M2-like tumour-associated macrophages (TAMs) have been demonstrated to promote the growth of anaplastic thyroid carcinoma (ATC). However, the underlying mechanism of M2-like TAMs in ATC remains unclear. Thus, in the present study, the role and mechanism of M2-like TAMs in ATC were investigated. M2-like TAMs were induced by treatment with PMA, plus IL-4 and IL-13, and identified by flow cytometry. Transwell and sphere formation assays were applied to assess the invasion and stemness of ATC cells. The expression levels of insulin-like growth factor (IGF)-1 and IGF-2 were examined by ELISA and reverse transcription-quantitative PCR. Proteins related to the epithelial-mesenchymal transition (EMT), stemness and the PI3K/AKT/mTOR pathway were examined via western blotting. Immunohistochemistry (IHC) was used to detect the expression of the M2-like TAM markers CD68 and CD206 in ATC tissues and thyroid adenoma tissues. It was found that treatment with PMA plus IL-4 and IL-13 successfully induced M2-like TAMs. Following co-culture with M2-like TAMs, the invasive ability and stemness of ATC cells were significantly increased. The expression levels of the EMT-related markers N-cadherin and Vimentin, the stemness-related markers Oct4, Sox2 and CD133, and the insulin receptor (IR)-A/IGF1 receptor (IGF1R) were markedly upregulated, whereas E-cadherin expression was significantly decreased. In addition, the production of IGF-1 and IGF-2 was significantly increased. Of note, exogenous IGF-1/IGF-2 promoted the invasion and stemness of C643 cells, whereas blocking IGF-1 and IGF-2 inhibited metastasis and stemness by repressing IR-A/IGF-1R-mediated PI3K/AKT/mTOR signalling in the co-culture system. IHC results showed that the expression of CD68 and CD206 was obviously increased in ATC tissues. To conclude, M2-like TAMs accelerated the metastasis and increased the stemness of ATC cells, and the underlying mechanism may be related to the section of IGF by M2-like TAMs, which activates the IR-A/IGF1R-mediated PI3K/AKT/mTOR signalling pathway.
Thyroid cancer is the most common malignancy of the endocrine system worldwide, and its incidence has shown a significant increase over the past 30 years (
Cancer stem cells (CSCs), also known as tumour-initiating cells, are a small subpopulation of cancer cells with the properties of multidirectional differentiation and metastasis, unlimited proliferation and self-renewal (
The insulin-like growth factor (IGF) system plays an important role in regulating the development and growth of mammals (
In the present study, the data showed that M2-like TAMs were enriched in human ATC tissues and that M2-like TAM-secreted IGF promoted the metastasis and stemness of ATC cells by activating the IR-A/IGF1R-mediated PI3K/AKT/mTOR signalling pathway. These data could improve the understanding of ATC progression and provide promising therapeutic targets for the treatment of ATC.
Tissues from 12 patients with thyroid adenoma (seven women and five men; mean age, 48.08±15.43 years old, range 22–68 years old), and tissues from 12 patients with ATC (six women and six men; mean age, 52.25±16.99 years old, range 19–73 years old) were acquired from the Third Affiliated Hospital of Kunming Medical University, also known as Yunnan Cancer Hospital (Kunming, China), collected from February 2019 to January 2020. Informed consent was obtained from all participants and the Research Ethics Committee of the Yunnan Cancer Hospital approved this study (approval no. KY2020220). The inclusion criteria were undifferentiated thyroid carcinoma patients with complete data. Patients with severe chronic diseases (such as renal insufficiency) and severe liver disease were excluded. Tissue samples were collected during surgery and immediately stored in liquid nitrogen and 4% paraformaldehyde.
The human monocyte cell line THP-1 was purchased from the American Type Culture Collection. The human anaplastic thyroid cancer cell line C643 was purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. Both cell lines were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% foetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Beijing Solarbio Science & Technology Co., Ltd.) in a humidified atmosphere at 37°C with 5% CO2.
