Purpose: Targeting tumor-associated macrophages with colony-stimulating factor 1 receptor (CSF-1R) inhibition reveals a strategy for cancer therapy. Here, we studied the impact of CSF1R germline genetic variant on CSF-1R signaling and the susceptibility to CSF-1R inhibitors.

Experimental designs:CSF1R germline genetic variants were studied in 140 cancer patients. CSF-1R phosphorylation, endocytosis, and macrophage polarization were measured as the response to CSF-1 stimulation. Tumor-associated macrophages in surgical specimens and sensitivity to CSF-1R inhibitors were used to determine macrophage function.

Results: A CSF1R c.1085A>G genetic variant causing the change of histidine to arginine in the domain of receptor dimerization was identified as a high allele frequency in Eastern Asian population. Cancer patients with this variant allele had less M2-like tumor-associated macrophages accompanied by low VEGF expression in tumor tissues. Importantly, CSF1R genetic variant was significantly associated with disease-free survival in colorectal, endometrial, and ovarian cancer. In terms of differentiation, macrophages with CSF1R c.1085A>G genetic variant displayed a refractory response to CSF-1 stimulation and macrophage survival was sensitive to CSF-1R inhibitors with IC50 of 0.1 to 1 nmol/L range. On contrast, CSF-1 induced a prominent phosphorylation and rapid endocytosis of CSF-1R, leading to an M2-like dominant polarization in macrophages with CSF1R c.1085 genotype A_A, in which CSF-1R inhibitors of PLX3397, BLZ945, and GW2580 inhibited macrophage survival with IC50 of 10 to 100 nmol/L range.

Conclusions: The CSF1R c.1085A>G genetic variant regulates tumor immunity by altering the polarization and function of macrophages. This genetic variant confers the sensitivity to CSF-1R inhibitors, implying as a biomarker in targeting CSF-1R signaling for cancer treatment. Clin Cancer Res; 23(20); 6021–30. ©2017 AACR.

Translational Relevance

Colony-stimulating factor 1 receptor (CSF-1R) signaling regulates the survival, proliferation, and differentiation of macrophages which plays an important role in innate immunity, inflammatory disease, and cancer. Multiple approaches to target CSF-1R signaling for cancer treatment have been tested clinically. However, the impact of CSF1R genetic variants on CSF-1R signaling and predicting the susceptibility to CSF-1R inhibitor is not clear. We analyzed the germline genetic variants of CSF1R gene and identified a nonsynonymous variant c.1085A>G, a high allele frequency in Eastern Asian, which causes the change of amino acid from histidine to arginine in the domain of receptor dimerization. Macrophages with CSF1R c.1085A>G genetic variant displayed a refractory response to CSF-1 stimulation and susceptibility to CSF-1R inhibitors. The different ethnic frequency of genetic variant together with differential sensitivity to CSF-1R inhibitors suggests this genetic variant is a potential biomarker to predict the response in targeting CSF-1R signaling for cancer treatment.

Colony stimulating factor 1 receptor (CSF-1R) is the receptor for CSF-1, a cytokine that controls the production, differentiation, and function of macrophages (1). Ligand binding activates the CSF-1R kinase through a process of oligomerization and transphosphorylation. Two different polarized subpopulations of macrophages, classically activated (M1) or alternatively activated (M2) macrophage, have been identified. Exposure of macrophages to granulocyte/macrophage colony-stimulating factor (GM-CSF) leads to an M1-like state that produces the pro-inflammatory cytokines, such as tumor necrosis factor (TNF), IL6, and IL12, whereas exposure to CSF-1 leads macrophages to be maintained in an M2-like polarized state (2). The M2-like macrophages produce cytokines that could promote tumor progression, angiogenesis, and matrix remodeling. Tumor-associated macrophages are often found to have M2-like phenotype that is associated with a poor outcome in different types of cancers (3). Thus, targeting CSF-1R signaling by monoclonal antibody or small molecular inhibitors to manipulate macrophage function has been studied in the treatment of cancer, such as glioma (4), breast cancer (5), and pancreatic cancer (6). Different responses to CSF-1R inhibition are observed in the early-phase clinical trials and several clinical studies of immunotherapy are undergoing to evaluate the combination of CSF-1R inhibition and blockade of PD-1/PD-L1 (7).

