Background
Colorectal cancer (CRC) is the third most common malignancy and the second leading cause of cancer-related mortality worldwide [
1]. Metastasis, a multi-step complex process involving multiple factors, is still the main cause for CRC-related deaths [
2]. Circulating tumor cell (CTC), originating from primary tumor or metastatic sites, is considered to be the precursors of metastases [
3]. Previously, our group reported several methods for CTC capture and identification, and demonstrated CTCs detection was closely associated with multiple clinicopathological factors that predicted high metastatic risk in different solid cancers, including gastric, colorectal and hepatocellular cancer [
4‐
7]. Subsequently, we found that only quantifying the CTC count is not sufficient to explain the important role of CTC in the metastasis process, nor can it understand the mechanisms of CTC-mediated metastasis. Meanwhile, we also found that CTC could undergo epithelial-mesenchymal transition (EMT). Moreover, numerous studies demonstrated mesenchymal CTC (
MCTC) had more prognostic value than total CTC, which was positively associated with tumor progression and poor patient’s survival in CRC, and knowing about the phenotype traits of CTC could give more information about CRC development [
8,
9]. Currently, EMT in cancer, as known to increase cell motility and invasive potential, has been proposed to play the critical role in CTC generation [
10]. CTC, which gains more mesenchymal traits by EMT, is easy to survive and metastasize [
11,
12]. Therefore, exploring the underlying mechanisms of CTC EMT have great significance for further understanding the metastatic process in CRC.
Tumor microenvironment (TME) represents the necessary prerequisite for cancer progression and metastasis [
13]. Macrophages in the TME, referred to as tumor-associated macrophages (TAMs), are one of the most abundant types of cells, and exhibit different phenotypes and functions in response to various microenvironmental signals generated from tumor and stromal cells [
14]. At present, numerous studies have showed that the localization and density of TAMs are associated with poor clinical outcome in several kinds of solid cancers, including bladder, breast, renal, prostate and gastric cancer [
15‐
19]. In terms of CRC, the exact roles of TAMs are seem to be somewhat contradictory [
20,
21]. Noteworthy, emerging studies have suggested that TAMs play important roles in tumor metastases by regulating EMT of cancer cells. In hepatocellular carcinoma (HCC), HCC-derived IL-8 stimulated the M2 polarization of TAMs, which promoted the EMT and invasive potential of HCC cells [
22]. Additionally, Wang and colleagues revealed that pancreatic cancer (PC) cells activated macrophages to the M2 phenotype, which then promoted EMT progress to increase migration and invasion of PC cells [
23]. Reciprocally, Su et al. showed that cancer cells that have undergone EMT secreted GM-CSF to promote macrophages recruitment, thereby mediating breast cancer metastasis [
24]. However, the roles and mechanisms of the crosstalk between TAMs and cancer cells in EMT of CRC are still unclear.
Given the crucial roles of TAMs, EMT and CTC in dictating CRC metastasis, we speculated that the crosstalk between TAMs and tumor cells could promote MCTC-mediated tumor metastasis by regulating the EMT program. In the present study, our results showed that CD163+ TAMs at invasive front were significantly correlated with EMT status, MCTC ratio, and patients’ prognosis in CRC. In vitro and in vivo experimental evidences also showed a significant increase in tumor EMT to enhance migration, invasion and metastasis in the presence of TAMs, confirming their pro-tumor functions in CRC. Further mechanistic studies revealed that TAMs induce EMT in CRC cells by regulating the STAT3/miR-506-3p/FoxQ1 axis, which in turn lead to the production of CCL2 to favor macrophages recruitment. These findings demonstrate a positive feedback loop between cancer cells and TAMs promotes CRC metastasis by regulating the EMT program of CTC, contributing to new insight concerning TME and CRC progression.
Methods
Patients and tissue samples
Primary CRC tissue samples were obtained from 81 patients who underwent curative resection at Zhongnan Hospital of Wuhan University (Wuhan, China). All included patients were identified as adenocarcinoma of colorectal by histopathology and had available preoperative CTC and survival data. Moreover, all patients were devoid of neoadjuvant chemotherapy or radiotherapy before surgical resection and did not be diagnosed with autoimmune diseases. Peripheral blood (PB) samples with a volume of 2.5 ml from all patients were collected in EDTA-containing tubes (BD, USA) at the time of one day before surgery. Formalin-fixed, paraffin-embedded (FFPE) cancer tissue specimens were obtained from these patients after surgery. All samples were collected with informed consent from patients, and all related procedures were performed with the approval of the internal review and ethics boards of Zhongnan Hospital of Wuhan University.
