CD11b deficiency suppresses intestinal tumor growth by reducing myeloid cell recruitment

Mac-1 (CD11b) is expressed on bone marrow-derived immune cells. CD11b binds to ligands to regulate leukocyte adhesion and migration across the endothelium or epithelium. Here, we employed CD11b knockout mice and an ApcMin/+ spontaneous intestinal adenoma mouse model to clarify the function of CD11b in intestinal tumorigenesis. We showed that CD11b deficiency may contribute to the inhibition of myeloid cell trafficking to the tumor microenvironment and inactivated Wnt/β-catenin pathway to suppress tumor growth. This effect was partly mediated by inhibiting the myeloid cell-mediated decrease in TNF-α secretion, which inhibits the recruitment of myeloid-derived suppressor cells to the tumor microenvironment and subsequently induces IFN-γ and CXCL9 production. This work provides evidence for the mechanism by which CD11b may function as an important oncogene and highlights the potential of CD11b as a therapeutic target in CRC.

Scientific RepoRts | 5:15948 | DOi: 10.1038/srep15948 of cells: cells with a granulocytic phenotype (Ly6G + Ly6C low ) and cells with a monocytic phenotype (Ly6G -Ly6C high ) 18,19 . Only the Ly6G + Ly6C low granulocytic MDSCs have been shown to expand in most tumor models, and these cells may play an important role in the general process of angiogenesis and tumor progression [19][20][21] . Moreover, the number of circulating MDSCs was also significantly increased in cancer patients and correlated with the clinical cancer stage 19 . Recent studies demonstrated that myeloid cells in tumor hosts can be transported to the spleen, peripheral blood and tumor sites and produce cytokines and chemokines that activate MDSCs and allow them to infiltrate the tumor sites 17 . Therefore, we hypothesized that CD11b-deficient myeloid cells may likely regulate MDSCs infiltration into the tumor environment and thereby inhibit the angiogenesis and tumor growth of colorectal carcinoma.
Wnt/β-catenin signaling is constitutively activated in nearly all colorectal tumors and leads to a loss of E-cadherin and the nuclear translocation of β-catenin. This signaling pathway then modulates the expression of a broad spectrum of target genes to promote tumor cell proliferation. Inflammatory cells in CRC tumors have been shown to be associated with tumor progression. Moreover, cytokines that are secreted by the inflammatory cells may interact with the Wnt signaling pathway in the tumor microenvironment and lead to an accumulation of β-catenin in the nucleus 22 . However, the ability of MDSCs that infiltrate the tumor environment to activate Wnt/β-catenin signaling in CRC is unknown.
In this study, we used the Apc Min/+ CRC mouse model and Mac-1-deficient (CD11b −/− ) mice to define the possible role and the underlying regulatory mechanisms of Mac-1 in intestinal tumorigenesis.
Patients and tissue samples. A total of 10 cases of colonic tumor tissues and their matched non-tumorous colonic tissues were collected from the Department of Pathology, The Third Affiliated Hospital of Sun Yat-Sen University. Written informed consent from each patient was obtained prior to the initiation of this study. Pathologic diagnosis was performed by two independently pathologists. All methods were performed in accordance with the guidelines approved by the Ethics Committee of Medicine, Guangdong Pharmaceutical University. Animals and treatment. C57BL/6 (C57) mice were purchased from the Guangdong Medical Laboratory Animal Center. APC Min/+ mice (strain: C57BL/6J-Apc Min /J, stock number: 002020) and CD11b −/− mice (strain: B6.129S4-Itgam tm1Myd /J, stock number: 003991) on the C57BL/6J background were purchased from the Jackson Laboratory (Bar Harbor, ME). APC Min/+ ;CD11b −/− mice were obtained by crossbreeding the APC Min/+ mice and CD11b −/− mice to the F2 generation. The founder mice were viable and exhibited normal growth. Genotyping was performed by PCR using genomic DNA prepared from mouse tail according to the genotyping protocol. The primers for CD11b genotyping were 5′ -TAG GCT ATC CAG AGG TAG AC-3′ (for the wild-type and targeted alleles); 5′ -CAT ACC TGT GAC CAG AAG AGC-3′ (for the wild-type allele); and 5′ -ATC GCC TTC TTG ACG AGT TC-3′ (for the targeted allele). The primers for APC Min/+ genotyping were 5′ -GCC ATC CCT TCA CGT TAG-3′ , 5′ -TTC CAC TTT GGC ATA AGG C-3′ and 5′ -TTC TGA GAA AGA CAG AAG TTA-3′ (for both the wild-type and targeted alleles). Eleven mice from each group were used for the studies, unless otherwise indicated. All mice (15-week old) that were used for analyses were maintained in a climate-controlled room at a temperature of 24 ± 2°C and a relative humidity of 60 ± 5% under a 12-h light/dark cycle. All surgical procedures were performed under diethylether anesthesia. BrdU (0.1 mg/g body weight) was intraperitoneally injected into the mice, which were sacrificed 2 hours later. All experiments were performed in accordance with the protocols approved by the Ethics Committee of the Center of Laboratory Animals at Guangdong Pharmaceutical University.
