Macrophages produce and functionally respond to interleukin-34 in colon cancer

In colorectal cancer (CRC), macrophages represent a major component of the tumor mass and exert mostly functions promoting tumor cell survival, proliferation, and dissemination. Interleukin-34 (IL-34) is a cytokine overproduced by colon cancer (CRC) cells and supposed to make a valid contribution to the growth and diffusion of CRC cells. The biological functions of IL-34 are mediated by the macrophage colony-stimulating factor receptor (M-CSFR-1), which controls monocyte/macrophage differentiation, growth, and survival. We here investigated whether, in CRC, tumor-associated macrophages (TAMs) express M-CSFR-1 and functionally respond to IL-34. By flow-cytometry analysis of tumor-infiltrating cells (TICs) and lamina propria mononuclear cells (LPMCs) isolated from normal, adjacent mucosa of CRC patients, we showed that CD68/HLA-DRII-expressing TICs and LPMCs expressed M-CSFR-1. Both these cell types produced IL-34 even though the expression of the cytokine was more pronounced in TICs as compared to normal LPMCs. Moreover, in CRC samples, there was a positive correlation between IL-34-producing cells and CD68-positive cells. Stimulation of LPMCs and TICs with IL-34 resulted in enhanced expression of CD163 and CD206, two markers of type II-polarized macrophages, and this was evident at both RNA and protein level. In the same cell cultures, IL-34 stimulated expression and production of IL-6, a cytokine known to promote CRC cell growth and diffusion. Finally, knockdown of IL-34 in TICs with specific antisense oligonucleotides with: a specific antisesne oligonucleotide decreased IL-6 production and the number of TAMs producing this cytokine. This is the first to show a positive role of IL-34 in the control of TAMs in CRC, further supporting the notion that IL-34 sustains colon tumorigenesis.


Introduction
Colorectal carcinoma (CRC), one of the most common forms of malignancy in the western world, is supposed to be caused by a complex interaction between environmental carcinogens and genetic alterations, which ultimately results in the uncontrolled growth of transformed cells 1 . Approximately 2% of CRC arise in patients with long-standing inflammatory bowel disease (IBD), while most CRC develop in individuals who are not affected by IBD 2 . However, even in these latter patients, the cancer tissue is infiltrated with various immune cells, which can either promote or inhibit CRC cell growth 3 . The promoting effect of immune cells on colon carcinogenesis is largely mediated by cytokines and growth factors, which either directly or indirectly trigger proliferative signals in CRC cells 4 . Within the tumor tissue, other cell types (i.e., stromal cells and cancer cells themselves) can secrete factors promoting CRC cell growth and survival 5 . For instance, we have recently shown that interleukin-34 (IL-34), a cytokine that regulates monocyte/macrophage functions, is highly produced in CRC 6,7 . Immunohistochemistry analysis of CRC sections showed that cancer cells were the major source of IL-34, even though it was also produced to a lesser extent by lamina propria mononuclear cells (LPMCs) 6 . CRC cells expressed both the macrophage colony-stimulating factor receptor (M-CSF1-R) and PTP-z, two functional IL-34 receptors, suggesting that IL-34 can act as an autocrine and/or paracrine factor that targets cancer cells in vivo 6 . Indeed, stimulation of CRC cells with IL-34 enhanced cell growth and invasion through an ERK1/2 MAP kinase-dependent mechanism 6 .
The tumor mass contains many cell types (i.e., neoplastic cells, fibroblasts, endothelial, and immune cells), which interact reciprocally with the downstream effect of sustaining cancer cell growth, survival, and diffusion 8 . Macrophages represent up to 50% of the tumor mass, and some subsets of them promote tumor cell survival, proliferation, and dissemination 9 . Indeed, high numbers of tumor-associated macrophages (TAMs) correlate often with a bad prognosis 10 . TAMs originate from blood monocytes, which are recruited at the tumor tissue, where they differentiate mostly in type 2-polarized (or alternatively activated) macrophages 11 . Factors/mechanisms involved in the differentiation and activation of type 2polarized TAMs in CRC are not fully understood. We here investigated whether, in CRC, TAMs produce and functionally respond to IL-34.