Immunohistochemistry
Paraffin-embedded samples were serially sectioned at 4 μm thickness. Antigen retrieval was performed by a pressure cooker for 30 min in 0.01 M citrate buffer (pH 6.0), followed by treatment with 3% hydrogen peroxide for 5 min. Specimens were incubated with monoclonal antibodies against human CD68 (1:500; Abcam, USA), CD163 (1:50; Abcam, USA), E-cadherin (1:200; CST, USA), Vimentin (1:200; CST, USA), IL6 (1:100; CST, USA) and FoxQ1 (1:100; Sigma-Aldrich, USA) overnight at 4 degree. Immunostaining was performed using DAB or Permanent Red (Dako) according to the manufacturer’s instructions. For negative control, isotype-matched antibodies were applied. Cells stained with indicated antibody were calculated calculated per field of view, with at least 10 view-fields per section were evaluated at 400× magnification. The expression levels of CD68, CD163, E-cadherin, Vimentin, IL6 and FoxQ1 were scored semiquantitatively based on staining intensity and distribution using the immunoreactive score (IRS) as described elsewhere [
25,
26]. Briefly, Immunoreactive score (IRS) = SI (staining intensity) × PP (percentage of positive cells). SI was assigned as: 0 = negative; 1 = weak; 2 = moderate; 3 = strong. PP is defined as 0 = 0%; 1 = 0–25%; 2 = 25–50%; 3 = 50–75%; 4 = 75–100%. All of the included patients were dichotomized into two groups (high expression group: >median score; low expression group: ≤median score) based on the median score of CD68 and CD163 expression.
CTC isolation and identification
CTC was enriched using the CTCBIOPSY
® device (Wuhan YZY Medical Science and Technology Co., Ltd., Wuhan, China) as described in our previous study [
7]. The samples were processed according to the manufacturer’s instructions. In brief, 2.5 ml blood sample of included patient was diluted up to 8 ml with 0.9% physiological saline containing 0.2% paraformaldehyde and left for 10 min at room temperature, then transferred to ISET tubes with an 8 μm diameter aperture membrane. After filtered by positive pressure from 12 mmHg to 20 mmHg, candidate CTC was adhered to the membrane and were identified by three-color immunofluorescence staining. Immunofluorescence staining was performed as described in our previous study [
7]. In brief, membranes with CTC were transferred to glass slides, which were fixed with 4% PFA for 5 min. Wash the membrane by BD wash buffer (BD, USA) for three times. Then, add 100 ul Cytofix/ Permeabilization Kit (BD, USA) on the membrane for 20 min in order to allow for intracellular staining. After that, add 10% Goat serum to block for one hour. Then, discard the serum and add the primary mouse antibody to FITC-CK (1:100; Abcam, USA), rat antibody to PE-Vimentin (1:100; Abcam, USA) and rat antibody to AF647-CD45 (1:100; Santa, USA) for incubation overnight at 4 °C. On the next day, wash the membrane by BD wash buffer and add the secondary Alexa Fluor 488-conjugated goat anti-mouse IgG (1:100; Invitrogen, USA), Alexa Fluor 546-conjugated goat anti-rat IgG (1:200; Invitrogen, USA) and Alexa Fluor 647-conjugated goat anti-rat IgG (1:200; Invitrogen, USA). Nuclei was stained with Hoechst 33342 (1:500; Sigma, USA) and incubated for one hour, then wash the membrane three times with BD wash buffer. Finally, we imaged and enumerated CTC using a fluorescence microscopy (IX81; Olympus, Tokyo, Japan). CTC captured on membranes were photographed using IPP software (Media Cybernetics Inc., Silver Spring, MD, USA). CK+/Vimentin−/CD45−/Hoechst+ cell, CK−/Vimentin+/CD45−/Hoechst+ cell and CK−/Vimentin−/CD45−/Hoechst+ cell was defined as epithelial CTC (
ECTC), mesenchymal CTC (
MCTC) and white blood cell (WBC), respectively. In this study,
MCTC ratio refered to the ratio of the number of
MCTC to the total number of CTCs in 2.5 ml of peripheral blood per patient.