Scientific RepoRts | 5:15948 | DOi: 10.1038/srep15948 Cell Culture. The human HCT-116 and mouse CT-26 colorectal carcinoma cell lines were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μ g/mL streptomycin and incubated in a humidified chamber containing 5% CO 2 at 37 °C. TNF-α was added to the cells at a dose of 50 ng/mL for 48 hours, and the total proteins were prepared for an immunoblotting analysis.
Isolation of myeloid cell and co-culture. The single suspension of bone marrow derived all nucleated cells from femurs and tibias was collected and maintained in DMEM supplemented with 100 μ g/mL PHA, 10% FBS, 100 U/mL penicillin and 200 μ g/mL streptomycin and incubated in a humidified chamber containing 5% CO 2 at 37 °C for 2 days.
CT-26 cells and HCT-116 cells were seeded into 24-well plates at a density of 8 × 10 4 per well and 2 × 10 5 per well respectively without FBS for 24 hours, and then re-added FBS and co-cultured with myeloid cells for 24 hours. Myeloid cells were loaded into the upper compartment of the transwell chambers at a density of 1:10 (myeloid cell: tumor cell) with or without TNF-α antibody (mouse anti-TNF-α , 1:500, BA0131, Boster, China). Then the tumor cells were collected for further detection.

Analysis of intestinal tumors.
After the APC Min/+ mice and APC Min/+ ;CD11b −/− mice were sacrificed, the entire gastrointestinal tract was excised and separated into the colon and 3 segments of the small intestine: proximal, medial, and distal. All regions were opened longitudinally, flattened between sheets of filter paper, immersed in 10% phosphate-buffered formalin, and then stained with 10% methylene blue. The tumor numbers and sizes were determined using dissecting microscope (OLYMPUS, Japan), and the tumor volume (V) was calculated according to the following equation: V = (L × W 2 ) × 0.5236 (L: length; W: width). The intestinal neoplasias were classified using microscope as described previously 23 (Supplementary Figure 1).
Microvessel Density. Microvessel density (MVD) was recorded as the number of point counts of endothelial cells with the specific antibody to CD34 per field at × 200 magnification. Ten fields were randomly selected in a section of tumors were examined. MVD counts were recorded independently by two observers in sections from three mice of each group.
Immunoblotting. The intestines were sliced longitudinally, and the macroscopic tumors were cut off from the intestines. The total proteins from the tumors and cells were prepared using RIPA buffer, and immunoblotting assays were performed as previously described 26 .

Flow cytometry (FACS). A single cell suspension of blood cells, bone marrow cell, splenocytes or
tumor digests that had been treated as described above was subjected to flow cytometry using the following MDSC surface markers: CD45, CD11b, Ly6C, and Ly6G. To analyze the inflammatory cell infiltrates in the tumor tissue, the tumors were mechanically dissociated on a wire mesh by crushing with the plunger of a 10-mL syringe and then incubated in tissue-digestion buffer at 37 °C for 25 min. The cells were filtered through 70-μ m nylon strainers (BD Biosciences, Bedford, MA), stained with specific antibodies and analyzed by flow cytometry. The FACS data were acquired using a Beckman Coulter Gallios flow cytometer and were analyzed using the FlowJo software package (Tree Star, Ashland, OR, USA).
