Tumor-associated macrophages correlate with the clinicopathological features and poor outcomes via inducing epithelial to mesenchymal transition in oral squamous cell carcinoma

One hundred twenty-seven primary OSCC specimens and 23 peritumoral tissue specimens were obtained from the Department of Oral Pathology and the Department of Oral Maxillofacial Surgery, Affiliated Hospital of Stomatology, Nanjing Medical University. The patients with primary OSCC had been treated with a wide excision surgery, with simultaneous an elective dissection of the regional lymph nodes or a classical radical neck dissection from August 2007 to April 2013 (74 men and 53 women; mean age, 61 years; rang, 34–88 years). None of the patients had received any preoperative radiation-chemotherapy. Peritumoral tissue samples were obtained from unaffected tissue surrounding the tumors. All the primary tumors were located in the tongue (n = 52), gingiva (n = 26), buccal mucosa (n = 31), lip (n = 4), floor of the mouth (n = 2), and others (n = 12). The tumor nest is the place at which tumor cells originates, accumulates, or develops, as the center on which a tumor forms. The tumor stroma is the microenvironment around the tumor cells. The TNM classification and clinical staging were carried according to the International Union Against Cancer (UICC). The pathological grade was classified into groups according to the WHO classification. The follow-up period ranged from 2 to 91 months (average: 41.2 months; median: 39 months). A total of 99 patients had at least a 2-year follow-up after treatment. This study protocol was approved according to the Ethics Committee of the Nanjing Medical University.

The specimens were fixed in a 10 % formaldehyde solution and embedded in paraffin. Then, Paraffin tissue sections (4–5 μm) with xylene dewaxing and with graded alcohols rehydrating. Endogenous peroxidases were blocked for 10 min using 3 % hydrogen peroxide. Antigen retrieval with 0.01 M sodium citrate retrieval buffer (pH 6.0) was carried out by microwave heating for 20 min. The sections were then incubated overnight with antibodies for E-cadherin (Cell Signaling Technology, Danvers, MA, USA), vimentin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD68 (Abcam, Cambridge, MA, USA), CD163 (Abcam) at 4 °C, and then incubated with appropriate secondary antibodies. Subsequently, the reaction product was visualized by incubation with 3,3’-diaminobenzidine and then counterstained with haematoxylin. Positive and negative controls were used in each staining run.

The immunoreactivity of the E-cadherin, vimentin, CD68 and CD163 in cancer cells was evaluated using the same method which was described in our previous studies [13]. Immunoreactivity was semiquanitatively evaluated by using immunoreactive score. The final immunoreactivity was divided into negative (score = 0), low (1 ≤ score ≤ 4) and high (score > 4). CD68-positive and CD163-positive macrophages were quantified by two pathologists who were blinded to the clinical patient data. They detected each section at low magnification (100×) and identified the areas with the highest macrophage density. The number of macrophages was counted in ten random high-power fields (HPFs, 400×), the mean number of macrophages per HPF was evaluated after two counts. On the basis of the number of macrophages, the samples were divided into low and high groups by using cut-off values of 6/HPF in tumor nest and 8/HPF in tumor stroma.

Human monocyte/macrophage cell line THP-1 was obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China. Human SCC9 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Human HN4 and HN6 were kindly provided from the Shanghai Ninth People's Hospital, Shanghai, China. The HN4, HN6 and SCC9 cancer cells were maintained in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (Invitrogen, Burlington, Ontario, Canada) supplemented with 10 % fetal bovine serum (FBS, Invitrogen), 400 ng/mL hydrocortisone (Sigma-Aldrich, St Louis, MO, USA) and 1 % penicillin/streptomycin solution (Invitrogen). The THP-1 cells were maintained in RPMI‑1640 medium (Invitrogen) supplemented with above FBS and antibodies. To obtain TAMs cells, we treated the THP-1 cells with phorbolmyristate acetate (PMA, 10 ng/mL, Sigma, USA) for 48 h, and then added the macrophage colony-stimulating factor (M-CSF, 10 ng/mL Sigma, USA) into the medium for 24 h. After a thorough wash with PBS for two times, the adherent cells were cultured in RPMI‑1640 medium supplemented with FBS and antibodies. Five days later, the cells were collected and detected the expression of CD68 and CD163 (experimental procedure was detailed in immunofluorescence microscopy), and the conditioned medium (CM) from macrophages was harvested, clarified by centrifugation, used for mass spectral protein analysis (Invitrogen) and treating with the cancer cells. The HN4, HN6 and SCC9 cancer cells treating with CM were regarded as the experimental group (HN4-M, HN6-M and SCC9-M cells) for subsequent studies.

