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 (24).

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 (25). GAPDH was used as an internal normalization control. The primer sequences used were as follows: IGF1 forward, 5′-CAGCAGTCTTCCAACCCAAT-3′ and reverse, 5′-CCACACACGAACTGAAGAGC-3′; IGF2 forward, 5′-CGGACAACTTCCCCAGATAC-3′ and reverse, 5′-GTCTTGGGTGGGTAGAGCAA-3′; and GAPDH forward, 5′-CCAGGTGGTCTCCTCTGA-3′ and reverse, 5′-GCTGTAGCCAAATCGTTGT-3′.

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 (26). To investigate the role of M2-like TAMs in ATC, the THP-1 cells were first treated with PMA, and then IL-4 and IL-13 were continuously used. As shown in Fig. 1A, M2-like macrophages were larger and more stacked in morphology. The THP-1 macrophages treated with PMA plus IL-4 and IL-13 exhibited notable expression of M2-like TAM surface markers, including CD68, CD206 and CD163, whereas the expression of CD14 (a monocyte marker) was markedly decreased (Fig. 1B). These results indicated that the M2-like TAM model was successful. M2-like TAMs were then used in co-culture with C643 cells. The results revealed that after co-culture with M2-like TAMs, the invasion capacity of ATC cells was significantly increased (Fig. 1C and D). Furthermore, a sphere formation assay and western blotting were performed to assess cancer stemness. The results revealed that sphere formation and the expression of stemness-related markers Oct4, Sox2 and CD133 in ATC cells were significantly increased following co-culture with M2-like TAMs (Fig. 1E-H). EMT is considered to be an essential part of the process of tumour cell metastatic dissemination (27). Thus, the present study investigated whether M2-like TAMs were involved in EMT. As shown in Fig. 1G and H, co-culture with M2-like TAMs significantly decreased the expression of the epithelial marker E-cadherin and upregulated the expression of the mesenchymal markers N-cadherin and Vimentin in ATC cells. Taken together, these results indicated that M2-like TAMs accelerated the invasion and increased the stemness of ATC cells.

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 (Fig. 2A and B). ELISA results also confirmed that the secretion of IGF-1 and IGF-2 was significantly increased in the supernatant of the co-cultured cells compared with that of C643 cells alone (as a control group) (Fig. 2C and D). In addition, the western blotting results showed that ATC cells exhibited higher expression of p-IR-A and p-IGF1R after co-culture with M2-like TAMs (Fig. 2E and F), indicating that the M2-like TAMs activated IR-A/IGF-1R signalling in the ATC cells.

Next, exogenous IGF-1 or IGF-2 were used to treat C643 cells. As shown in Fig. 3A-D, it was found that exogenous IGF-1 or IGF-2 promoted the invasion and stemness in C643 cells. Furthermore, exogenous IGF-1 or IGF-2 increased the protein expression levels of N-cadherin, Vimentin, Oct4, Sox2 and CD133, and decreased the protein expression level of E-cadherin in C643 cells (Fig. 3E and F). Taken together, these data suggested that exogenous IGF-1/IGF-2 promoted the invasion and stemness of C643 cells.

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 (Fig. 4A-D). In addition, blockade of IGF-1 and IGF-2 reversed the increase in N-cadherin, Vimentin, Oct4, Sox2 and CD133 protein expression and the decrease in E-cadherin protein expression induced by M2-like TAMs in C643 cells (Fig. 4E and F). These data indicated that blocking IGF-1/IGF-2 suppressed the alteration of invasion and stemness of C643 cells induced by M2-like TAMs.

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 Fig. 5A and B, blockade of IGF1 and IGF2 significantly attenuated the protein expression of p-IR-A, p-IGF1R, p-AKT and p-mTOR. Although the expression of IR-A, IGF1R, AKT and mTOR showed no obvious change in any group, the ratios of p-IR-A/IR-A, p-IGF1R/IGF1R, p-AKT/AKT and p-mTOR/mTOR were markedly decreased following the blockade of IGF1/IGF2 in the co-culture system. These results indicated that IR-A/IGF-1R-mediated PI3K/AKT signalling was activated in the co-culture system and that blocking IGF1/IGF2 inhibited its activation.

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 (Fig. 6A-D). In addition, inhibition of the PI3K/AKT pathway reversed the increase in N-cadherin, Vimentin, Oct4, Sox2 and CD133 protein expression and the decrease in E-cadherin protein expression in the co-culture system (Fig. 6E and F). These results indicated that inhibition of the PI3K/AKT pathway inhibited the M2-like TAM-induced invasion and stemness of ATC cells.

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 (Fig. 7), indicating that the M2-like TAMs clustered with the ATC tissues.