To detect the cell cycle progression, the tumor cells in co-culture system were collected and fixed the cells with 75% ethanol for 40 min at 4 °C, centrifuged, washed twice in phosphate buffered saline, and stained with PI solution (#550825, BD Biosciences, USA) at 37 °C for 15 min. The analysis was performed using a FACS Calibur flow cytometer (Becton Dickinson) and analyzed using the Modfit software, version 3.0 (Verity Software House).
Real-time quantitative PCR arrays. The total RNA was extracted from the blood or spleen of mice using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The total RNA (500 ng) was reverse transcribed using an PrimeScript TM RT Reagent Kit (TaKaRa, Japan), and the real-time quantified PCR was performed on a LightCycler480 PCR machine (Roche) using the SYBR ® Premix Ex Taq ™ II (Tli RNaseH Plus) PCR Kit (TaKaRa, Japan), to the manufacturer's instructions. The data were analyzed using the 2 −∆∆CT methodology as described 27 .
Statistical analysis. The data are presented as the mean ± standard deviation (SD) and the differences between groups were analyzed using a Student's t test. Differences were considered statistically significant at P < 0.05. The protein expression levels in the IHC slices were determined by measuring the cumulated integrated optical density (IOD) using IPP software (Media Cybernetics, Inc., USA). The densitometric analysis of the immunoblotting bands was performed using Quantity One software (Bio-Rad, USA), and the protein band intensities were quantitated and normalized to those of GAPDH.

Results
Intestinal tumor development is accompanied by infiltrating myeloid cells. Infiltrating inflammatory myeloid cells often reportedly heavily infiltrate solid tumors 7,28 . To determine the level of myeloid cell infiltration, we stained the tumors with a CD11b antibody to detect myeloid cells using immunofluorescence analysis (IF) and further stained a single cell suspension of blood cells, bone marrow cell and splenocytes with CD11b antibody to detect myeloid cells (gated on CD45 + cells (all leukocytes)) using FACS assay 11 . To study the tumor microenvironment, we immunofluorenscently stained the human tumor tissues (Tumor) and their matched surrounding non-cancerous colonic tissues (Normal) with CD11b antibody. We observed that the levels of the CD11b + myeloid cells were increased in the tumor tissues compared with the non-cancerous colonic tissues (Fig. 1a). The Apc Min/+ mouse is a model for the development of CRC. This transgenic mouse model has been widely used to study the development of intestinal tumorigenesis 29 . We examined the infiltration of myeloid cells in the spontaneous adenomatous tissues of Apc Min/+ mice by IHC staining. Many CD11b + myeloid cells had infiltrated the mesenchyme of the tumor region (Fig. 1b). In addition, we detected the number of CD11b + myeloid cells (gated on CD45 + cells) in the bone marrow, spleen and peripheral blood by FACS to determine the myeloid cell contribution to intestinal tumorigenesis. With the exception of the peripheral blood, the CD11b + myeloid cell populations were substantially enriched in the bone marrow and spleen of the Apc Min/+ mice compared with the C57 mice ( Fig. 1c-e). These results suggest that CD11b + myeloid cells are activated during CRC development.  (Fig. 2e, left panel). Moreover, we also observed the development of invasive carcinoma in the intestines of 15-week-old Apc Min/+ mice, albeit less frequently. However, invasive carcinoma was not observed in the Apc Min/+ ;CD11b −/− mice at the same age (Fig. 2e, right panel).

CD11b deficiency inhibits intestinal tumor growth in vivo.
Reports indicated that myeloid cells infiltrate the tumor microenvironment can support tumor growth and promote tumor angiogenesis 4,17 . The results showed that depletion of CD11b + cells in the Apc Min/+ mice abrogated leukocyte cell infiltration in the tumor microenvironment but not in the adjacent normal villus tissue (Fig. 2f and Supplementary Figure 2g). We further detected tumor cell proliferation and angiogenesis by IHC staining. Most tumors in the experimental CD11b-deficient mice displayed significantly fewer BrdU-positive cells (Fig. 2g) and CD34 point counts as well as significantly reduced VEGF expression (Fig. 2h,i). These data suggest that the CD11b-deficient myeloid cells significantly inhibited the growth of intestinal tumors.

