Chemokine receptor CX3CR1 contributes to macrophage survival in tumor metastasis

Because the treatment and prognosis of colorectal cancer patients depends on the tumor grade (degree of primary tumor differentiation) and tumor-node-metastasis stage (how widespread the cancer is at the time of diagnosis), we first measured the CX3CR1 expression levels in normal colon tissues and human colon cancer tissue. As shown in Figure 1A, CX3CR1 was rarely detected in normal tissues, whereas CX3CR1 was observed in the inflammatory-like cells located in carcinoma tissue. CX3CR1 expression was significantly elevated in poorly differentiated colon carcinoma, which was much higher than that of moderately differentiated or well-differentiated colon carcinoma (Figure 1A). The clinical colon carcinoma stages were characterized by extensive invasion of the primary tumor into the submucosa, muscularis propria, or through the wall of the colon, and by invasion to nearby lymph nodes at stage III and to distant metastases in stage IV tumors. CX3CR1 was expressed in a high percentage of stage III and IV tumors (Figure 1A). Moreover, analysis of CX3CR1 expression in primary tumors and formation of metastases revealed that patients who had advanced clinical stage disease, metastasis, or recurrent lesion within 3 years showed increased CX3CR1 expression in colon cancer tissue (Figure 1A). Thus, CX3CR1 expression is associated with the aggressiveness of human colon cancer.

In addition, the advanced colon carcinoma tissues were markedly and extensively infiltrated by CD68-positive macrophages, particularly along the tumor cell-invasive front; however, the number of macrophages in normal colon tissues was low (Figure 1A). CX3CR1-positive cells within the area colocalized with CD68-positive macrophages, indicating a high concordance of CD68 and CX3CR1 expression in human primary colon carcinoma during malignant progression.

To further analyze the expression of CX3CR1 in the tumor stroma, we utilized a murine model in which mouse colon cancer cells (SL4) were injected into the spleen and tumors developed in the liver. Previous studies have shown that SL4 cells form tumors in the liver of 100% of syngeneic mice when injected intrasplenically. This model is not a pathological metastatic tumor model; however, SL4 cells are a useful tool to study how injected colon carcinoma cells seed and develop into a new microenvironment in the liver [16]. Liver tumor sections were immunostained with the macrophage marker F4/80 and an anti-CX3CR1 antibody. Immunofluorescence staining showed that CX3CR1 was abundantly expressed in macrophages in hepatic metastatic tumors of WT mice; as expected, there was no CX3CR1 expression in CX3CR1^−/− control mice (Figure 1B). These results indicate that expression of CX3CR1 in TAMs is increased during malignant progression, which might have predictive value in determining tumor outcome.

To investigate the role of CX3CR1 in tumor development of colon carcinoma cells in the liver, we injected CX3CR1^−/− mice and WT controls with SL4 cells intrasplenically. The SL4 cells generated tumors within the spleen (primary) and liver (metastasis). Multiple hepatic tumor nodules, detectable by gross inspection, were evident by 2 weeks. Livers were isolated and the incidence of hepatic metastasis was evaluated. Livers from CX3CR1^−/− mice showed significantly smaller tumor foci, fewer metastatic tumors, and decreased tumor-occupied area compared with those from tumor-bearing WT mice (Figure 2A).

To further evaluate the anti-metastasis effect in CX3CR1 deficiency, micrometastasis to livers were quantified in H&E-stained sections. WT mice exhibited massive and developed metastatic nodules in their livers, whereas CX3CR1^−/− mice had several small metastasis nodules (Figure 2B). Histologic analysis showed that the number of hepatic micrometastasis was significantly decreased in CX3CR1^−/− mice compared with the number in WT mice. These results indicate that CX3CR1 plays a critical role in promoting metastasis.

The relative proportions of leukocytes that infiltrated into hepatic metastatic tumors were analyzed and compared between CX3CR1^−/− and WT mice. As shown in Figure 3A, the proportion of CD45-positive leukocytes that infiltrated into metastatic tumors was significantly decreased in CX3CR1^−/− mice compared to that in WT mice (9.61 ± 0.87% versus 19.16 ± 1.12% for CD45, P<0.01). In particular, the proportion of infiltrating CD45⁺F4/80⁺ cells in metastatic tumors was significantly lower in CX3CR1^−/− mice than that in WT mice (31.22 ± 2.10% versus 51.79 ± 2.66% for CD45⁺F4/80⁺ cells, P<0.01; Figure 3A). Furthermore, immunohistochemical staining of metastatic foci further demonstrated that the expression of Mac-2 (a macrophage marker) was markedly decreased in CX3CR1^−/− mice relative to that in WT mice (Figure 3B). Thus, CX3CR1 plays an important role in promoting macrophage infiltration in metastatic tumors.