The total RNA was extracted from cells by using the TRIzol reagent (Invitrogen), and mRNA was reverse transcribed into cDNA synthesis by using the 5 × PrimeScript RT Master Mix (TaKaRa, Otsu, Shiga, Japan) at 37 °C for 15 min and 85 °C for 5 s, according to the corresponding manufacturer’s protocol. Quantitative PCR (qPCR) was performed to evaluate the gene expression using 2 × SYBR Premix Ex Taq (TaKaRa) with a 7300 ABI Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) following the conditions: 95 °C for 30 s, 95 °C for 5 s, and 60 °C for 31 s for 40 cycles. The relative mRNA levels were checked by the 2 (−ΔΔCT) method and were normalized to expression of GAPDH. GAPDH (5’-GAAGGTGAAGGTCGGAGTC-3’, 5’-GAGATGGTGATGGGATTTC-3’), E-cadherin (5’-TACACTGCCCAGGAGCCAGA-3’, 5’-TGGCACCAGTGTCCGGATTA-3’), vimentin (5’-TGAGTACCGGAGACAGGTGCAG-3’, 5’-TAGCAGCTTCAACGGCAAAGTTC-3’).

Western blotting was performed as described in our previous study [14]. The blots were first probed with primary antibodies, such as anti-β-actin as control (1:1000, Santa Cruz Biotechnology), anti-E-cadherin (1:1000, Cell Signaling Technology), anti-vimentin (1:500, Santa Cruz Biotechnology), and then probed with appropriate secondary antibodies. The membranes were then developed by Immun-Star WesternC Kit (Bio-Rad, Hercules, CA, USA) products and bands were analyzed by exposure to film (Kodak, Japan).

Cells were grown on glass coverslips, fixed with 4 % paraformaldehyde (PFA) for 20 min at room temperature, permeabilised in 1 % Triton X-100 for 15 min, blocked with goat serum albumin for 30 min at 37 °C, then incubated with antibodies specific for CD68 (1:100, Abcam), CD163 (1:50, Abcam), E-cadherin (1:100, Cell Signaling Technology) and vimentin (1:100, Santa Cruz Biotechnology) followed by the appropriate secondary antibodies (1:50) and then further stained with DAPI (1:1000, Invitrogen) for 2 min. Cells were visualized using a Zeiss LSM-710 confocal microscope.

The cells were grown in 6-well plates to 90 % confluence and scraped with a pipette tip across the cell surface. After wounding, the suspension cells and debris were carefully removed, and the cells were incubated with serum-free medium. Migrating cells at the wound front were photographed after 24 h. Cells invasion assay was carried using Transwell filters (6.5-mm diameters, 8-μM pore sizes, Costar, Lowell, MA, USA). The filters were pre-coated for 30 min at 37 °C with Matrigel Basement Membrane Matrix (20 μL per square centimeter, BD Biosciences, MA, USA) mixed with serum free medium (1:3). Cells (4 × 10⁵) were washed with PBS, resuspended with 100 μL serum-free medium and added to the upper chamber, while a medium containing 10 % FBS was placed as a chemoattractant in the lower chamber. After incubating for 24 h at 37 °C in 5 % CO2, the chambers were fixed in 4 % PFA and stained with crystal violet (Sigma-Aldrich) for 30 min. The remaining cells and matrigel on the upper chamber were removed using cotton swabs, and the migratory cells present on the lower surface of the membrane were counted in ten random fields and photographed at 100x magnification (Olympus).

In this study, we assessed the expression of two TAMs markers CD68 and CD163 in tumor or peritumoral tissue specimens. Of note, CD68-positive macrophages were detected only in peritumoral stroma (Fig. 1 a1, a2, a3), where had no CD163-positive macrophages (Fig. 1 b1, b2, b3). However, CD68 and CD163 were detected on the plasma membrane or in the cytoplasm of the macrophages both in tumor nest and stroma (Fig. 1 c1, c2, c3; 1d1, d2, d3).

