, lower grade glioma; moDc, monocyte-derived dendritic cells; PD-1, programmed cell death protein-1; PD-L1, programmed death-ligand 1; TCGA, the cancer genome atlas; THCA, thyroid carcinoma; TSH, thyroid-stimulating hormone; TSHR, TSH receptor; TSHR-OE, TSHR-overexpressed; WT, wild type.

Article Snippet: TSH (T9265, Sigma-Aldrich) and TSHR inhibitor (TSHRi) (ML224, MedChemExpress) were supplemented into the culture medium in the following experiments.

Techniques: Control, RNA Sequencing, Expressing, Flow Cytometry, Gene Expression, Cell Culture, Pore Size, Diffusion-based Assay, Migration, Fluorescence, Immunofluorescence, Staining, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: TSH-TSHR axis promotes tumor immune evasion

doi: 10.1136/jitc-2021-004049

Figure Lengend Snippet: Mechanism of TSH-induced tumor PD-L1 expression (A) KTC1 (left) and U87 (right) were treated with the culture supernatant of moDCs from patients with DTC and/or TSHR inhibitor (ML224). Whole-cell lysates were subjected for immunoblot analysis of PD-L1, HIF1α, β-actin, phosphorylated, and total AKT, ERK, JNK, P38, P65. Representative immunoblot picture (up) and quantitative histogram (down) are shown. Expression of PD-L1 and HIF1α was calculated as the ratio between band intensity of these genes and β-actin. Phosphorylation of five kinases was calculated as the ratio between band intensity of phosphorylated protein and total protein. (B–C) KTC1 (up) and U87 (down) were treated with TSH (B) or culture supernatant of moDCs (C) and four inhibitors. Representative histograms (left) and PD-L1 MFI (right) were shown. (D) Transcription factors (TF) enrichment in KTC1 treated with culture supernatant of moDCs based on RNA sequencing data sets. The scatter plot ranked TF from first with increasing enrichment and decreasing ChIP-X enrichment analysis 3 scores. TF from the AP-1 family (blue) and other PD-L1-related TF (red) were colored. (E) KTC1 (up) and U87 (down) were treated with the culture supernatant of moDCs from patients with DTC and/or TSHR inhibitor (ML224). Whole-cell lysates were subjected for immunoblot analysis of β-actin, phosphorylated and total c-JUN, and STAT1. (F) Immunofluorescence staining of KTC1 (left) and U87 (right) for phosphorylated (down) and total (up) c-JUN. DTC, differentiated thyroid cancers; MFI, median fluorescence intensity; moDC, monocyte-derived dendritic cells; PD-L1, programmed death-ligand 1; TSH, thyroid-stimulating hormone; TSHR, TSH receptor.

Article Snippet: TSH (T9265, Sigma-Aldrich) and TSHR inhibitor (TSHRi) (ML224, MedChemExpress) were supplemented into the culture medium in the following experiments.

Techniques: Expressing, Western Blot, Phospho-proteomics, RNA Sequencing, Immunofluorescence, Staining, Fluorescence, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: TSH-TSHR axis promotes tumor immune evasion

doi: 10.1136/jitc-2021-004049

Figure Lengend Snippet: TSHR inhibitors boost antitumor immunity in vivo (A–E) C57BL6 mice were subcutaneously injected with 500,000 wild types (WT) or TSHR-overexpressed (TSHR-OE) B16-F10 tumor cells. Once tumors were palpable, mice were injected intraperitoneally (i.p.) with PBS or TSHR inhibitor (TSHRi, 10 mg/kg) on days 14, 16, 18, 20, and 24. Tumors were collected on day 24 after tumor inoculation and processed as described in methods (A). Each group was marked by a different color (A–E). The mean tumor volume of the tumor-bearing mice was shown in the right panel (A), n=6 mice/group. Data are presented as mean values±SEM. Representative histograms showing IFNγ expression in CD4 + T cells (B) and Granzyme B (GzmB) expression in CD8 + T cells (C). (D) Graphs showing PD-L1 MFI in macrophage, moDCs, plasmacytoid DCs (pDCs), neutrophils, eosinophils, and tumor cells. (E) Representative flow cytometry plots showing expression of FoxP3 vs CD25 in CD4 +T cells (left) and boxplots were showing percentage CD25 + FoxP3 + Treg cells of total CD4 + cells. (F–G) C57BL6 mice were subcutaneously injected with 500,000 GL261 cell lines. (F) Survival analysis of GL261-bearing mice treated with TSHRi. (G) Tumor volume change in mice treated with TSHRi and anti-PD1. IFNγ, interferon γ; MFI, median fluorescence intensity; moDC, monocyte-derived dendritic cells; PBS, phosphate buffered saline; PD-L1, programmed death-ligand 1.

Article Snippet: TSH (T9265, Sigma-Aldrich) and TSHR inhibitor (TSHRi) (ML224, MedChemExpress) were supplemented into the culture medium in the following experiments.

Techniques: In Vivo, Injection, Expressing, Flow Cytometry, Fluorescence, Derivative Assay, Saline

Journal: Medicine

Article Title: Production of parathyroid-like cells from thyroid stem cells in co-culture environment

doi: 10.1097/MD.0000000000032009

Figure Lengend Snippet: Immunofluorescence staining of proteins in thyroid cells using confocal microscopy before differentiation protocol. (A) TSHR (membrane) and (B) TTF1 (nuclear), Thyroid cells stained in green for all proteins and counterstained in blue for nuclei. The cytoskeleton was stained with phalloidin red to observe cell morphology. Objective 40 × original magnification, scale bars represent 50 µm. TSHR = thyroid stimulating hormone receptor, TTF1 = thyroid transcription factor 1.

Article Snippet: Immunohistochemistry were performed using primary antibodies against calcium-sensing receptor (CaSR, Abcam®; 1:100 dilution), parathyroid hormone [rPTH/911] (PTH, Abcam®; 1:100 dilution), TSH Receptor antibody (TSHR, Abcam®; 1:100 dilution), and TTF1 (Abcam®; 1:100 dilution).

Techniques: Immunofluorescence, Staining, Confocal Microscopy