To investigate the mechanism by which CX3CR1 promotes macrophage infiltration into metastatic foci, we analyzed macrophage apoptosis in metastatic tumors. Both the number of TUNEL- and F4/80-positive (macrophage marker) cells was significantly increased in metastatic tumors in CX3CR1^−/− mice compared with those in WT mice (Figure 4A). Moreover, we analyzed the role of CX3CR1 on apoptotic pathways. As shown in Figure 4B, CX3CR1 knockout significantly increased the Bax/Bcl-2 ratio in metastatic tumors compared with that in WT mice. Similarly, caspase 3 activation was markedly unregulated in the metastatic tumors of CX3CR1^−/− mice versus WT mice (Figure 4B). However, no alteration of FOXO3a phosphorylation was detected in metastatic foci in either CX3CR1^−/− or WT mice (Figure 4B).

To understand the role of CX3CR1 in macrophage survival during hepatic metastasis of colon cancer, we cocultured macrophages derived from CX3CR1^−/− or WT mice with murine SL4 colon cancer cells in a 3D peptide gel. A peptide gel was chosen because it more accurately resembles the extracellular matrix milieu of carcinoma compared to Matrigel, which models the laminin-rich basement membrane-type extracellular matrix [17]. As shown in Figure 4C, the number of both TUNEL- and F4/80-positive (macrophage marker) cells was significantly increased in CX3CR1^−/− macrophages cocultured with SL4 cells, but not WT macrophages. Therefore, CX3CR1 is responsible for promoting macrophage survival within the tumor microenvironment, which is typically associated with TAMs that contribute to tumor development.

We investigated whether the decreased metastatic ability of tumor cells in CX3CR1^−/− mice could be caused by inhibition of angiogenesis. Thus, we stained slices of tumors of comparable size, grown either in WT or CX3CR1^−/− mice, with an anti-CD31 antibody (a marker of endothelial cells). As shown in Figure 5A, the total vessel area and size were significantly smaller in metastatic foci grown in the livers of CX3CR1^−/− mice than those in WT mice. Immunohistochemistry and western blot analysis showed that the levels of the proangiogenic cytokines VEGF and TGF-β were significantly decreased in metastatic liver tumors in CX3CR1^−/− mice compared with those in WT mice (Figure 5A and B). To further explore the relationship between CX3CR1 expression in macrophages and tumor angiogenesis, we assayed the proangiogenic function of macrophages from metastatic tumors. Flow cytometric analysis revealed that macrophages from metastatic tumors in CX3CR1^−/− mice expressed significantly less CCR2, VEGFR2, and CXCR4 (proangiogenic markers) compared to WT mice (40.72 ± 3.89% versus 75.78 ± 3.08% for CCR2, P<0.01; 36.58 ± 3.97% versus 55.08 ± 4.13% for VEGFR2, P<0.05; 39.04 ± 2.46% versus 70.78 ± 3.07% for CXCR4, P<0.01; Figure 5C).

To further confirm the in vivo proangiogenic role of CX3CR1 expression in macrophages, Matrigel plugs containing WT or CX3CR1^−/− macrophages were subcutaneously implanted in WT or CX3CR1^−/− mice. After 7 days, the mice were sacrificed and the plugs were microscopically analyzed by staining the blood vessels with an anti-CD31 antibody. As shown in Figure 5D, plugs containing WT macrophages in WT mice were well vascularized with well-formed and branched vessels, whereas plugs containing CX3CR1^−/− macrophages in WT mice displayed poorly organized vessels. In addition, plugs containing WT macrophages in CX3CR1^−/− mice showed well-formed vessels compared to those containing CX3CR1^−/− macrophages in CX3CR1^−/− mice. Taken together, these results suggested that CX3CR1 was necessary for TAMs-induced angiogenic responses during tumor development.