The number of CD68-positive macrophages in the nest and stroma of tumor was increased in 59 % (75 out of 127) and 66 % (84 out of 127) of samples, respectively. Interestingly, the increase of CD68-positive macrophages in the nest but not stroma correlates with the tumor location (P = 0.034) and mortality (P = 0.005, Table 1). In contrast, the density of CD163-positive macrophages in the tumor nest and stroma was higher in 59 % (75 out of 127) and 57 % (73 out of 127) of total samples (Table 2). Importantly, the density of CD163-positive macrophages in tumor nest was significantly associated with the sex (P = 0.037) and mortality (P = 0.044), while that in the tumor stroma was significantly correlated with the recurrence (P = 0.008) and mortality (P = 0.04). In addition, we analyzed the expression of CD68 and CD163 in 23 peritumoral tissue samples (Table 3). 52 % (12 out of 23, P = 0.374) of samples showed relatively high density of CD68-positive macrophages in peritumoral stroma, however, the density of CD163-positive macrophages (P < 0.001) was none in all samples. Based on these results, we conclude that the infiltration CD163-positive macrophages have a prognostic significance.

In addition to TAMs, tumor cells also expressed CD68 (Fig. 2 a1, a2, a3; 2c1, c2, c3) and CD163 (Fig. 2 b1, b2, b3;2 d1, d2, d3) on the plasma membrane and in the cytoplasm as detected by the immunohistochemical staining. CD68-positive tumor cells were detected in 83 % of samples, of which 79 cases (Fig. 2 a1, a2, a3) and 27 cases (Fig. 2 c1, c2, c3) showed relatively high and low expression of CD68. In contrast, CD163-positive tumor cells were found in 62 % of samples, of which 36 cases (Fig. 2 d1, d2, d3) and 43 cases (Fig. 2 b1, b2, b3) have increased and decreased expression of CD163. The expression levels of CD68 in tumor cells were only associated with lymph node metastasis (P = 0.044, Table 4), while the expression of CD163 was significantly associated with the recurrence (P = 0.013) and mortality (P = 0.001). This is the first time showed that a fraction of OSCC tumor cells exhibited definite CD68 and CD163 expression. More important, the high expression of CD163 was correlated with poor survival.

It has been reported that the number of TAMs is associated with EMT in tumor samples [16]. So we are wondering whether this relationship also exists in OSCC. Therefore, E-cadherin and vimentin, markers of EMT were detected by IHC. As shown in Fig. 3, the absence or reduced expression of CD68-positive macrophages (Fig. 3 a1, a2, a3) and increased expression of CD163-positive macrophages (Fig. 3 b1, b2, b3) was observed in tumor tissue, where have no expression of E-cadherin (Fig. 3 c1, c2, c3), but strong cytoplasmic staining of vimentin (Fig. 3 d1, d2, d3). Otherwise, Increased expression of CD68 (Fig. 4 a1, a2, a3) and CD163 (Fig. 4 b1, b2, b3) was also observed in tumor cells, whereas there was absence or reduced expression of E-cadherin (Fig. 4 c1, c2, c3), but strong cytoplasmic expression of vimentin (Fig. 4 d1, d2, d3) at the invasive front of the tumor. In the cancerous tissue, 99 out of 127 tumors express E-cadherin (78 %), the low expression, 36 cases; the high expression, 63 cases; and the negative expression, 28 (22 %) cases. Then, we check the expression levels of the tumors invasive front, 92 out of 127 (72 %) exhibit E-cadherin expression, the low expression, 52 cases; the high expression, 40 cases; and the negative expression, 35 (28 %) cases. As shown in Table 5 and Table 6, there was no significant relationship between the expression of E-cadherin and clinical variables, including sex, age, tumor location, tumor size, lymph node, metastasis, clinical stage, pathological grade, recurrence and mortality. Meanwhile, of the 127 tumors, 71 (56 %) cases express vimentin, the low expression, 39 cases; the high expression, 32 cases; and the negative expression, 56 (44 %) cases. Besides, high cytoplasmic staining of vimentin was also observed at the invasive front of tumors in 35 % (45 out of 127) patients. Despite no significant correlation with sex, age, and location and size of tumor, the expression of vimentin was associated with the metastasis into lymph nodes, clinical stage, pathological grade, recurrence and mortality. Especially at the tumor invasion front, the increased expression of vimentin was observed in 55 % (23 out of 42) patients with recurrence, and in 61 % (20 out of 33) patients with poor survival.