Because CSF-1R signaling controls the differentiation and survival of macrophages, a critical question that remains to be answered is whether mutations or nonsynonymous variants of CSF1R gene would change macrophage function. For example, mutations in CSF1R gene affecting the tyrosine kinase domain were found to interfere with the autophosphorylation of CSF-1R and cause hereditary diffuse leukoencephalopathy (8). By the genome-wide association study, SNP of CSF1R gene, rs10079250, was significantly associated with lung cancer in never-smoking females (9). This rs10079250 polymorphism of CSF1R was located in the proximity of the binding sites of monoclonal antibody RG7155 and also correlated with a trend toward a reduced response to the anti-CSF-1R antibody, RG7155 (10).

Here we surveyed the CSF1R germline genetic variants in colorectal, ovarian, and endometrial cancer and studied the impact of the variants on macrophage function. The results showed that macrophages with CSF1R c.1085A>G genetic variant, a high incidence in Eastern Asian population, displayed a refractory response to CSF-1 stimulation and susceptibility to CSF-1R inhibitors.

Databases for analyzing CSF1R genetic variants

Germline CSF1R genetic variants were analyzed from the database of an ongoing prospective study and a pilot study investigating the association of germline genetic variants and chemotherapy-induced peripheral neuropathy (CIPN; ref. 11). This clinical study was approved by the institutional review board of National Cheng Kung University Hospital (NCKUH) and registered in ClinicalTrial.gov. All patients provided written informed consent. This study was conducted in accordance with the Declaration of Helsinki. In this CIPN study, patients with stage III colorectal cancer receiving the standard adjuvant chemotherapy with mFOLFOX6 and patients with ovarian and endometrial cancers receiving the postoperative chemotherapy with the regimen of carboplatin and paclitaxel were enrolled in NCKUH in Taiwan. Among these patients, DNA was isolated from peripheral blood and the whole genome was sequenced by next-generation sequencing on Illumina HiSeq 2500 to analyze the germline genetic variants. Till December 2016, whole genome data of 140 subjects, including 90 cases of colorectal cancer, 28 cases of ovarian cancer, and 22 cases of endometrial cancer, were available for analysis of CSF1R genetic variants. In addition, to study the association between CSF1R genetic variant and clinical outcome, germline DNA was extracted from normal tissue and whole exome was sequenced by using Ion Proton Sequencer (Thermal Fisher Scientific) in another 18 cases of advanced stage endometrial cancer. Whole genome sequencing data of 499 normal Taiwanese were provided by Taiwan Biobank to compare the distribution and frequency of CSF1R variant between cancer patients and normal population (12). Publicly available data from 1,000 Genomes Project was used to compare the worldwide distribution of CSF1R c.1085 genetic variant (13).

PCR and Sanger sequencing

Sanger sequencing was used to determine the CSF1R c.1085 genotypes of normal volunteers who provided peripheral blood for isolation of monocytes and confirm the CSF1R c.1085 genotype of the 140 subjects from the CIPN study. Briefly, genomic DNA was extracted from buffy-coat of peripheral blood using QIAGEN genomic DNA Purification Kit. DNA pellets were dissolved in MQ water for PCR. Coding exons containing CSF1R c.1085 were PCR-amplified using the forward and reverse primers: 5′-ACAGTGGTCAACGTAGGCGA-3′ and 3′-ATGAATGTCCATATGACGCTTACC-5′. Reactions were amplified with the following protocol: 95°C denaturation for 5 minutes, followed by 40 cycles of denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 1 minute, followed by a 6-minute extension at 72°C. PCR products were sequenced to determine the genotype of CSF1R c.1085.