Cell culture and reagents
The human monocyte cell line THP-1, HEK 293 T cells, human normal colon epithelial cell line NCM460 and CRC cell lines (HCT116, DLD-1, HT29, SW480, SW620 and Lovo) were purchased from the Chinese Academy of Sciences in Shanghai. Cells were cultured in RPMI 1640 medium (Gibco, USA) with 10% fetal bovine serum (FBS) (Gibco, USA) at 37 °C in a humidified atmosphere with 5% CO2. For macrophage generation, 3 × 105 THP-1 cells were seeded in 0.4 μm sized pores inserts treated with 200 nM PMA (Sigma-Aldrich, USA) for 24 h and polarized into macrophages. To obtain TAMs, THP-1 macrophages were cultured by the addition of conditioned media from CRC cell lines (HCT116 or HT-29) for another 24 h. Morphologies of treated macrophages were observed and photographed under an inverted microscope (ZEISS, German). Macrophages and CRC cell lines co-cultivation was conducted using the non-contact co-culture transwell system (Corning, USA). Inserts containing TAMs or THP-1 macrophages were transferred to 6-well plate seeded with CRC cells (1 × 105 cells per well) in advance and co-cultured. After 48 h of co-culture, TAMs or CRC cells were harvested for further analyses.
Recombinant human IL6 (R&D Systems) was dissolved in PBS containing 0.1% BSA and used at a final concentration of 50 ng/ml. STATTIC (STAT3 inhibitors), an anti-human neutralizing IL-6 antibody and an anti-human neutralizing CCL2 antibody were purchased from Med Chem Express, China.
Plasmid constructs, siRNAs, miRNAs, and transfections
The STAT3 eukaryotic expression vector (NM_003150) and FoxQ1 plasmid vector (NM_033260) were chemically synthesized, constructed, sequenced and identified by Shanghai GeneChem Chemical Technology, Co. Ltd., China. Vectors of STAT3-siRNA (NM_003150), FoxQ1-siRNA (NM_033260), IL-6-siRNA (NM_000600) or negative control RNA (si-control) were also chemically synthesized, constructed, sequenced and identified by Shanghai GeneChem Chemical Technology, Co. Ltd., China. CRC cells (HCT116, HT-29) and TAMs were transfected with siRNAs or or negative control RNA using X-treme GENE siRNA Transfection Reagent (Roche, USA) according to the manufacturer’s instructions. Forty-eight hours after transfection, cells were plated for a functional assay or harvested for RNA and protein analyses. miR-506-3p mimics and inhibitor were obtained from RiboBio Co. Ltd., China. The RNA was transfected using Lipofectamine 2000 (Invitrogen, USA), following the manufacturer’s instructions. Stably transfected HCT116 and HT-29 cells were derived from the parental cells by puromycin (Sigma-Aldrich, USA) selection.
Quantification of cytokines by enzyme-linked immunosorbent assay (ELISA)
The concentrations of cytokines were estimated for each experimental condition by ELISA, using commercial kits purchased from R&D Systems (Minneapolis, MN, USA), according to the manufacturer’s instructions. The cytokine kits included IL-10 (DY217B), IL-12 (DY1240), IL-1β (DY201), TNF-α (DY210), IFN-γ (DY285), and IL-6 (DY206). Positive controls were supplied in the kit.
Flow cytometry
Macrophages were processed into single cell suspensions, incubated with antibodies (PE Mouse anti-Human CD163, APC Mouse anti-Human CD206, FITC Mouse anti-Human HLA-DR, APC-Cy7 Mouse anti-Human CD80, all from BD Biosciences, USA) for 1 h at 4 °C. The cells were then washed twice with 4 ml of flow buffer, then centrifuged, and resuspended in 0.5 ml of flow buffer for analysis. Flow cytometry was performed using a FACSCalibur flow cytometer (BD Biosciences, USA). Flow cytometric analysis was performed on FlowJo software (FlowJo, USA).
