A TAM model was established according to Lin et al (37), and the protocols for the treatment of animals were approved by the Ethics Committee of Tongji University prior to the study. The C57 mice were euthanized, and their hind legs were removed and placed in 75% alcohol for 5 min. Soft tissues were removed, a 26-G needle attached to a 1-cc syringe was inserted into the bone marrow cavity to wash out cells with RPMI-1640 (Gibco, Foster city, CA, USA) containing 10% fetal bovine serum (FBS; Gibco) until the bone became white. The medium with bone marrow cells was passed through a cell strainer with 70-µm pores (Merck Millipore, Billerica, MA, USA) into a 50-ml centrifuge tube. The cells were centrifuged for 10 min at 1,350 rpm, and the supernatant was subsequently discarded. Next, the cells were resuspended in 5 ml RPMI-1640 supplemented with 10% FBS and centrifuged for 10 min at 1,350 rpm. The cells were resuspended in RPMI-1640 containing 10% FBS and 1% penicillin-streptomycin (Gibco) to a final concentration of 5×10⁶ cells/ml. Then, 1×10⁷ cells/well were plated in 6-well plates. M-CSF (10 ng/ml; R&D Systems, Minneapolis, MN, USA) was added to the medium for M0 cell formation, or M-CSF, IL-4, IL-13, and IL-10 (10 ng/ml; R&D Systems) were added to the medium for TAM formation. The cells were incubated at 37°C in a humidified, 5% CO₂ incubator, and medium containing cytokines was changed daily for 4 days.

ID8 mouse EOC cells were purchased from Fuheng Biotechnology Co., Ltd. (Shanghai, China) and maintained in DMEM (Gibco) medium supplemented with 10% FBS and 1% penicillin-streptomycin. The cells were cultured at 37°C in a humidified, 5% CO₂ incubator.

The TAM CM transfer was performed according to Richards et al (9). TAM medium was centrifuged at 2,500 rpm for 30 min to remove cell debris and then mixed with complete DMEM at the ratio of 1:1. Complete RPMI-1640, and DMEM was mixed at the ratio of 1:1 as normal medium. TAM CM or normal medium was transferred to plates with ID8 cells for coculture daily. IGF1 neutralizing antibody (monoclonal, rabbit to mouse; Abcam, Cambridge, MA, USA) was dissolved in PBS and linsitinib (Selleck, Houston, TX, USA) was dissolved in DMSO before use.

Total RNA was extracted from macrophages and ID8 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Reverse transcription was performed using a Prime Script™ II 1st Strand cDNA Synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's instructions. qRT-PCR was performed using Talent qPCR PreMix (SYBR-Green) (Tiangen Biotech Co., Ltd., Beijing, China) and the StepOnePlus™ Real-time PCR system (Invitrogen) according to the manufacturer's instructions. The following primer sequences were used: CD204 forward, 5′-TGGAGGAGAGAATCGAAAGCA-3′ and reverse, 5′-CTGGACTGACGAAATCAAGGAA-3′; IGF1 forward, 5′-CACATCATGTCGTCTTCACACC-3′ and reverse, 5′-GGAAGCAACACTCATCCACAATG-3′; and GAPDH forward, 5′-AGGTCGGTGTGAACGGATTTG-3′ and reverse, 5′-GGGTCGTTGATGGCAACA-3′. The final concentration of all reagents was 2X Talent qPCR PreMix (with SYBR-Green I), 1X; 50X ROX Reference Dye, 5X; and forward and reverse primers: 0.3 µM. The PCR reactions cycling conditions included an initial cycle at 95°C for 15 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 15 sec. The data were calculated using the 2^−∆∆Cq method, with GAPDH as an internal normalization control.