Chemicals and antibodies

PLX3397, BLZ945, and GW2580 were purchased from Selleckchem. Recombinant human CSF-1 was obtained from R&D systems. The antibodies used for immunofluorescent staining were: rabbit anti-TNF-α (Abcam, ab6671), mouse anti-VEGF (GeneTex, A7-E11-G2), mouse anti-CD68 (GeneTex, GTX41865), mouse anti-CD163 (GeneTex, GTX42365), rabbit anti-iNOS (Cell Signaling Technology, 13120), and Hoechst 33258 (Sigma-Aldrich, 861405). Alexa Fluor 594- (A11012) and Alexa Fluro 488-conjugated second antibody (A11001) were purchased from Thermo Fisher Scientific Inc. The antibodies used for analysis of flow cytometry were: FITC mouse anti-human CD14 (#555397), PE mouse anti-human CD80 (#557227), PE-Cy5 mouse anti-human CD206 (#551136), FITC mouse IgG2a κ isotype control (#555573), PE mouse IgG1 κ isotype control (#555749), and PE-Cy5 mouse IgG1 κ isotype control (#555750) from BD Pharmingen.

Immunofluorescence, confocal images, and tissue scanning

Tumor specimens of 52 colorectal, 20 ovarian, and 16 endometrial cancer patients enrolled in the CIPN study were available for immunofluorescent staining. We used anti-iNOS, anti-CD163, and anti-CD68 to stain M1-like, M2-like, and total macrophages, respectively. Anti-TNFα and anti-VEGF were used to stain M1- and M2-associated cytokines, respectively. Alexa Fluor 594-conjugated second antibody was used for iNOS and TNFα; and Alexa Fluor 488-conjugated second antibody was used for CD68, CD163, and VEGF. Tissue was co-stained with Hoechst 33258 to detect the nucleus. The fluorophores were excites by laser at 405, 488, and 594 nm, respectively, and detected by a scanning confocal microscope (FV-1000, Olympus). The numbers of M1- and M2-like macrophages were counted in five different fields with 40× oil immersion lens and M1/M2 ratio was calculated. The iNOS expression level of macrophages in confocal imaging was analyzed by FV10-ASW 4.0 microscopy software. To investigate the total macrophages and cytokine expression, whole tissue was scanned with TissueGnostics GmbH FACS-like Tissue Cytometry (TissueFAXS Plus). Series of separate images per fluorescence channel and field of view were acquired automatically and merged. HistoQuest software was used to analyze the positive staining area of TNFα and VEGF in total tumor area. In each sample, the number of macrophages (CD68-positive) cells was quantitated in 10 fields of tumor microenvironment. Each field was 1.2 × 1.2 μm2.

Culture of human monocyte-derived macrophages

Monocytes were enriched from whole blood by negative selection using the Lymphoprep (STEMCELL) according to the manufacturer's instruction. To differentiate these monocytes into monocyte-derived macrophages, 1 × 107 cells were plated in 6-cm dish and maintained in RPMI plus 10% FBS with recombinant human CSF-1 10 ng/mL for 6 days.

Analysis and cell sorting by flow cytometry

For detection of cell surface markers and cell sorting, 1 μg of monoclonal FITC mouse anti-human CD14 (BD Biosciences), PE mouse anti-human CD80 (BD Biosciences), PE-Cy5 mouse anti-human CD206 antibodies (BD Biosciences), or the relevant isotypes were incubated with samples containing 2 × 105 cells for 15 minutes at 4°C. Following incubation, samples were washed and resuspended in PBS. Flow cytometric analysis was performed by using a four color flow cytometer CytoFLEX (Beckman Coulter). Forward and side scatter light measuring the size and granularity of the cells, respectively, was used to gate the population of macrophages. CD80 and CD206 were used to characterize M1- and M2-like macrophages, respectively. Ten thousand events were recorded and the data were analyzed using FlowJo software, version 10.1 (Tree Star, Inc.). To prevent the contamination of nonmacrophage cells, CD14 and CD206 were used to purify the mature macrophages by using a Beckman CoulterMoFlow XDP cell sorting system (Beckman Coulter). Macrophages with or without purification were used for analysis of CSF-1R phosphorylation and endocytosis.

CSF-1R phosphorylation

After the human monocytes were differentiated into macrophages by 6-day incubation with CSF-1, the CSF-1-containing medium was replaced by medium without serum and CSF-1 to serum starve the macrophages for 18 hours. After serum starvation, the cells were pretreated with CSF-1R inhibitors for 2 hours and then stimulated with CSF-1 (100 ng/mL) for 5 minutes. Cell lysates were generated with RIPA buffer containing protease and phosphatase inhibitor cocktails. DC protein assay (Bio-Rad) was used to determine the protein concentrations and lysates at 0.2 mg/mL were used to quantify the phosphorylated CSF-1R by the PathScan Phospho-M-CSF-Receptor sandwich ELISA Kit, according to manufacturer's instruction.