C643 cells were incubated with serum-free medium containing 100 ng/ml IGF-1 or IGF-2 (Sigma-Aldrich; Merck KGaA) for 24 h at 37°C. To block IGF-1 and IGF-2 signals, cells were treated with serum-free containing anti-IGF-1 (cat. no. ab40657, Abcam) and anti-IGF-2 (cat. no. ab63984, Abcam) antibodies for 2 h at 37°C, and then co-cultured with M2-like TAMs. To block the PI3K/AKT pathway, cells were pre-incubated with serum-free medium containing PI3K/AKT pathway inhibitor LY294002 (20 µM; Sigma-Aldrich; Merck KGaA) for 2 h, and then co-cultured with M2-like TAMs for 24 h at 37°C.
THP-1 cells (1×106/well) were cultured in 6-well plates. To generate M2-like TAMs, 320 nM PMA (Sigma-Aldrich; Merck KGaA) was used to treat THP-1 cells for 6 h, and then the cells were treated with PMA plus 20 ng/ml IL-4 (Sigma-Aldrich; Merck KGaA) and 20 ng/ml IL-13 (Sigma-Aldrich; Merck KGaA) for another 18 h (
Following washing, trypsin digestion and centrifugation (1,000 × g, 4°C, 5 min), the M2-like TAMs were resuspended in 100 µl PBS (1×106 cells) and stained with 5 µl mouse anti-human CD14-FITC, CD68-FITC, CD206-PE and CD163-FITC antibodies for 30 min at 4°C. The stained cells were then analysed using a FACSCalibur flow cytometer (BD Biosciences) and Cell Quest 3.3 software (BD Biosciences). Antibodies against CD14-FITC (cat. no.367115; 1:500), CD68-FITC (cat. no. 333805; 1:500), CD206-PE (cat. no. 321105; 1:500) and CD163-FITC (cat. no. 333617; 1:500) were purchased from BioLegend, Inc.
A 0.4-µm Transwell chamber (Corning, Inc.) was used in the co-culture assay. Briefly, M2-like TAMs (2×105) were seeded into the upper chamber and co-cultured with C643 cells (2×105/well) in 6-well plates. After 24 h of co-culture at 37 °C, the upper chamber was discarded, and C643 cells in the lower chamber were collected and used for subsequent experiments.
A 24-well Transwell cell culture chamber (Corning, Inc.) coated with Matrigel (BD Biosciences) for 30 min at 37°C was used to investigate cell invasion ability. To assess invasion, C643 cells were harvested after co-culture with M2-like TAMs for 24 h at 37°C. Then, the C643 cells were diluted to 1×105/ml in 200 µl serum-free RPMI-1640 medium and added to the upper chamber. RPMI-1640 medium (600 µl) containing 10% FBS was placed in the lower chamber. After incubation for 24 h at 37°C, the invaded cells in the lower chamber were fixed with 4% paraformaldehyde at 25°C for 30 min and stained with 0.1% crystal violet at 25°C for 15 min. The invaded cells were imaged and counted at ×200 magnification using a light microscope in five different fields for each chamber.
C643 cells were harvested after co-culture with M2-like TAMs for 24 h. Then, C643 cells (4×104/well) were plated in ultra-low-attachment 24-well plates (Corning, Inc.) and maintained in serum-free DMEM-F12 (Sigma-Aldrich; Merck KGaA) containing B27 supplement minus vitamin A (Gibco; Thermo Fisher Scientific, Inc.), 20 ng/ml epidermal growth factor (R&D Systems) and 20 ng/ml basic fibroblast growth factor (R&D Systems). After 2 weeks, cell spheroids with a diameter >75 µm were counted at ×100 magnification using a light microscope.