RNA isolation and quantitative real-time PCR (qRT-PCR)
The total RNA from CRC cells and primary CRC xenograft tumor cells was isolated using the Trizol Reagent (Invitrogen, USA) according to the manufacturer’s instructions. After detection of RNA concentration, 1 μg of total RNA was reverse transcribed into cDNA using the PrimeScript™ RT reagent kit (Toyobo, Osaka). cDNA was used for subsequent qRT-PCR using the SYBR-Green PCR Master Mix (Takara, Osaka). Each reaction was run on the BioRad IQ5 Real time PCR machine (BioRad, USA). Relative expression was calculated using the 2
-ΔΔCt method. The sequences of primers used in the study are shown in Additional file
1: Table S3.
Luciferase reporter assay
For miRNA target report assays, the 3′-UTR sequences of FoxQ1, and miRNA binding sites were amplified from the genomic DNA and sub-cloned into the psi-CHECK2 (Promega, USA). For the FoxQ1 promoter assay, a 2000-bp DNA fragment containing STAT3 binding sites upstream from the FoxQ1 promoter was cloned into pGL3-Basic plasmid (Promega, USA). For the miRNA promoter assay, miR-506-3p promoter (− 2000/+ 1) and its truncation (− 1753/+ 1, − 1298/+ 1, − 1137/+ 1 and − 856/+ 1) were amplified from genomic DNA by PCR, and inserted into pGL3-Basic (Promega, USA). The mutant constructs of STAT3 binding sites in the miR-506-3p promoter were generated using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, USA) and also cloned into pGL3-Basic vector. Cells (5 × 104/well) were seeded at about 70% confluence in 24-well plates. For the miRNA target reporter assay, HEK293T were co-transfected with psi-CHECK-2 vectors and miRNA mimics, miRNA inhibitor or negative control using Lipofectamine 2000. For the STAT3-mediated miR-506-3p expression, pGL3-Basic luciferase reporters were transfected into HCT116 and HT29 cells after treated with IL-6 using Lipofectamine 2000. Renilla luciferase reporter vector pRL-SV40 (Promega, USA) was provided as an internal transfection control. The total cell lysates were harvested 48 h after transfection, and luciferase activities were determined using Dual-Luciferase reporter system (Promega, USA) according to the manufacturer’s instructions.
Western blot
Cells were lysed using a RIPA buffer, including a protease inhibitor cocktail (Thermo Scientific, USA). The proteins were separated by SDS-PAGE gels and transferred to PVDF membranes (Millipore, USA). After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies at 4 °C overnight. The HRP-conjugated secondary antibodies were used to incubate the membranes for 2 h at room temperature. The membranes were washed and incubated for 1 h at room temperature with HRP-conjugated secondary antibodies. Proteins were detected using a Bio-Rad ChemiDoc XRS + System. Bio-Rad Image Lab software was used for densitometric analysis. The following primary antibodies were purchased: anti-E-cadherin (1:1000; Cell Signaling, USA), anti-Vimentin (1:1000; Proteintech, USA), anti-p-JAK2 (1:1000; phosphor Y1007 + Y1008) (1:1000; Abcam, USA), anti-JAK2 (1:1000; Abcam, USA), anti-p-STAT3 (phosphor Y705) (1:1000; Cell Signaling, USA), anti-STAT3 (1:1000; Cell Signaling, USA), anti-p-AKT (phosphor S473) (1:1000; Abcam, USA), anti-AKT (1:1000; Abcam, USA), anti-p-ERK1/2 (phosphor T202 + T204) (1:1000; Cell Signaling, USA), anti-ERK1/2 (1:1000; Cell Signaling, USA), anti-FoxQ1 (1:1000; Sigma-Aldrich, USA), anti-GAPDH (1:5000; Santa Cruz, CA), anti-β-actin (Santa Cruz, CA).
For colony formation detection, 500 cells were planted in 6-well plates and cultured for 2 weeks. Cells were then fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. The assay was performed three times for each treatment. A wound-healing assay was used to evaluate the ability of CRC cells to migrate following culture with TAMs. Cells were grown to 80–90% confluence in 24-well plates, and a wound was made by dragging a plastic pipette tip across the cell surface. The remaining cells were washed three times in PBS to remove cellular debris and incubated at 37 °C with serum-free medium. Migrating cells at the wound front were photographed after 24 h. All experiments were performed in triplicate. The area of the wound was measured with Image J software (NIH, USA).