CSF-1R endocytosis

Monocytes, which were enriched from whole blood by using the Lymphoprep (STEMCELL), were seeded on 20 mm cover glass and induced differentiation into macrophages by the stimulation of CSF-1 (10 ng/mL) for 6 days. Cells were blocked with CAS-Block Histochemical reagent (Thermo Fisher Scientific Inc.) at 37°C for 1 hour. After that, cells were stained with CSF-1R-GFP antibody for 1 hour and washed by filtered PBS. Dynamics of CSF-1R-GFP was acquired with an APO 100×/1.65 NA oil immersion objective at 37°C using a total internal reflection fluorescence (TIRF) microscopy (Olympus). After the region of interest was selected, the 488-nm excitation laser was angled until reflection was observed on the cover glass. The evanescent penetration depth of 80 nm was used and images were continuously acquired once per 30 seconds for 45 minutes to monitor the endocytosis of CSF-1R.

Cell viability

Monocytes were enriched from whole blood by using the Lymphoprep (STEMCELL) and 7 × 105 cells were seeded into the wells of 96-well cell culture plate. CSF-1 10 ng/mL was used to induce the differentiation of macrophages after seeding and different concentrations of CSF-1R inhibitor were added at the same day of CSF-1 stimulation. After incubation of CSF-1 and CSF-1R inhibitor for 8 days, CellTiter-Glo Luminescent Cell Viability Assay was used to determine the cell viability according to manufacturer's instruction.

Statistical analysis

All values were reported as mean ± SEM. Fisher's exact test, Chi-square test, and unpaired t test were used to compare the difference between groups. Pearson correlation was used to determine the correlation between the M1/M2 ratio and VEGF expression. Kaplan–Meier survival analysis and log-rank test were used to estimate the survival and compare the difference between groups. A P value <0.05 was considered statistically significant.

Global distribution of CSF1R c.1085A>G genetic variant

The germline genetic variants of CSF1R from 140 cancer patients were analyzed, including 90 cases of colorectal cancer, 28 cases of ovarian cancer, and 22 cases of endometrial cancer. In these 140 cancer patients, 496 genetic variants of CSF1R, including the single nucleotide variants (SNV) and small insertion/deletions (Indel), were identified in 3′ untranslated region (UTR), 5′UTR, introns, and exons. Among the 496 genetic variants, 14 variants were located in exons and only 7 of 14 exonic variants were nonsynonymous substitution leading to a change in amino acid (Fig. 1A and B). The frequency of these nonsynonymous variants was low (<2%), except for the genetic variant c.1085A>G with a high allelic frequency of 42.86%. However, there was no significant difference in this allele frequency between cancer types (Supplementary Fig. S1A). We also checked the frequency and distribution of CSF1R genetic variants in Taiwan Biobank (12) that enrolled healthy Taiwanese, the same ethnic group with those 140 cancer patients. The c.1085A>G variant was the only nonsynonymous genetic variant of CSF1R that could be identified in the exonic regions with the allele frequency more than 2% (Fig. 1B and C). There was no significant difference in the frequency of CSF1R genetic variants between healthy Taiwan population and cancer patients. We further studied the worldwide distribution of c.1085A>G genetic variant of CSF1R based on 1,000 Genomes Project. The high frequency of c.1085A>G variant (about 38%) was also observed in the population of East Asia (Fig. 1D and Supplementary Fig. S1B). In contrast, only 7% to 11% alternative allele frequency was observed in Africa, America, South Asia, and European population (Fig. 1D).

Figure 1.