Cellular lysates were prepared using radioimmunoprecipitation assay lysis buffer (Wuhan Boster Biological Technology, Ltd.) supplemented with protease inhibitors (Roche Diagnostics) according to the manufacturer's protocols. A bicinchoninic acid protein assay kit (Wuhan Boster Biological Technology, Ltd.) was used to determine the protein concentration. Equal amounts of protein (30 µg of lysates) were separated via 10% SDS-PAGE, and then separated proteins were transferred onto PVDF membranes (EMD Millipore). The membranes were then blocked with 5% non-fat milk at room temperature for 1 h, followed by incubation at 4°C overnight with primary antibodies against E-cadherin (cat. no. ab15148; 1:500), N-cadherin (cat. no. ab18203; 1:500), Vimentin (cat. no. ab137321; 1:1,000), CD133 (cat. no. ab19898; 1:1,000), Oct4 (cat. no. ab18976; 1:500), Sox2 (cat. no. ab97959; 1:500), IGF1R (cat. no. ab131476; 1:500), phosphorylated (p)-IGF1R (cat. no. ab39398; 1:1,000), IR (cat. no. ab137747; 1:1,000), p-IR (cat. no. ab60946; 1:1,000), AKT (cat. no. ab18785; 1:500), p-AKT (cat. no. ab38449; 1:500), mTOR (cat. no. ab2732; 1:2,000), p-mTOR (cat. no. ab84400; 1:1,000) and GAPDH (cat. no. ab9485; 1:2,000). All antibodies were purchased from Abcam. After the membrane was incubated with HRP-conjugated goat anti-rabbit immunoglobulin G secondary antibody (cat. no. BA1054; 1:2,000; Wuhan Boster Biological Technology, Ltd.) at room temperature for 1 h, the signals were developed using an enhanced chemiluminescence reagent (EMD Millipore) according to the manufacturer's instructions. Image-Pro Plus 6.0 software (Media Cybernetics, Inc.) was used to semi-quantify the protein bands, and GAPDH was used as a loading control.
Isolation of total RNA from cultured cells was performed with TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Briefly, RT was performed according to the manufacturer's protocol and cDNA was generated using a PrimeScript™ RT reagent kit (Takara Biotechnology Co., Ltd.). qPCR was conducted using SYBR Green P0.remix Ex Taq II (Takara Biotechnology Co., Ltd.) in an Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was performed under the following conditions: 95°C for 30 sec, followed by 40 cycles of 95°C for 3 sec and 60°C for 30 sec. The 2−∆∆Cq method was used for comparative quantitation (
C643 cells were co-cultured with M2-like TAMs and C643 cells alone were used as a control group. After 24 h of co-culture at 37°C, the supernatant was collected and centrifuged at 200 × g for 5 min at 4°C. Then, following the instructions of the Human IGF Signaling Antibody Array (cat. no. ab197446; Abcam), the optical density of the collected supernatant was detected at 450 nm using a microplate reader, and the concentration of IGF-1 or IGF-2 was calculated.
The tissues were fixed in 4% paraformaldehyde for 24 h at 4°C and dehydrated with a gradient of ethanol (100, 95, 80 and 70%). The tissues were embedded in paraffin and sectioned at a thickness of 3 µm. The sections were soaked in 3% H2O2 for 10 min and blocked with 5% non-immune goat serum (cat. no. 5425; Cell Signaling Technology, Inc.) at room temperature for 10 min, followed by incubation with anti-CD68 (cat. no. ab213363; 1:2,000; Abcam) or anti-CD206 (cat. no. 91992; 1:400; Cell Signaling Technology, Inc.) antibody at 4°C for ~12 h. Subsequently, the sections were washed with PBS buffer three times and incubated with secondary antibodies (cat. no. 8114; 1:2,000; Cell Signaling Technology, Inc.) at 25°C for 30 min. Then, the sections were stained with DAB reagent (Sigma-Aldrich; Merck KGaA) for 5 min at room temperature and stained with haematoxylin for 2 min at room temperature. The histomorphological changes were observed and imaged under a light microscope (Leica DM LB2; Leica Microsystems, Inc.).
Statistical evaluation was performed using SPSS 20.0 software (IBM Corp.). The values are presented as the mean ± standard deviation. Differences among multiple groups were compared by one-way analysis of variance followed by Tukey's post hoc test and differences between two groups were compared by unpaired Student's t-test (for parametric data). P<0.05 was considered to indicate a statistically significant difference.