Transwell migration and invasion assay
Cell migration assays were performed using 24-well Transwells (8 μm pore size; Corning, USA) uncoated with Matrigel. Cell invasion assays were performed using 24-well Transwells (8 μm pore size; Corning, USA) pre-coated with Matrigel (Falcon 354,480; BD Biosciences, USA). In total, 1 × 105 cells were suspended in 500 μl RPMI 1640 containing 1% FBS and added to the upper chamber, while 750 μl RPMI 1640 containing 10% FBS was placed in the lower chamber. After 48 h of incubation, Matrigel and the cells remaining in the upper chamber were removed using cotton swabs. Cells on the lower surface of the membrane were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet. Cells in 5 microscopic fields (at × 200 magnification) were counted and photographed. All experiments were performed in triplicate.
Chromatin immunoprecipitation (ChIP) assay
ChIP assays were performed using a SimpleChIP® Enzymatic Chromatin IP Kit (Cell Signaling, #9003, USA) according to the manufacturer’s instructions. The resulting precipitated DNA specimens were analyzed by using PCR to amplify a 106-bp region (CHIP 1) of the miR-506-3p promoter with the primers 5′-ACC CAT GAA ATC ATC CCC TA-3′ (forward) and 5′-TGT GCA GAA GAC CGA AAA TG-3′ (reverse) and a 146-bp region (CHIP 2) of the miR-506-3p promoter with the primers 5′-TGT GTG TAT GAG CAT GTG TTT G-3′ (forward) and 5′-GAT TTA GGG GAT GAT TTC ATG G-3′ (reverse). The negative control is an encoding region of miR-506-3p, which was amplified by PCR with the primers 5′-GTG CCA TTT TAC TTT CCT ACC-3′ (forward) and 5′-TAG GGA AAT GCA ACC AAA ACC-3′ (reverse). The PCR products were resolved electrophoretically on a 1% agarose gel and visualized with use of ethidium bromide staining.
Animal experiments
All animal experiments were performed according to our institutions’ guidelines for the use of laboratory animals and were approved by the Institutional Animal Care and ethical committee of Zhongnan hospital of Wuhan University. For the tumor growth assay, the 6–8 weeks old nude mice were divided into four randomized groups (n = 6 per group), and HCT116 cells alone (5 × 105), TAMs alone (5 × 105), HCT116 cells (5 × 105) and TAMs/si-control (5 × 105), or HCT116 cells (5 × 105) and TAMs/si-IL-6(5 × 105) in 200 μl were subcutaneously injected into the flank of each mouse. After 10 days, we began measuring the tumor size every 5 days using digital vernier calipers, and calculated the tumor volume according to the following formula: volume = 1/2 × (width2 × length). Thirty days after cell injection, 1 ml of blood was collected via cardiac puncture into EDTA-containing tubes (BD, USA), the mice were sacrificed to collected the tumors and visually examined. For the liver and lung metastasis experiment, the 6–8 weeks old nude mice were divided into three randomized groups (n = 6 per group), and HCT116 cells alone (5 × 105), HCT116 cells (5 × 105) and TAMs/si-control (5 × 105), or HCT116 cells (5 × 105) and TAMs/si-IL-6(5 × 105) in 100 μl were injected into the mice via tail vein. Thirty days after cell injection, the mice were euthanized and were necropsied to assess metastatic burden. The tumor tissues, liver and lung tissues of mice were further examined by H&E, IHC staining, or RT-PCR assay.
Statistics analysis
All statistical analyses were performed with SPSS statistical software (version 22.0, IBM SPSS, USA) and GraphPad Prism software (version 6.0, GraphPad Software, USA) for Windows. Pearson’s correlation analysis was performed to assess the relationship between CD68, CD163 expression and MCTC ratio in the PB of patients. Chi-square test was applied to analyze the relationship between CD68 and CD163 expression and clinicopathological status. Groups of discrete variables were compared by means of the Mann-Whitney U test or Kruskal-Wallis nonparametric analysis of variance. Kaplan–Meier method was used for survival analysis and drawing the survival curves, and difference among patients’ subgroups was calculated by log-rank test. Univariate and multivariate Cox-regression analyses were applied to identify the independent factors of prognosis. All experiments for cell cultures were performed independently at least three times and in triplicate each time. In all cases, P values < 0.05 were considered statistically significant.
Discussion
In this study, we found that increased CD163+ TAMs infiltration in the tumor invasive front was significantly associated with EMT, MCTC ratio and dismal prognosis in CRC. Further studies confirmed that TAMs-derived IL6 induced EMT to enhance migration and invasion of CRC cells by regulating the STAT3/miR-506-3p/FoxQ1 pathway, and elevated CCL2 expression in TAMs-educated CRC cells significantly promoted the recruitment of macrophages in a feedback way.