Total protein was collected from M0 cells, TAMs, ID8 cells treated with or without TAM CM or linsitinib using RIPA lysis buffer (Beyotime, Shanghai, China). A BCA kit (Thermo Fisher, Rockford, IL, USA) was used for protein quantification according to the manufacturer's instructions. Protein was separated on 10% SDS-PAGE gels (EpiZyme, Shanghai, China) at a consistent voltage of 80 V in the stacking gel and 120 V in the separating gel and subsequently transferred onto polyvinylidene difluoride membranes at consistent current of 300 mA for 80 min. The membranes were blocked in TBST buffer with 5% bovine serum albumin (BSA) for 2 h at room temperature. Then, the membranes were incubated with the following primary antibodies at 4°C overnight: IGF1 (cat. no. ab9572; 1:1,000, monoclonal, rabbit to mouse; Abcam), CD204 (cat. no. ab15707; 1:1,000, polyclonal, rabbit to mouse; Abcam), IGF1R (cat. no. 9750; 1:1,000, monoclonal, rabbit to mouse; Cell Signaling Technology, Inc., Danvers, MA, USA), phospho-IGF1R (cat. no. 3918; 1:1,000, monoclonal, rabbit to mouse; Cell Signaling Technology, Inc.), Akt (cat. no. 4685; 1:1,000, monoclonal, rabbit to mouse; Cell Signaling Technology, Inc.), phospho-Akt (cat. no. ab81283; 1:1,000, monoclonal, rabbit to mouse; Abcam), Erk (cat. no. 4695; 1:1,000, monoclonal, rabbit to mouse; Cell Signaling Technology, Inc.), phospho-Erk (cat. no. 4370; 1:1,000, monoclonal, rabbit to mouse; Cell Signaling Technology, Inc.), GAPDH (cat. no. AB0036; 1:5,000; AB2000, monoclonal, rabbit to mouse; Abways, Shanghai, China), and the HRP-conjugated rabbit secondary antibody (cat. no. 16402-1-AP; 1:5,000; Proteintech, Wuhan, China) was used to incubate the membranes at room temperature for 1 h. The protein signals were detected using enhanced chemiluminescent HRP substrate (Merck Millipore).

Ovarian benign tumor specimens (patient ages ranged from 21 to 70 years) and EOC specimens (patient ages ranged from 33 to 74 years) used for tissue microarrays were obtained from the specimen repository of Shanghai First Maternity and Infant Hospital, after obtaining consent from each patient and the Ethics Committee of Tongji University. IHC was performed using an IGF1 primary antibody (1:125, monoclonal, rabbit to mouse; Abcam). Specimens (4-µm thick) were subjected to heat-induced epitope retrieval and then dewaxed in xylene and hydrated through a graded series of alcohol. The specimens were incubated in 0.3% H₂O₂ for 30 min to inactivate the endogenous peroxidase. Next, the specimens were incubated with 1% goat serum (Invitrogen) for 20 min at room temperature, followed by incubation with IGF1 antibody overnight at 4°C and then rabbit secondary antibody for 1 h at 37°C. A Vectastain ABC Elite kit (Vector Laboratories, Burlingame, CA, USA) was used according to the manufacturer's instructions for color development. The specimens were counterstained with hematoxylin and then dehydrated through a graded series of alcohol, cleared in xylene and mounted. Tumor cells with immunohistochemical expression in the cytoplasm were regarded as IGF1-positive, and the expression level was determined using the IRS system by multiplication of the staining intensity (0, no; 1, weak; 2, moderate; and 3, strong staining) and the percentage of positively stained cells (0, no staining; 1, <10% of cells; 2, 11–50% of cells; 3, 51–80% of cells; and 4, >81% of cells stained), according to Remmele and Stegner (38). The total score per sample therefore ranged from 0 to 12; a score <6 indicates low expression, whereas a score of 6–12 indicates high expression. The slides were examined in a blinded manner by two experienced investigators.

ID8 cells (1.5×10³) were seeded onto 96-well plates and cultured with 100 µl of medium for 72 h. Then, 20 µl of MTS Solution Reagent (PR Omega Biosciences, USA) was added to each well followed by incubation at 37°C in 5% CO₂ for 2 h. The absorbance was measured at 490 nm using a 96-well plate reader. Each group had six duplicates.

Transwell chambers (Corning, Glendale, AZ, USA) containing 8-µm inserts were used to measure the migration of tumor cells. ID8 cells (5×10⁴ cells) in 200 µl DMEM containing 2% FBS was plated in the top chambers. The bottom of the wells was filled with 800 µl complete DMEM containing 10% FBS. Calcein AM (Invitrogen) was used to stain the cells in the bottom of the filter membrane. The images of cells were captured using a fluorescence microscope at ×100. ImageJ software was used to count the cells that had migrated to the bottom of the filter membrane. Each group had two duplicates, and five images of different fields for each duplicate were captured.