CD11b deficiency inactivates the Wnt/β-catenin pathway during intestinal tumorigenesis.
The canonical Wnt/β-catenin signaling pathway is critical for the homeostasis and neoplastic transformation of the intestinal tract 31,32 . APC mutation results in the activation of the Wnt pathway. Constitutively active Wnt/β-catenin signaling is associated with CRC initiation, and leads to an accumulation of β-catenin in the nucleus and a loss of E-cadherin. Whether CD11b-deficient myeloid cells that infiltrate the tumor microenvironment inhibit intestinal tumorigenesis by inactivating the Wnt/β-catenin signaling has not yet been determined. Compared with the Apc Min/+ mice, IHC staining (Fig. 3a-c and supplementary Figure 3) and immunoblotting assay (Fig. 3d) indicated a significant increase in the expression of E-cadherin and decreased expression levels of β-catenin and cyclin D1, the downstream target of the Wnt/β-catenin pathway, in the tumor tissues but not in the adjacent normal tissue of the Apc Min/+ ;CD11b −/− mice. Decreased nuclear translocation of β-catenin was observed in the tumor cells of the Apc Min/+ ;CD11b −/− mice by IF staining (Fig. 3e). These results indicate that activated Wnt/β-catenin signaling was partially inhibited by deficient CD11b + myeloid cells infiltration into the tumor microenvironment.

CD11b deficiency inhibits intestinal tumor growth by reducing TNF-α release. Myeloid cells
that infiltrate the tumor microenvironment stimulate cancer initiation, malignant progression and angiogenesis by releasing a number of potent pro-tumorigenic cytokines. Therefore, the Inflammatory Cytokines & Receptors PCR Array (#APM-011, SUPERARRAY, USA) was used to identify the cytokines that might affected by CD11b + myeloid cells in CRC using a quantitative RT-PCR assay (supplementary Figure 4a). The results indicated that the CD11b-deficent tumor tissue exhibited a robust 1.36-fold decrease in TNF-α expression. We detected the concentration of TNF-α in the peripheral blood with an ELISA assay, and found that the TNF-α level in peripheral blood was up-regulated in the Apc Min/+ mice compared with the C57 mice. However, the increase in the TNF-α level was reversed in CD11b-deficient Apc Min/+ mice (Fig. 4a). The total RNAs of the peripheral blood and spleen were extracted, and the quantitative RT-PCR assay showed the same effect as the ELISA (Fig. 4b). TNF-α expression was also decreased in the tumor tissues of the Apc Min/+ ;CD11b −/− mice compared with Apc Min/+ mice, as analyzed by immunoblotting   . 4c). We further detected the effect of TNF-α on tumor growth and Wnt/β-catenin signaling activity in HCT-116 cells (this cell line shows low Wnt/β-catenin signaling activity) and found that TNF-α significantly induced cell proliferation (Fig. 4d) and promoted the activation of Wnt/β-catenin signaling by inhibiting the expression of E-cadherin and up-regulating the expression of β-catenin and cyclin D1 (Fig. 4e). Further confocal microscopy studies also confirmed that TNF-α significantly induced the nuclear translocation of β-catenin in HCT-116 cells (Fig. 4f). Meanwhile, we observed the expression of NF-κ B, TNF-α downstream targets, and found that the expression of p65 showed no difference in the tumor tissues between the Apc Min/+ ;CD11b −/− mice and Apc Min/+ mice. However, the expression of pp65 (Ser276) was significantly inhibited in the tumor tissues of the Apc Min/+ ;CD11b −/− mice compared with Apc Min/+ mice (Supplementary Figure 4b). Moreover, the myeloid cells were isolated from the bone marrow of C57 mice and co-culture with CT-26 and HCT-116 cells. Compared with the negative control (NC) group, CT-26 (Fig. 4g) and HCT-116 (Fig. 4h) cells significantly accumulated in the G0/G1 peak and arrested the cell cycle at the G1/S transition in TNF-α antibody treated co-culture system. These results demonstrated that CD11b deficiency suppressed tumor growth by reducing the levels of TNF-α secreted by myeloid cells.