The frequency and distribution of germline CSF1R genetic variants. A, Germline CSF1R genetic variants in 140 cancer patients. The whole genome data of 140 cancer patients from a clinical study evaluating the association between genetic variants and chemotherapy-induced neurotoxicity were used to analyze the distribution and allele frequency of germline CSF1R genetic variants in this study. The variants located at exons were shown here. Solid and open symbols indicate nonsynonymous and synonymous variants, respectively. Ig, immunoglobulin domain; Tyr kinase, tyrosine kinase domain. B, Comparison of the distribution of CSF1R genetic variants between cancer patients and normal population. The allele frequency of germline CSF1R variants was compared between 140 cancer patients and 499 normal Taiwanese by Fisher's exact test. Bold indicates the nonsynonymous variants. NS, no significance. C, Germline CSF1R genetic variants in Taiwan general populations. The database of Taiwan Biobank, which included 499 normal Taiwanese, was used to analyze the distribution of germline CSF1R genetic variants in Taiwan general population. D, The alternative allele frequency in CSF1R c.1085 in different ethnic groups. The allele frequency of variant allele in CSF1R c.1085 was compared between this study, normal Taiwanese, and different ethnic groups enrolled in the 1,000 Genomes Project.

Figure 1.

The frequency and distribution of germline CSF1R genetic variants. A, Germline CSF1R genetic variants in 140 cancer patients. The whole genome data of 140 cancer patients from a clinical study evaluating the association between genetic variants and chemotherapy-induced neurotoxicity were used to analyze the distribution and allele frequency of germline CSF1R genetic variants in this study. The variants located at exons were shown here. Solid and open symbols indicate nonsynonymous and synonymous variants, respectively. Ig, immunoglobulin domain; Tyr kinase, tyrosine kinase domain. B, Comparison of the distribution of CSF1R genetic variants between cancer patients and normal population. The allele frequency of germline CSF1R variants was compared between 140 cancer patients and 499 normal Taiwanese by Fisher's exact test. Bold indicates the nonsynonymous variants. NS, no significance. C, Germline CSF1R genetic variants in Taiwan general populations. The database of Taiwan Biobank, which included 499 normal Taiwanese, was used to analyze the distribution of germline CSF1R genetic variants in Taiwan general population. D, The alternative allele frequency in CSF1R c.1085 in different ethnic groups. The allele frequency of variant allele in CSF1R c.1085 was compared between this study, normal Taiwanese, and different ethnic groups enrolled in the 1,000 Genomes Project.

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CSF1R variant is associated with clinical outcome

To study the clinical relevance, the total macrophage content, expression patterns of M1- and M2-like tumor-associated macrophages, and associated cytokines were examined in the surgical specimens of colorectal, ovarian, and endometrial cancer (Fig. 2A–E and Supplementary Fig. S2). CD68 was used to stain the total macrophages, and M1 and M2 macrophages were monitored using cluster of inducible nitric oxide synthase (iNOS) and CD163 staining, respectively. As shown in Fig. 2A and B, the number of tumor-associated macrophage is significantly higher in tumor tissues derived from the genetic background of CSF1R c.1085 genotype A_A. Compared with that of reference group, the tumor M1/M2 ratio was significantly higher in the surgical specimens of c.1085A>G genetic variant of CSF1R (Fig. 2C and D and Supplementary Fig. S2). In addition, the M1/M2 ratio of tumor-associated macrophages inversely correlated with VEGF expression in tumor tissues (Fig. 2F, r2 = 0.37, P < 0.0001). A higher M1/M2 ratio of tumor-associated macrophages accompanied by lower VEGF expression in tumor tissues was noted in the surgical specimens from cancer patients with CSF1R c.1085 A>G genetic variant (Fig. 2C–E). More importantly, CSF1R genetic variant is significantly associated with disease-free survival in stage III colorectal cancer (Fig. 2G and Supplementary Table S1) and overall survival in endometrial cancer (Supplementary Fig. S3A and S3B). For patients with ovarian cancer, there was also a trend showing poor outcome in the group of CSF1R c.1085 genotype A_A than genotype A_G (Supplementary Fig. S3C and S3D, P = 0.059). These results imply the impact of CSF1R genetic variant on the clinical outcome of cancer patients.

Figure 2.