TAMs express IL-34 receptors and produce IL-34
To begin to examine whether, in CRC, TAMs are a cell target of IL-34, we isolated TICs from CRC tissues and LPMCs from normal, adjacent mucosal areas of CRC patients and examined them by flow cytometry. By gating on live CD45 + cells, we showed that M-CSF1-R was barely detectable in CD3 + cells and CD19 + cells (Fig.  1A, B), while the receptor was expressed by CD68 + cells (Fig. 1C). The percentage of CD45 + CD68 + cells expressing M-CSF1-R was significantly greater in TICs than in LPMCs (Fig. 1C, right inset). When the analysis was restricted to TICs, it was evident that more than 50% of CD68 + HLA-DRII + cells, which correspond to TAMs, expressed the receptor, and the percentage of such cells was significantly greater than the percentage of cells negative for M-CSF1-R (Fig. 1D).
Next, we determined the cell source of IL-34 in CRC tissue. Flow-cytometry analysis of TICs and LPMCs preparations showed that~2% of CD3 + cells and CD19 + TICs were positive for IL-34, while only a few CD3 + and CD19 + LPMCs expressed IL-34 ( Fig. 2A, B). A more prevalent expression of IL-34 was seen in CD68 + cells, and the percentage of CD68/IL-34-expressing cells was significantly greater in TICs than in LPMCs (Fig. 2C).
Immunofluorescence analysis confirmed that IL-34 is produced by CD68 + cells in the tumoral area of CRC patients (Fig. 3). Further analysis showed that the percentages of CD68 + HLA-DRII + IL-34+ either expressing or not M-CSF1-R were significantly greater in TICs than in LPMCs (Fig. 4A, B) even though in both cell preparations there was variability in the percentage of IL-34-expressing cells (Fig. 4A, B). Finally, by immunohistochemistry of CRC sections, we showed a positive correlation between the number of CD68 + cells and the number of IL-34-producing cells (Fig. 4C).
Altogether these data indicate that macrophages infiltrating CRC tissue produce IL-34 and express IL-34 receptor.
IL-34 enhances the expression of markers of type 2-polarized macrophages In subsequent experiments, we assessed whether IL-34 stimulates the synthesis of molecules, which are typically expressed by TAMs in CRC. To this end, we cultured both TICs and LPMCs with graded doses of recombinant human IL-34. At the end, cells were collected and assessed for the expression of CD163 and CD206, two markers of type 2-polarized macrophages 12 , INOS, a marker of type 1-polarized macrophages 12 and IL-10, a cytokine involved in the control of type 2-polarized macrophages 13 . IL-34 significantly enhanced CD163, CD206, and IL-10 expression and decreased INOS expression in both TICs and LPMCs (Fig. 5A, B). Flow-cytometry analysis showed that, in unstimulated conditions, the percentage of CD68 + /HAL-DRII + cells expressing CD163 and CD206 was significantly greater in TICs (15 ± 1.1) than in LPMCs (5 ± 1; P < 0.05) (Fig. 6A, B). In line with the RNA results, stimulation of TICs and LPMCs with IL-34 increased significantly the fractions of CD163/CD206-expressing CD68 + HLA-DRII + cells (Fig. 6A, B).

Discussion
The molecular events that stimulate CRC cell growth are not fully understood. However, in recent years, a large body of evidence has been accumulated to show that the tumor microenvironment provides a variety of signals that sustain colon tumorigenesis. For instance, immune cells and stromal cells produce huge amounts of cytokines, which exert proliferative and survival effects on CRC cells 2 . In this context, we have recently shown that CRC cells synthesize IL-34, a factor that expands CRC cell   6 . Interestingly, both neoplastic cells and nontumoral cells infiltrating CRC tissue express IL-34 receptors raising the possibility that IL-34 can mediate the cross-talk between cancer cells and other cells during colon carcinogenesis process 7 . This study was undertaken to examine whether IL-34 regulates the function of TAMs, as these cells are known to facilitate CRC progression 15 . Through flow-cytometry analysis of single cells isolated from both tumoral and non-tumoral specimens of CRC patients, we initially showed that both TAMs and normal mucosal CD68/HLA-DRII-positive macrophages expressed M-CSFR-1, a functional IL-34 receptor 16 . These findings confirm and expand on the data of previous studies showing that monocytes/macrophages are the main targets of IL-34 17 . Our data are also in line with studies documenting the expression of IL-34 receptors in TAMs in other settings 7 . Next, we showed that CD68/ HLA-DRII-positive cells expressed IL-34 in both TICs and LPMCs preparations, even though the fraction of IL-34-positive cells was significantly higher in cancer   cancer 19,20 . Moreover, it has been demonstrated that IL-34 induces the differentiation of monocytes into IL-10 high IL-12 low immunoregulatory macrophages, which are similar to TAMs seen in ovarian cancer 17 . IL-34treated macrophages switch non-Th17 committed memory CD4 + T cells into conventional CCR4 + CCR6 + CD161 + Th17 cells, a phenomenon which is typically seen in many cancer tissues 21 . In mammary cancer and other tumors (i.e., teratoma, hepatocellular carcinoma), the pro-tumoral effect of IL-34 has been linked to the ability of the cytokine to promote the function of type 2 macrophages [22][23][24] . Taken together, these findings suggest that, in CRC tissue, IL-34 contributes to maintain the function of type 2-polarized TAMs, with the downstream effect of sustaining colon carcinogenesis. The demonstration that IL-34 stimulates IL-6 induction in both TICs and LPMCs supports further the protumorigenic role of IL-34, as IL-6 can target directly cancer cells and activate signaling pathways that promote CRC cell growth and survival 25,26 .
In conclusion, this is the first to show a positive role of IL-34 in the control of TAMs in CRC, further supporting the hypothesis that IL-34 sustains colon tumorigenesis.