CD11b deficiency reduces MDSC recruitment in the tumor environment. The Ly6G + Ly6C low subset of MDSCs exhibits pro-inflammatory activity and often enriched in tumor models 16,19 . Previous reports indicated that TNF-α is secreted by myeloid cells and enriched in the tumor microenvironment, which can drive MDSC accumulation [33][34][35] . Moreover, MDSCs can contribute to angiogenesis and facilitate tumor growth 17 . The Ly6G + Ly6C low subset of MDSCs was significantly larger during CD11b+ myeloid cell-mediated tumor growth. We demonstrated that Gr-1 + CD11b + MDSCs accumulated in the tumor cells in the tumor microenvironment of the Apc Min/+ mice, but the number of Gr-1 + cells decreased in the tumor microenvironment of CD11b-deficient tumor-bearing mice (Fig. 5a). We further detected the difference in production of the Ly6G + Ly6C low subset of MDSCs (gated on CD45 + cells) in the bone marrow of CD11b −/− mice and CD11b −/− tumor-bearing mice (wild type mice were used as controls) and found that the CD45 + leukocyte and granulocytic Ly6G + Ly6C low MDSC populations did not differ in the bone marrow of the C57, CD11b −/− , Apc Min/+ and Apc Min/+ ;CD11b −/− mice (Fig. 5b). However, CD11b deficiency significantly inhibited the number of granulocytic Ly6G + Ly6C low MDSCs that were recruited to peripheral blood, spleen and tumor microenvironment in the Apc Min/+ ;CD11b −/− mice compared with the Apc Min/+ mice (Fig. 5c).
MDSCs inhibit IFN-γ production by specifically inhibiting the CD8-mediated Ag-specific T cell response 36,37 . Moreover, IFN-γ can induce CXCL9 production 38,39 . Reports indicated that IFN-γ and CXCL9 can attenuate angiogenesis and tumor growth [40][41][42] . We further detected the expression of IFN-γ and CXCL9 in tumor site, and found that IFN-γ and CXCL9 were significantly up-regulated in the tumor tissues of the Apc Min/+ ;CD11b −/− mice compared with the Apc Min/+ mice (Fig. 5d). These results demonstrate that CD11b deficiency suppressed tumor growth by reducing the amount of TNF-α secreted by myeloid cells and inhibiting MDSCs recruitment to the tumor microenvironment, which further prevented the inhibition of IFN-γ production and promoted the production of CXCL9 in CRC.

Discussion
In this study, we demonstrate an oncogenic role for CD11b during CRC tumorigenesis. Our findings demonstrated that CD11b expression on the myeloid cells promotes myeloid cell migration to the peripheral blood, spleen and tumor microenvironment. The myeloid cells also secrete cytokines, which promote MDSC recruitment to the tumor sites, further induce IFN-γ and CXCL9 production and activate Wnt/β-catenin signaling in CRC (Fig. 6).
Tumorigenesis is a complex process that involves many factors. A large number of reports indicated that chronic inflammation is an important factor for tumor development 43 . The tumor microenvironment is characterized by the chronic overexpression of inflammatory mediators that are produced by tumor-infiltrating immune cells, particularly bone marrow-derived cells. Therefore, the importance of the inflammatory tumor microenvironment cannot be overlooked. Recently, cytokines in the tumor microenvironment have been shown to contribute to angiogenesis, tumor cell proliferation, invasion and resistant to chemotherapy and radiotherapy. In CRC, tumor cells interact with cytokines in the tumor environment to activate the Wnt pathway 22 . Our work shows that the inhibition of myeloid cells infiltration into the CRC tumor environment can reduce the release of inflammatory factors and consequently activate the Wnt/β-catenin signaling pathway. The data generated in our current study further demonstrated the significance of the myeloid cells that infiltrate the tumor environment during tumorigenesis.