CSF1R c.1085A>G genetic variant was associated with the polarized tumor-associated macrophages, related cytokine, and clinical outcome. A and B, Representative images and quantitative analysis of tumor-associated macrophages in 52 tumor tissues from colorectal cancer. Scale bar, 100 μm. C, Representative confocal images co-stained with iNOS (for M1 macrophages), CD163 (for M2 macrophages) and Hoechst 33258 (for nucleus) in tumor specimens of colorectal cancer were shown in top and those co-stained with TNFα (M1-related cytokine), VEGF (M2-related cytokine), and Hoechst 33258 were shown in bottom (n = 52). Scale bar, 40 μm. D, The number of M1- and M2-like macrophages quantified in five different fields with 40× oil immersion lens was used to calculate the M1/M2 ratio. The M1 to M2 ratio was compared between the group of CSF1R c.1085 A_A and CSF1R c.1085 A_G. E, The percentage of positive staining area for VEGF relative to total tumor area was measured and analyzed by TissueGnostics GmbH FACS-like Tissue Cytometry (TissueFAXS Plus). The percentage of positive staining area to total tumor area was compared between the group of CSF1R c.1085 A_A and CSF1R c.1085 A_G. Solid lines, mean ± SEM. Parentheses indicate case number in each group. ***, P < 0.001 by unpaired t test. F, Correlation between the positive staining area of VEGF and M1/M2 ratio in tumor specimens was determined by Pearson correlation coefficient. G, The Kaplan–Meier curves of disease-free survival in colorectal cancer patients with CSF1R c.1085 genotype A_A (n = 21) and A_G (n = 31) were shown and compared by log-rank test.

Figure 2.

CSF1R c.1085A>G genetic variant was associated with the polarized tumor-associated macrophages, related cytokine, and clinical outcome. A and B, Representative images and quantitative analysis of tumor-associated macrophages in 52 tumor tissues from colorectal cancer. Scale bar, 100 μm. C, Representative confocal images co-stained with iNOS (for M1 macrophages), CD163 (for M2 macrophages) and Hoechst 33258 (for nucleus) in tumor specimens of colorectal cancer were shown in top and those co-stained with TNFα (M1-related cytokine), VEGF (M2-related cytokine), and Hoechst 33258 were shown in bottom (n = 52). Scale bar, 40 μm. D, The number of M1- and M2-like macrophages quantified in five different fields with 40× oil immersion lens was used to calculate the M1/M2 ratio. The M1 to M2 ratio was compared between the group of CSF1R c.1085 A_A and CSF1R c.1085 A_G. E, The percentage of positive staining area for VEGF relative to total tumor area was measured and analyzed by TissueGnostics GmbH FACS-like Tissue Cytometry (TissueFAXS Plus). The percentage of positive staining area to total tumor area was compared between the group of CSF1R c.1085 A_A and CSF1R c.1085 A_G. Solid lines, mean ± SEM. Parentheses indicate case number in each group. ***, P < 0.001 by unpaired t test. F, Correlation between the positive staining area of VEGF and M1/M2 ratio in tumor specimens was determined by Pearson correlation coefficient. G, The Kaplan–Meier curves of disease-free survival in colorectal cancer patients with CSF1R c.1085 genotype A_A (n = 21) and A_G (n = 31) were shown and compared by log-rank test.

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CSF1R c.1085A>G genetic variant regulates the dynamics of receptor endocytosis

We further explored the mechanism underlying the impact of CSF1R c.1085 genetic variant on macrophage function. CSF-1 stimulates CSF-1R dimerization which results in CSF-1R activation, followed by kinase inactivation, dephosphorylation, and internalization of the receptor–ligand complex (14). Because the CSF1R c.1085 genetic variant is located in the domain of dimerization, we studied whether CSF1R c.1085 genetic variant is involved in the regulation of CSF-1-induced internalization and endocytosis of CSF-1R. The mononuclear cells were isolated from the peripheral blood, seeded on 20 mm cover glass, and differentiated into macrophages by incubation with 10 ng/mL CSF-1 for 6 days. To study the endocytosis of CSF-1R, anti-CSF-1R-GFP antibody was used to stain CSF-1R and the dynamics of CSF-1R endocytosis in macrophage were real-time monitored by the total internal reflection fluorescence microscopy (TIRFM) with an evanescent penetration depth of 80 nm (Fig. 3A). It took 35–45 minutes from the exposure to CSF-1, activation of CSF-1R signaling, clustering, internalization, endocytosis, to final degradation of CSF-1R (15). Accordingly, we monitored the CSF-1-induced changes of fluorescent signals for 40 minutes to visualize the dynamics of CSF-1R endocytosis. When macrophages with CSF1R genotype A_A were stimulated by CSF-1, the fluorescent signals first became brighter in the first 5 minutes, suggesting the clustering of CSF-1R. After that, the intensity of fluorescent signals remarkably decreased by 70% in the following 5 minutes and then recovered gradually in the next 5 minutes which indicated the rapid endocytosis of CSF-1R induced by CSF-1 (Fig. 3B and C; Movie 1). By striking contrast, when macrophages with the CSF1R genotype A_G were stimulated by CSF-1, the fluorescent intensity initially surged and gradually decreased by 10% to 20% in the whole period (Fig. 3B and D; Movie 2). It seemed that CSF-1 induced CSF-1R clustering which was followed by slower internalized process in the genotype of CSF1R c.1085 A_G. These results indicate that CSF1R c.1085 genetic variant, located in the domain of dimerization, is involved in the regulation of CSF-1-induced endocytosis of CSF-1R.