The antibodies for CX3CR1, CD68, CD31, VEGF, TGF-β, Mac-2, GAPDH, and IgG were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); the antibodies for phospho-FOXO3a, FOXO3a, Bax and Bcl-2 were from Cell Signaling Technology (Beverly, MA, USA); the antibodies for F4/80 and A-Caspase3 were from Abcam (Cambridge, MA, USA); and ChemMate TM EnVision System/DAB Detection Kits were from Dako (Glostrup, Denmark). Antibodies for PerCP/Cy5.5-conjugated CD45.2, phycoerythrin (PE)-conjugated F4/80, APC-conjugated CD11b and isotype control were from Biolegend (San Diego, CA, USA). Antibodies for PE-conjugated CCR2, PE-conjugated VEGFR2, PE-conjugated CXCR4 and isotype control were from R&D Systems (Minneapolis, MN, USA). DeadEnd™ fluorometric TUNEL system was from Promega (Madison, WI, USA).

CX3CR1 knockout (CX3CR1^−/−) mice and wild-type (WT) littermates were used for all experiments. CX3CR1 knockout (CX3CR1^−/−) mice were harbored a target replacement of the CX3CR1 gene by a GFP gene [36]. CX3CR1^−/− mice were obtained from the Jackson Laboratory and maintained in a Specific Pathogen-Free atmosphere at the Beijing Anzhen Hospital affiliated to the Capital Medical University, China. The investigations conformed to the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication no. 85–23, 1996) and were approved by the Animal Care and Use Committee of Capital Medical University.

The specimens from 30 cases of human colon carcinoma tissue/adjacent normal colon tissues and the clinicopathologic data were obtained from the Second Affiliated Hospital to Nanchang University gastrointestinal tumor bank. The specimens were isolated at the time of surgery, formalin-fixed and paraffin-embedded, and stained with hematoxylin and eosin, then examined by 2 experienced pathologists. The clinicopathologic stage was determined according to the TNM classification system of the International Union against Cancer. Human specimens use for research had been approved by the Second Affiliated Hospital to Nanchang University Research Ethics Committee.

A highly metastatic murine colon adenocarcinoma cell line, colon SL4, was used for the in vivo experiments [16]. SL4 cells were derived from C57BL/6 mice on the same background as the CX3CR1 deficient mice and WT control mice. SL4 cells were maintained in 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (DMEM/F12) containing 10% heat-inactivated fetal calf serum (FCS) in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. For in vivo model, after anaesthetizing mice, a transverse incision in the left flank was made, exposing the spleen, then 1.0 × 10⁶ SL4 tumor cells in 100 μl DMEM/F12 medium were intrasplenically injected with use of a 26-gauge needle. 14 days after inoculation, mice were sacrificed, and the tissues were processed as described below. After the initial dissection, the left ventricle was flushed by puncture with 40 ml normal saline to remove blood samples. The spleen and liver were removed, wet spleen and liver weights were measured, and the incidence of liver tumor development was examined.

Livers in mice were fixed for 24 hrs with 10% buffered formalin before embedding in paraffin. Serial sections of 5 μm thick were obtained for histologic analysis. Hematoxylin&eosin (HE) staining involved standard procedures.

For immunohistochemistry, sections were incubated with the primary antibody for CX3CR1(1:200), CD68(1:200), CD31(1:200), VEGF(1:200), TGF-β(1:200), or Mac-2(1:200), then incubated with the Dako ChemMate™ EnVision System (Dako, Glostrup, Denmark) for 30 mins. Staining was visualized with use of diaminobenzidine and counterstaining with hematoxylin. Negative controls were omission of the primary antibody, non-immune IgG or secondary antibody only; in all cases, negative controls showed insignificant staining. Images were captured with use of a Nikon Labophot 2 microscope equipped with a Sony CCD-Iris/RGB color video camera attached to a computerized imaging system and analyzed by use of ImagePro Plus 3.0 (ECLIPSE80i/90i; Nikon, Japan) with blinding to treatment. The expression of CX3CR1, CD68, CD31, VEGF, TGF-β, or Mac-2 was calculated as proportion of positive area to total tissue area for all measurements of the section.