Clinically, elevated level of CD163
+ TAMs localized at the invasive front was correlated with EMT phenotype,
MCTC ratio, and poor prognosis, indicating their potential roles in facilitating CRC dissemination and invasion. Recently, growing clinical evidences have suggested TAMs and EMT are related. Our results were in accordance with previous study, which comprehensively demonstrated that CCL18
+ TAMs infiltration in the tumor invasive front might establish an aggressive TME and could regulate breast cancer cells an EMT shift to increase metastatic ability [
33]. Lai et al. demonstrated that CD68
+ TAMs could both decrease Snail expression and inhibit tumor buds which negatively related with EMT phenotype in CRC [
34]. Different markers were used to identify TAMs in CRC, and CD68 had been widely recommended as a pan-macrophage marker, making this protein unspecific to the TAMs correlated with tumor growth, which might explain these discrepant effects of TAM subtypes on the EMT regulation of CRC. Furthermore, CTC disseminate from primary tumor by undergoing EMT that allow them to penetrate blood vessels [
35], and
MCTC was thought to have stronger invasive and metastatic ability [
36]. Qi et al. showed high ratio of
MCTC prior to resection was significantly associated with early recurrence, multi-intrahepatic recurrence, and lung metastasis in HCC [
11]. Our previous study also demonstrated
MCTC count in baseline level was significantly correlated with patients’ prognosis in CRC (unpublished data). Currently, our results further found higher ratio of
MCTC was detected in peripheral blood of patients with CD163
+ TAMs infiltrated in invasive front. Tumor invasive front is the most important area for the infiltration of cancer tissues and the immune response of cancerous hosts to cancer. The biological behavior of cancer cells in this location could best reflect the invasive ability of cancer tissues. At present, the clinical associations of high CD163
+ TAMs infiltration with poor clinical outcomes had been widely shown in numerous human cancers [
37,
38], however, whether CD163
+ TAMs, especially infiltrated in invasive front, contribute to better or poorer prognosis still remains contradictory in terms of CRC [
39,
40]. Herrera, et al. reported that infiltration of CD163
+ macrophages in CRC tissues was related to the shorter survival time [
41]. In contrast, Algars et al. showed that stromal infiltration of CD163
+ macrophages in CRC was correlated to a significantly improved survival [
40]. TAMs are distributed in the different microanatomical locations of CRC tissues, such as tumor center and invasive front, and TAMs in different locations could involve variations with different biological and prognostic properties. Combined the previous and our present results, we therefore supposed that this discrepancy could be the result of macrophages heterogeneity in distinct microanatomical locations, which allowed them to exert antagonistic functions-protumor or antitumor. Above results indicated CD163
+ TAMs infiltrated in invasive front may promote the production of
MCTC by regulating the EMT process of primary tumor cells, thereby affecting tumor progression and prognosis. This was the first study, to our knowledge, where assessment of different TAMs was used in purpose to explore the associations of their sub-localization with EMT phenotype and ratio of
MCTC in CRC.
In our study, characterization of in vitro-generated macrophages revealed that HCT116 or HT29-conditioned macrophages exhibited a mixed M1/M2 phenotype, with increased expression of the M2 markers CD163, as well as increased expression of the inflammatory cytokines, IL-1β, IFN-γ, and TNF-α. At present, regarding the roles of TAMs in EMT and tumor metastasis, most studies mainly focused on M2-polarized phenotype [
39,
42]. Nevertheless, because polarization of monocytes/macrophages was driven by environmental factors, it was likely that TAMs were not purely polarized M1- or M2- macrophages when facing the plethora of CRC released mediators, but rather exhibited both pro- and anti-inflammatory properties. Indeed, Penny and colleagues also reported that pancreatic ductal adenocarcinoma-generated TAMs expressed both M1 (IL-1β, IL6, and TNF-α) and M2 (CD163, CD206 and Arg1) markers [
43]. Moreover, as illustrated by our in vitro co-culture experiments and in vivo animal model, in the presence of mixed phenotype-TAMs, the growth, migration, invasion and metastasis of CRC cells were increased accompanied by EMT phenotype. These results firmly established that mixed phenotype-TAMs, especially with CD163 high expression, was a functional mediator of CRC tumorigenesis in vitro.