Figure 3.

CSF1R c.1085A>G genetic variant regulates the endocytosis of CSF-1R. A, Schematic showing TIRFM monitored the endocytosis of CSF-1R by selective excitation of fluorophores at plasma membrane and submembrane region near the interface of the cell and cover glass. Cells were stained with anti-CSF-1R-GFP antibody and endocytosis of CSF-1R was monitored by imaging the changes of fluorescent signals with the evanescent penetration depth of 80 nm continuously. B, Representative TIRFM images showing the changes of fluorescent signals after stimulation by CSF-1. Macrophages differentiated from peripheral blood mononuclear cells were seeded on 20 mm cover glass and stained by anti-CSF-1R-GFP antibody. CSF-1 20 ng/mL was used to stimulate macrophages and the changes of fluorescent signals were real-time monitored by TIRFM once per 30 second for 40 minutes. C and D, Changes of CSF-1R-GFP fluorescent intensity in macrophages with CSF1R c.1085 A_A and CSF1R c.1085 A_G, respectively. Y axis, normalized fluorescent intensity in TIRFM. ROI, region of interest.

Figure 3.

CSF1R c.1085A>G genetic variant regulates the endocytosis of CSF-1R. A, Schematic showing TIRFM monitored the endocytosis of CSF-1R by selective excitation of fluorophores at plasma membrane and submembrane region near the interface of the cell and cover glass. Cells were stained with anti-CSF-1R-GFP antibody and endocytosis of CSF-1R was monitored by imaging the changes of fluorescent signals with the evanescent penetration depth of 80 nm continuously. B, Representative TIRFM images showing the changes of fluorescent signals after stimulation by CSF-1. Macrophages differentiated from peripheral blood mononuclear cells were seeded on 20 mm cover glass and stained by anti-CSF-1R-GFP antibody. CSF-1 20 ng/mL was used to stimulate macrophages and the changes of fluorescent signals were real-time monitored by TIRFM once per 30 second for 40 minutes. C and D, Changes of CSF-1R-GFP fluorescent intensity in macrophages with CSF1R c.1085 A_A and CSF1R c.1085 A_G, respectively. Y axis, normalized fluorescent intensity in TIRFM. ROI, region of interest.

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CSF1R genetic variant determines macrophage polarization