All experiments were independently performed three or more times. Statistical analysis was performed using SPSS 19.0 (SPSS, Inc., Chicago, IL, USA). The data are expressed as the means ± SEMs or means ± SDs and analyzed using Student's t-test or the Mann-Whitney test. The correlation of IGF1 expression and clinical and pathological factors was analyzed using the Chi-square test or the Fisher's exact test. P-value <0.05 was considered statistically significant.

TAMs have been characterized as a popularized M2 macrophage phenotype different from the M1 type, which differentiates through a classic pathway. In the present study, a TAM model was established using isolated primary mouse bone marrow cells. Monocytes were induced to become undifferentiated M0 cells using MCSF. To induce M0 cells to differentiate into TAMs, IL-10, IL-13, and IL-4 were continuously used.

To determine whether the TAM model was successful, analyses of cell morphology and the expression level of the TAM-specific marker CD204 was used. As shown, TAMs were larger and more stacked in morphology (Fig. 1A). The expression of CD204 was higher in TAMs at both the mRNA (Fig. 1B) and protein levels (Fig. 1C).

As TAMs are associated with the enhanced progression of various cancers, including OC, and the number of TAMs in OC specimens is correlated with poor prognosis, we assessed whether TAMs similarly affect the physiological activity of mouse EOC cells. We first studied the effect of TAMs on the proliferation of ID8 cells, observing that TAM CM promoted the proliferation of ID8 cells (Fig. 2A). We next determined whether TAMs could change the migration of ID8 cells, and the results showed that the migration of ID8 cells treated with TAM CM was significantly increased compared with the control group (Fig. 2B). Taken together, these data showed that TAMs could accelerate the proliferation and migration of ID8 cells.

In a previous study, we performed a gene chip analysis to compare the expression profile of human TAMs and M0 cells, and observed that TAMs expressed significantly higher levels of IGF1 than M0 cells. We identified this result at both the mRNA and protein levels in mouse TAMs. Consistent with the gene chip analysis, the expression level of IGF1 was higher in mouse TAMs at both the mRNA (Fig. 3A) and protein levels (Fig. 3B).

The IGF1 level is higher in prostate cancer and in gastric cancer compared with corresponding benign tumors (24,25); thus, we examined whether EOC would have a similar phenomenon. First, we used western blot analysis to assess the level of IGF1 in fresh ovarian specimens, and observed that EOC specimens expressed higher levels of IGF1 compared with levels in the benign ovarian tumors (Fig. 4A).

Next, we used IHC staining to assess the expression level of IGF1 in tissue microarrays of benign ovarian tumors and EOC. The results showed that the mean IRS score of EOC specimens was significantly higher than that of the benign ovarian tumors (Fig. 4B). Then, we classified the EOC specimens into high and low expression groups, and used the Chi-square test or Fisher's exact test to identify the correlation between clinical and pathological factors and IGF1 expression levels. The median age of the patients was 56 years. Patients with a high IGF1 expression were in a more advanced stage and tended to have undergone liver metastasis more frequently (Table I). These results indicated that high IGF1 expression may enhance the progression of EOC.

Based on the preceding results, we determined whether TAMs could alter the proliferation and migration of mouse OC cells by upregulating the tumor-promoting gene IGF1. Therefore, we treated ID8 cells with TAM CM. As expected, the expression level of IGF1 in ID8 cells increased following culture with TAM CM at both the mRNA (Fig. 5A) and protein levels (Fig. 5B).

After observing that TAMs accelerate the proliferation and migration of ID8 cells and simultaneously upregulate IGF1, we finally assessed whether the blockade of IGF1 in TAM CM would reverse the changes in proliferation and migration. An IGF1 neutralizing antibody was added to TAM CM to block the IGF1 pathway. We observed that the blockade of IGF1 reduced the proliferation of ID8 cells (Fig. 5C). We then determined whether the IGF1 neutralizing antibody had the same effect on the migration of ID8 cells, observing that blockade of IGF1 reversed the increase in ID8 cell migration (Fig. 5D).

To validate the value of IGF1 pathway inhibition in EOC therapy, we used the IGF1R inhibitor linsitinib to inhibit the IGF1 pathway. As expected, the phosphorylation of IGF1R, Akt and Erk was significantly inhibited (Fig. 6A). Additionally, the proliferation and migration of ID8 cells were both significantly suppressed by linsitinib (Fig. 6B and C).

Taken together, these data suggest that IGF1 may be a key regulator by which TAMs promote the proliferation and migration of OC cells, indicating the potential of IGF1 inhibition in EOC therapy.