Given the key role of cytokines in cell-cell interactions, we screened the changes of a panel of inflammatory cytokines in the TAMs co-cultured with CRC cells, and IL6 was identified as the most significantly upregulated cytokine. Subsequent functional assays confirmed that IL6 was accountable for the TAMs-induced EMT, invasion, and metastasis in CRC. As a key cytokine linked to inflammation-associated cancer, IL6 is implicated in the facilitation of angiogenesis, tumorigenesis and progression by complicated mechanisms, such as increasing expression of invasion-related genes (Twist and MMP-1) and anti-apoptotic factors (Bcl-2 and Bcl-xL), and activation of PI3K, ERK, and STAT3 signaling pathway [
44,
45]. Herein, we revealed that IL6 phosphorylated STAT3, which led to EMT of CRC cells. EMT is orchestrated by several transcription factors. Among them, FoxQ1 plays an important role in the invasion and metastasis of many cancers [
46,
47]. Our study demonstrated FoxQ1 was the most upregulated transcription factors in CRC cells cocultured with TAMs, and silencing FoxQ1 abrogated TAMs-mediated EMT change and invasion/metastasis, indicating a driving role of FoxQ1 in the TAMs-induced EMT and aggressiveness of CRC. Consistently, Guo et al. reported that FoxQ1 was essential for TAMs-induced EMT and metastasis in gastric cancer cells [
48]. Notably, our study also found a novel reciprocal activation between cancer cells and TAMs that FoxQ1 expression in CRC cells co-cultured with TAMs promoted macrophage attraction in a CCL2-dependent manner. Previously, a study also reported FoxQ1 expression could promote macrophage infiltration through the VersicanV1/CCL2 axis in HCC [
31]. Additionally, Wolf et al. showed that CCL2 produced by CRC cells could also foster vascularization and intravasation [
49]. In breast cancer, inflammatory monocytes could be continually recruit by CCL2 produced by cancer cells and differentiate into TAMs that facilitate the subsequent growth of metastatic cells [
50]. At present, our group is conducting an in-depth study to explore the specific role of recruited macrophages in the CRC microenvironment. On the basis of our data and previous studies, we proposed that FoxQ1 expressed in cancer cells was closely involved in development of the TME by inducing invasion, metastasis, and chemotactic activity.
MiRNAs are small non-coding RNAs that affect tumor progression, achieved by binding to the target gene. Here, we found an inverse correlation between miR-506-3p and FoxQ1 expression in CRC cell lines, and further proved miR-506-3p targets FoxQ1 mRNA by binding to the FoxQ1 3’UTR to inhibit TAMs-induced EMT of CRC cells. The results of our study were consistent with the finding in previous reporters that miR-506-3p could significantly inhibit cell growth, invasion and enhance the chemotherapeutic response in CRC [
51,
52]. In the present study, we also found that FoxQ1 expression was regulated by STAT3, but this regulation was indirect and involved miR-506-3p as an intermediary, which indicated a potential link between FoxQ1 and IL6/STAT3 signaling. STAT3 directly bound two sites in the miR-506-3p promoter regions and was required for transcriptional regulation of this miRNA. STAT3-induced transcription of protein-coding genes has been widely reported in various types of cancers; however, the role of STAT3 in the transcription of non-coding genes, such as miRNAs, is relatively less studied. Recent results found that STAT3 played an important role in miRNA regulation. For example, STAT3 activated by IL6 directly upregulated the miR-21 and miR-181b-1, which was shown to be necessary for the epigenetic switch linking inflammation to cancer [
53]. Furthermore, STAT3-mediated activation of miR-182-5p had been shown to upregulate the proliferative and invasive capacities by directly targeting PCDH8 in glioma cells [
54]. STAT3-repressed miR-34a were also required for invasion and metastasis of human CRC cells [
55]. These data cumulatively demonstrated that STAT3-miRNAs interactions were emerging as key regulators of the malignant phenotype of cancer cells. Moreover, we also provided strong evidence that the activation of FoxQ1 induced by STAT3/miR-506-3p was important for CRC cell growth, migration, and invasion, which indicated that STAT3-miR-506-3p-FoxQ1 signal axis played an imperative role in the TAMs-medicated CRC progression.