We found that the number of CD11b + myeloid cells increased substantially in the bone marrow and spleens of the Apc Min/+ mice compared with the C57 mice. However, we did not observe a difference in the CD11b + myeloid cells in the peripheral blood. The bone marrow and spleen are the important immune organs in which myeloid cells are generated and localized to activate an immune program. However, the peripheral blood is a part of the circulation system by which myeloid cells translocate from the central immune organs to the peripheral immune organ. Therefore, the differences in the CD11b + myeloid cells may not be detected in the mobile phase of the peripheral blood. However, the peripheral blood of the Apc Min/+ mice tended to contain more CD11b + myeloid cells than that of the C57 mice. Tumor necrosis factor-α (TNF-α ) is a key inflammatory cytokine that is primarily produced by myeloid cells 44 . However, it plays paradoxical roles in carcinogenesis. Reports indicated that TNF-α possesses both anti-tumor and pro-tumor activities 45 . It was reported that TNF-α can inhibit tumor growth in a breast cancer xenograft model 46 . Recently, a large number of studies demonstrated that TNF-α produced in tumor microenvironment may promote cancer development [47][48][49][50] . TNF-α produced by leukocyte was up-regulated in the colorectal carcinoma, and blocking the expression of TNF-α in mice can reduces colorectal carcinogenesis 51 . TNF-α is produced primarily by activated macrophages and also by other cell types including monocytes and lymphoid cells. We observed no differences between the macrophages from the Apc Min/+ mice and those in the Apc Min/+ ;CD11b −/− mice (Supplementary Figure 5). However, the inhibition of TNF-α secretion in myeloid cells can significantly suppress HCT-116 cell proliferation. It is suggested that CD11b deficiency could inhibit the secretion of TNF-α by other type of myeloid cells, but not macrophage. Our results demonstrated that TNF-α , produced by myeloid cells, acts as a tumor-promoting factor that can significantly promote tumor growth in the Apc Min/+ mice.
CD11b, which is expressed on the surface of myeloid cells, has been widely implicated in mediating leukocyte adhesion and transendothelial migration 10,12,13 . CD11b inhibition can reduce myeloid cell recruitment in the tumor environment 11 . MDSCs are a population of bone marrow-derived immature myeloid cells, which constitute approximately 5% of the total cell population within CRC tumors 20 . However, granulocytic MDSC (Ly6G + Ly6C low ) are often expanded in the tumor sites 16,19 . Our study has confirmed that CD11b deficiency cannot affect the production of CD45 + leukocytes and granulocytic Ly6G + Ly6C low MDSCs in the bone marrow. However, the number of the Ly6G + Ly6C low subset of MDSCs was dramatically decreased in the peripheral blood, spleen and tumor sites. Our work suggests that CD11b may not mediate the production of these cells, but only regulates the transendothelial migration of the Ly6G + Ly6C low subset MDSCs. These data are consistent with previous reports showing that CD11b deficiency is characterized by defects in leukocyte adhesion and migration across the endothelium 52 .
In the tumor, CD11b inhibition led to a significant enhancement of the tumor response to irradiation by suppressing vasculogenesis, with no effect on non-irradiated tumors from hypopharyngeal carcinoma cells that were transplanted into immunodeficient mice 11 . However, our data showed that CD11b deficiency can decrease the number of myeloid cells in tumor environment, and thereby inhibit angiogenesis and tumor growth over the protracted course (15 weeks) of tumor development in Apc Min/+ mice under normal conditions, which were not consistent with previously published results 11 . MDSCs suppress T cell function, which contributes to tumor growth. The defective T cells evoke the functional disruption of myeloid cells in the immunodeficient mice with transplanted tumor. Our work was performed in mice with a normal immune system, which may not affect MDSC suppression of T cell function. In support of our data, previous report indicated that myeloid cells promote tumor growth by stimulating tumor angiogenesis and suppressing tumor immunity 17 . The data generated in our current study not only support the published data that CD11b deficiency can reduce myeloid cell recruitment in tumor environment, but also indicate that CD11b deficiency is likely to inhibit angiogenesis and tumor growth in CRC tumor-bearing mice.
In summary, our data have demonstrated that CD11b is critically involved in the transendothelial migration of bone marrow-derived immune cells to the tumor sites, resulting in intestinal tumorigenesis. Moreover, CD11b may serve as a potential biomarker for therapy of CRC treatment.