To investigate the role of TAMs in tumor progression, the relationship between the CD163, E-cadherin and vimentin was analyzed. As shown in Table 7, the levels of CD163 expression in tumor cells were positively associated with the expression of E-cadherin (P = 0.04 and P = 0.038, respectively) and vimentin (P < 0.001 and P < 0.001, respectively) in tumors and at the tumor invasion front. However, the CD163-positive macrophages have no association with E-cadherin and vimentin (data not shown).

To predict the prognostic value, survival analysis was carried by univariate Cox’s proportional hazard regression models in all the 127 patients with OSCC. The overall survival curve for the 127 samples was shown in Fig. 5a. The expression levels of CD68-positive macrophages (P = 0.009, Fig. 5b) and CD163-positive macrophages (P = 0.015, Fig. 5d) in tumor nest was significantly associated with poor prognosis. Furthermore, the expression levels of CD163 (Fig. 5g) in tumor cells (P = 0.001), vimentin both in tumor (P = 0.006, Fig. 5j) and at the tumor invasion front (P = 0.002, Fig. 5k) had a worse prognostic impact on the survival rate. However, the number of CD68-positive macrophages (Fig. 5c) and CD163-positive macrophages (Fig. 5e) in tumor stroma, and the levels of CD68 (Fig. 5f) and E-cadherin (Fig. 5h, 5i) expression in tumor cells did not shown any significant correlation with overall survival (Table 8).

Human THP-1 cells are always used as models to investigate the monocyte/macrophages differentiation [20]. In the current study, THP-1 cells quickly extended pseudopodia and became attached after the treatment with phorbolmyristate acetate (PMA, 10 ng/mL) for 48 h. Thereafter, the macrophage colony-stimulating factor (M-CSF) stimulates them to further differentiate into adherent macrophages (Fig. 6a). The expression of CD68 and CD163 on these macrophages was detected by immunofluorescence. As shown in Fig. 6b, these macrophages expressed not only CD68 (red) but also CD163 (green). Using mass spectral analysis, a variety of inflammatory cytokines and chemokines, including TGF-β, TNF, IL and so on (Fig. 6c) were present in the collected CM. Taken together, we confirmed that the TAMs were established and exhibited anti-inflammatory (M2) phenotype [20].

To clarity whether TAMs could induce the cells underwent EMT in OSCC, we treated the HN4, HN6 and SCC9 cells with the CM from TAMs and the morphology was observed. HN4-M, HN6-M and SCC9-M cells lose tight cellular junctions and became scattered and spindle shaped, appearing like fibroblasts (Fig. 7a). To further determine the EMT, the pattern of gene expression was analyzed by real-time PCR. The expression of E-cadherin decreased slightly, while vimentin expression increased sharply in HN4-M (3.46-fold, P < 0.05), HN6-M (1.99-fold, P < 0.05), and SCC9-M (2.22-fold, P < 0.05) cells (Fig. 7b). In line with the gene expression, Western blot analysis showed that the HN4-M, HN6-M and SCC9-M cells exhibited a significant decrease of E-cadherin, but an increase of the mesenchymal marker vimentin (Fig. 7c). In addition, we used double-staining immunofluorescence analysis to investigate the pattern of E-cadherin and vimentin. As Fig. 8 shown, the HN4-M, HN6-M and SCC9-M cells showed typical fibroblast-like and spindle-shaped appearance, with reduced E-cadherin but enhanced vimentin. Taken together, these results indicated that the TAMs could induce EMT in OSCC.

To verify the migration of cancer cells, a wound-healing assay was used. The CM promotes the cancer cells to migrate and the wound was closed easily after 24 h as compared to untreated cancer cells (Fig. 9a). Furthermore, Transwell assays were carried to measure the invasive ability of the cells. After 24 h, the invasive cells were quantified and captured with × 100 magnification in five randomly chosen fields (Fig. 9b). After the treatment of CM, the cancer cells showed an increase in invasion with HN4-M (4.34-fold, P < 0.001), HN6-M (2.76-fold, P < 0.001) and SCC9-M (6.55-fold, P < 0.001) cells (Fig. 9c). These results demonstrated that the TAMs could induce EMT and enhance the migration and invasion of OSCC.