Patients and samples
Paired tissue samples were taken from the tumoral area and the macroscopically and microscopically unaffected, adjacent colonic mucosa of 22   Hospital (Rome, Italy). All patients received neither radiotherapy nor chemotherapy prior to undergoing surgery. Each patient who took part in the study gave written informed consent, and the study protocol was approved by the local Ethics Committee (Tor Vergata University Hospital, Rome. Protocol number:171/16).
To assess whether IL-34 regulates the function of type II-polarized macrophages, both TICs and LPMCs were seeded at a concentration of 5 × 10 5 cells/ml into 48-well culture dishes with increasing doses of recombinant human IL-34 (25-100 ng/ml, Miltenyi Biotec, Bologna, Italy) for 6 or 24 h, and then analyzed by real-time PCR, ELISA, or flow cytometry.
In additional experiments, 5 × 10 5 TICs or LPMCs were plated into each well of a 48-well plate, and then either left untreated or transfected with IL-34 AS or scrambled antisense oligonucleotide (Src AS) (both used a 2 µg/ml, Exiqon, Woburn, USA,) for 24-48 h using Opti-MEM medium and Lipofectamine 3000 reagent according to the manufacturer's instructions (both from Life Technologies, Milan, Italy). The efficiency of the transfection was determined by western blotting after 24 h. To assess IL-6producing cells, PMA (40 ng ml/1), ionomycin (1 mg ml/ 1), and Brefeldin A (10 mg/ml) were added 4 h before the end of the culture (24 h), and the cells were then analyzed by flow cytometry. Moreover, IL-6 was evaluated in cellfree supernatants by ELISA after 48-h culture.

Immunohistochemistry
Immunohistochemistry was performed on formalinfixed, paraffin-embedded colon sections of five CRC patients. The sections were deparaffinized and dehydrated through xylene and ethanol, and the antigen retrieval was performed in Tris EDTA citrate buffer (pH 7.8) in a thermostatic bath at 98°C for 30 min. Immunohistochemical staining was performed using mouse monoclonal antibody directed against human IL-34 (final dilution 1:50000, Abcam, Cambridge, UK), and a mouse monoclonal antibody directed against human CD68 (final concentration 1:100, Dako, Milan, Italy) incubated at room temperature (RT) for 1 h followed by a biotin-free HRP-polymer detection technology with 3,3'diaminobenzidine (DAB) as a chromogen (MACH 4 Universal HRP-Polymer Kit, Biocare Medical). The sections were counterstained with hematoxylin, dehydrated, and mounted. Isotype control IgG-stained sections were prepared under identical immunohistochemical conditions as described above, replacing the primary antibody with a purified mouse normal IgG control antibody (R&D Systems). The IL-34 and CD68-positive cells were counted in at least six fields per section using IAS 2000 System (Delta Sistemi, Rome, Italy) in serial sections of the same CRC surgical samples and expressed as the number of cells for high-power field (hpf).

Immunofluorescence
Immunofluorescence was performed on frozen sections of mucosal samples taken from the colon sections of three CRC patients. Samples were embedded in a cryostat mounting medium (Neg-50 Frozen Section Medium, Thermo Scientific), snap-frozen, and stored at −80°C. Sections (6-µm thick) were mounted onto superfrost glass slides and fixed in 4% paraformaldehyde (PFA) for 10 min at 4°C. Slides were washed three times with PBS, treated with 0.1% Triton X-100 for 20 min at RT. The blocking procedure was performed with 10% normal goat serum in PBS solution for 1 h at room temperature. Slides were then incubated overnight at 4°C with mouse anti-human IL-34 (final dilution 1:100, Abcam, Cambridge, UK), rabbit anti-human CD68 (final dilution 1:100, BioSpring Germany, Frankfurt). After washing three times with PBS, slides were incubated for 1 h at room temperature with specific secondary antibodies coupled with Alexa Fluor Dyes (final dilution 1:2000 final dilution; Invitrogen, Milan, Italy). Coverslips were mounted on glass slides using ProLong Gold antifade reagent with DAPI (Invitrogen) to counterstain the DNA. Isotype control IgGstained sections were prepared under identical immunofluorescence conditions as described above, replacing the primary antibody with a normal IgG control antibody (Abcam). Samples were analyzed with a Leica DMI 4000 B fluorescence microscope (Leica, Wetzlar, Germany).

Enzyme-linked immunosorbent assay
Human IL-6 was measured using a sensitive commercial ELISA kit (R&D Systems) according to the manufacturer's instructions.

Statistical analysis
Differences between the two groups were compared using the Student's t test or Mann-Whitney U test. The significance of correlation was determined using Pearson's test. All the analyses were performed using Graph-Pad 6 software.