Human THP-1 cells are widely used as a model for macrophage differentiation (
To further investigate the underlying mechanisms by which M2-like TAMs regulate cancer stemness and invasion of human ATC, RT-qPCR, ELISA and western blot assays were performed to discover the potential molecular mechanism. Significant induction of IGF-1 and IGF-2 mRNA expression was found in the supernatants of the co-culture group (
Next, exogenous IGF-1 or IGF-2 were used to treat C643 cells. As shown in
After observing that M2-like TAMs accelerated the invasion and increased the stemness of C643 cells and simultaneously promoted the production of IGF-1 and IGF-2, the present study next assessed whether blockade of IGF-1/IGF-2 would suppress the M2-like TAM-induced alteration of invasion and stemness of C643 cells. An IGF-1-/IGF-2-neutralizing antibody was used to block the IGF pathway, and the blockade of IGF-1 and IGF-2 inhibited the invasion and stemness induced by M2-like TAMs in C643 cells (
Next, the effects of blocking IGF1/IGF2 on the IR-A/IGF-1R-mediated PI3K/AKT signalling pathway in the co-culture system were examined. As shown in
As the present study observed activation of the PI3K/AKT/mTOR signalling pathway in the co-culture system, it was next investigated whether the PI3K/AKT/mTOR pathway was involved in cell invasion and stemness in the co-culture system. The PI3K/AKT pathway inhibitor LY294002 was used, and inhibition of the PI3K/AKT pathway significantly inhibited the invasion and stemness of the co-culture system (
Next, IHC was performed to detect the expression of the M2-like TAM markers CD68 and CD206 in ATC tissues and thyroid adenoma tissues. The results showed that the expression of CD68 and CD206 in ATC tissues was markedly increased compared with that in thyroid adenoma tissues (
ATC remains one of the most fatal human malignancies, despite significant progress in diagnosis and treatment. Cancer stemness and distant metastasis are the main causes of poor survival in patients with ATC (
Macrophages have functional plasticity and can be differentiated into differentially polarized TAMs under different microenvironmental stimuli (
IGFs are often present in large quantities in the tumour microenvironment and can be secreted by tumour stroma and/or malignant cells. Elevated circulating IGF levels are associated with a higher cancer risk, and experimental evidence has suggested that IGFs contribute to the growth, metastasis and chemoresistance of various cancers (
Activation of the PI3K/AKT pathway has been linked to cancer cell proliferation, survival and apoptosis by regulating downstream molecules (
In summary, the present study revealed that M2-like TAM-secreted IGF-1 and IGF-2 promoted cancer invasion, stemness and EMT of ATC via the IR-A/IGF-1R-mediated PI3K/AKT/mTOR signalling pathway. These data provided novel insights into the molecular mechanism underlying ATC progression, and currently available IGF1/IGF2 inhibitors may be a therapeutic strategy for ATC intervention.
Not applicable.
This work was supported by a grant from the Joint Program of Yunnan Province and Kunming Medical University [grant no. 2017FE467(−080)] and the National Natural Science Foundation of China (grant no. 81860312).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
ZYD was the guarantor of integrity for the entire study. ZYD and ZPF conceived the study, and ZYD and JL designed the study. PJL, LJ, ZXY and FH performed the experiments. ZXY acquired the data, CL performed data analysis and FKC conducted the statistical analysis. JL prepared and edited the manuscript. ZYD and ZPF reviewed the manuscript. ZYD and CL confirmed the authenticity of all the raw data. All authors read and reviewed the final manuscript.
Informed consent was obtained from all participants and The Research Ethics Committee of the Yunnan Cancer Hospital approved this study (approval no. KY2020220; Kunming, China).
Not applicable.
The authors declare that they have no competing interests.
tumour-associated macrophages
anaplastic thyroid carcinoma
cancer stem cells
insulin-like growth factor
insulin receptor
epithelial-mesenchymal transition
Effects of M2-like TAMs on the invasion, stemness and EMT of anaplastic thyroid carcinoma cells. (A) THP-1 cell morphology was observed via microscopy after treatment with PMA plus IL-4 and IL-13. Scale bar, 100 µm. (B) M2-like TAM surface markers, including CD14 (monocytes), CD68 (M2 TAMs), CD206 (M2 TAMs) and CD163 (M2 TAMs), were identified by flow cytometry after treatment with PMA plus IL-4 and IL-13. (C and D) The invasive ability of C643 cells was determined by Transwell assay after co-culture with M2-like TAMs for 24 h. (E and F) The cancer stemness of C643 cells was determined by a tumour sphere-formation assay after co-culture with M2-like TAMs for 24 h. (G and H) EMT and cancer stemness markers of C643 cells were examined by western blotting after co-culture with M2-like TAMs for 24 h. C643 cells were cultured alone as the control groups. The experiment was repeated three times, and the results are shown as the mean ± standard deviation. **P<0.01. TAM, tumour-associated macrophage; EMT, epithelial-mesenchymal transition.