For double immunofluorescence, tumors were excised and fixed in 4% paraformaldehyde for 2 hrs, then dehydrated with 30% sucrose/PBS and frozen in compound. Frozen tissue sections, 7 μm, were permeabilized and blocked with 0.1% Triton X-100, 0.2% bovine serum albumin, and 5% normal donkey serum in PBS, then incubated with the primary antibodies F4/80 (1:100) and CX3CR1 (1:100)overnight at 4°C, then FITC or TRITC-conjugated secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA, USA) at room temperature for 1 hr in the dark, and coverslipped with DAPI-containing mounting medium.

Identification of apoptotic cells was performed using TUNEL system Kit (Promega). Briefly, frozen slides were permeabilized using 0.1% solution of Triton X-100 in sodium citrate. Consequently, slides were incubated with TUNEL reaction mixture for 1 hr at 37°C. To allow detection of apoptotic macrophages, frozen sections were incubated with the primary antibodies for F4/80 (1:100) at 4°C overnight and then with TRITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) at room temperature for 1 hr. Nuclei were stained with DAPI (Invitrogen). The apoptotic index of macrophages was assessed as the percentage of apoptotic TUNEL⁺ F4/80⁺ cells to the total number of F4/80⁺ cells.

For extracellular staining of immune markers, single-cell suspensions were prepared by mechanical dispersion and enzymatic digestion of tumor tissues as described [17]. Briefly, tumor tissues were cut into multiple small cubes and digested in an enzyme mixture for 45 mins at 37°C. The cell suspension was centrifuged and preincubated with Fc-γ block antibody (anti-mouse CD16/32; Pharmingen, San Diego, CA, USA) to prevent nonspecific binding. Cell staining involved different combinations of fluorochrome-coupled antibodies to CD45, F4/80, CD11b, CCR2, VEGFR2 or CXCR4 for 30 mins at room temperature in the dark. Fluorescence data were collected by use of an EPICS XL flow cytometer (Beckman Coulter) and analyzed by use of Cellquest (Beckman). Fluorescence minus one (FMO) controls were included to determine the level of nonspecific staining and autofluorescence associated with subsets of cells in each fluorescence channel.

Protein extracts were were diluted with loading buffer and separated by electrophoresis on 10% SDS-polyacrylamide gels before transfer to nitrocellulose membranes. The membranes were blocked in Odyssey blocking buffer (LI-COR Bioscience, Lincoln, NE) at room temperature for 1 hr, then incubated at 4°C overnight with primary antibody: Bax (1:500), Bcl2 (1:500), p-Foxo3a (1:800), Foxo3a (1:800), A-caspase3 (1:800) or GAPDH (1:1000). The membranes were washed and incubated with fluorescent secondary antibodies (Alexa Fluor 680 or IRDye 800, Rockland Immunochemicals, Gilbertsville, PA, US) for 1 hr at room temperature at 1:5000, blots were analyzed with the Odyssey infrared imaging system and Odyssey software.

Macrophages were isolated from tibias and femurs of 8-week-old WT and CX3CR1^−/− mice as previously described in our lab [17]. Three-dimensional peptide gel co-culture was also as described [17]. Macrophages and SL4 cells were mixed at a ratio of 1:2 in peptide gel at a final peptide gel concentration of 3%. Peptide gel co-culture was maintained in DMEM containing 10% FCS at 37°C in a humidified atmosphere containing 5% CO₂. At 48 hrs after co-culture, peptide gel cultures were fixed in 4% paraformaldehyde in PBS for 15 mins at room temperature and washed for 20 mins in PBS. Frozen sections, 7 μm, of peptide gel cultures were treated with 0.15 M glycine in PBS at 4°C overnight to reduce autofluorescence.

700 μl of Matrigel (Becton Dickinson) was injected subcutaneously in the ventral area in WT or CX3CR1^−/− mice. Matrigel plugs contained 5 × 10⁵ WT or CX3CR1^−/− macrophages. Plugs containing PBS were used as a negative control, whereas plug containing 100 ng/ml bFGF (R&D Systems), a known angiogenic factor, was used as a positive control. Each experimental condition was analyzed in triplicate. 7 days after plug implantation, plugs were collected, immediately fixed in 10% buffered formalin. Identification of angiogenesis was performed by immunohistochemistry with the primary antibody against CD31.

Data analysis involved use of GraphPad software (GraphPad Prism version 5.00 for Windows, GraphPad Software). Results are expressed as mean ± SEM. Differences were analyzed by t test or ANOVA, and results were considered significant at a P<0.05.