Effects of M2-like TAM-secreted IGF-1 and IGF-2 on the activation of IR-A/IGF-1R signalling in anaplastic thyroid carcinoma cells. (A and B) The IGF-1 and IGF-2 mRNA levels in C643 cells, M2-like TAMs and co-cultured cells were measured by reverse transcription-quantitative PCR. (C and D) The concentrations of IGF-1 and IGF-2 in supernatants obtained from C643 cells, M2-like TAMs and co-cultured cells were measured by ELISA. (E and F) The protein expression levels of IR-A, IGF-1R, p-IR-A and p-IGF-1R in C643 cells were measured by western blotting after co-culture with M2-like TAMs for 24 h. C643 cells were cultured alone as the control groups. The experiment was repeated three times, and the results are shown as the mean ± standard deviation. *P<0.05 and **P<0.01. TAM, tumour-associated macrophage; IGF, insulin-like growth factor; IR, insulin receptor; IGF-1R, IGF1 receptor; p-, phosphorylated.
Effects of exogenous IGF-1/IGF-2 on the invasion and stemness of C643 cells. (A and B) Invasive ability was determined using a Transwell assay in C643 cells treated with IGF-1 or IGF-2. (C and D) The cancer stemness of C643 cells was determined by a tumour sphere-formation assay. (E and F) epithelial-mesenchymal transition and cancer stemness markers of C643 cells were examined by western blotting. The experiment was repeated three times, and the results are shown as the mean ± standard deviation. *P<0.05 and **P<0.01 vs. control. TAM, tumour-associated macrophage; IGF, insulin-like growth factor.
Effects of IGF-1-/IGF-2-neutralizing antibody on invasion and stemness of co-cultured cells. The co-culture system of C643 cells and M2-like TAMs was cultured in RPMI-1640 medium containing IGF-1-/IGF-2-neutralizing antibody (2 µg/ml) for 24 h. (A and B) The invasive ability of C643 cells was determined by a Transwell assay. (C and D) The cancer stemness of C643 cells was determined by a tumour sphere-formation assay. (E and F) Epithelial-mesenchymal transition and cancer stemness markers of C643 cells were examined by western blotting. The experiment was repeated three times, and the results are shown as the mean ± standard deviation. *P<0.05 and **P<0.01. TAM, tumour-associated macrophage; IGF, insulin-like growth factor.
Effects of IGF-1-/IGF-2-neutralizing antibody on IR-A/IGF-1R-mediated PI3K/AKT/mTOR signalling in co-cultured cells. The co-culture system of C643 cells and M2-like TAMs was cultured in RPMI-1640 medium containing IGF-1-/IGF-2-neutralizing antibody (2 µg/ml) for 24 h. (A and B) The expression of IR-A/IGF-1R/PI3K/AKT signalling pathway proteins in C643 cells was examined by western blotting. C643 cells were cultured alone as the control groups. The experiment was repeated three times, and the results are shown as the mean ± standard deviation. *P<0.05 and **P<0.01. TAM, tumour-associated macrophage; IGF, insulin-like growth factor; IR, insulin receptor; IGF-1R, IGF1 receptor; p-, phosphorylated.
Effects of LY294002 on invasion, stemness and EMT in anaplastic thyroid carcinoma cells after co-culture with M2-like TAMs. C643 cells were pre-treated with LY294002 (20 µM) for 2 h and then co-cultured with M2-like TAMs for 24 h. C643 cells were cultured alone as the control groups. (A and B) The invasive ability of C643 cells was determined by a Transwell assay. (C and D) The cancer stemness of C643 cells was determined by tumour sphere-formation assay. (E and F) EMT and cancer stemness markers of C643 cells were examined by western blotting. The experiment was repeated three times, and the results are shown as the mean ± standard deviation. *P<0.05 and **P<0.01. EMT, epithelial-mesenchymal transition; TAM, tumour-associated macrophage.
Expression of CD68 and CD206 in ATC tissues. CD68 and CD206 expression levels were notably increased in ATC tissues. The M2-like tumour-associated macrophage markers CD68 and CD206 in ATC tissues and thyroid adenoma tissues were analysed by immunohistochemistry staining. Scale bar, 100 µm. ATC, anaplastic thyroid carcinoma.