The biological effects of CSF-1 are mediated by the auto-phosphorylation of CSF-1R, which triggers the downstream signaling and determines macrophage differentiation. Because CSF1R c.1085A>G genetic variant regulates the dynamics of CSF-1R endocytosis, it is likely that CSF-1 differentially activates CSF-1R phosphorylation and affects the subsequent macrophage function. Accordingly, we studied the effects of the CSF1R genetic variant on the CSF-1-induced phosphorylation of CSF-1R and the differentiation of M2-like macrophages in vitro. After peripheral blood mononuclear cells were differentiated into macrophages by incubated with CSF-1 (10 ng/mL) for 6 days, macrophages were serum starved for 18 hours, stimulated by CSF-1 for 5 minutes, and then harvested for collection of cell lysates. Phospho-MCSF-Receptor sandwich ELISA kit was used to determine the phosphorylation of CSF-1R. As shown in Figure 4A, there was a significant difference in CSF-1-induced CSF-1R phosphorylation between the reference (CSF1R c.1085A_A) and variant groups (CSF1R c.1085A_G). In macrophages from reference group (CSF1R c.1085 A_A), CSF-1 induced 2.2-fold increase in CSF-1R phosphorylation, in which 10 nmol/L PLX3397, a CSF-1R inhibitor, blocked 70% of CSF-1-induced phosphorylation. In contrast, in the variant groups, CSF-1 induced 1.4-fold increase in CSF-1R phosphorylation that was abolished by 10 nmol/L PLX3397. We further studied whether CSF1R genetic variant determined macrophage polarization (Fig. 4B–E). Cells differentiated from peripheral blood mononuclear cells were harvested after incubation with CSF-1 for 6 days. Forward scatter and side scatter were used to gate the population of macrophages (Fig. 4B) and polarized M2-like macrophages were characterized by the expression of CD206 (Fig. 4D). Compared to the variant group, the percentage of CSF-1-induced macrophage differentiation was significantly higher in the reference group (Fig. 4C, P < 0.05). When CD206 was used to characterize the M2-like macrophages, the percentage of M2-like macrophages after incubation with CSF-1 was also significantly higher in the reference group than in the variant group (Fig. 4E, P < 0.05). Accompanied with the higher percentage of M2-like macrophages, macrophages with CSF1R c.1085 genotype A_A had lower iNOS expression than those with CSF1R c1.085 genotype A_G (Supplementary Fig. S4, P < 0.05).

Figure 4.

CSF1R c.1085A>G genetic variant determines the phosphorylation of CSF-1R and the differentiation and polarization of macrophages. A, Quantitative analyses of CSF-1R phosphorylation induced by CSF-1 in macrophages with (open column) and without (solid column) CSF1R c.1085A>G genetic variant. Macrophages differentiated from peripheral blood mononuclear cells were serum starved for 18 hours followed by stimulation with CSF-1 100 ng/mL for 5 minutes with or without pretreatment with CSF-1R inhibitor, PLX3397. The phosphorylation of CSF-1R was measured by phospho-MCSF-receptor sandwich ELISA Kit. Y axis, normalized CSF-1R phosphorylation. Each value represents mean ± SEM from at least seven different samples in each group. B, Macrophages were differentiated from peripheral blood mononuclear cells by incubation with CSF-1 10 ng/mL for 6 days. Cells were harvested for flow cytometry analysis. Forward scatter (FSC) and side scatter (SSC) were used to gate the population of macrophages. C, The percentage of macrophage differentiation induced by CSF-1 was compared between reference (CSF1R c.1085 A_A) and variant group (CSF1R c.1085 A_G). D, The expression of CD206 was used to characterize the M2-like macrophages. E, The percentage of CD206 (+) cells was compared between the reference and variant group. Data were presented as means ± SEM. **, P < 0.01 by unpaired t test.

Figure 4.

CSF1R c.1085A>G genetic variant determines the phosphorylation of CSF-1R and the differentiation and polarization of macrophages. A, Quantitative analyses of CSF-1R phosphorylation induced by CSF-1 in macrophages with (open column) and without (solid column) CSF1R c.1085A>G genetic variant. Macrophages differentiated from peripheral blood mononuclear cells were serum starved for 18 hours followed by stimulation with CSF-1 100 ng/mL for 5 minutes with or without pretreatment with CSF-1R inhibitor, PLX3397. The phosphorylation of CSF-1R was measured by phospho-MCSF-receptor sandwich ELISA Kit. Y axis, normalized CSF-1R phosphorylation. Each value represents mean ± SEM from at least seven different samples in each group. B, Macrophages were differentiated from peripheral blood mononuclear cells by incubation with CSF-1 10 ng/mL for 6 days. Cells were harvested for flow cytometry analysis. Forward scatter (FSC) and side scatter (SSC) were used to gate the population of macrophages. C, The percentage of macrophage differentiation induced by CSF-1 was compared between reference (CSF1R c.1085 A_A) and variant group (CSF1R c.1085 A_G). D, The expression of CD206 was used to characterize the M2-like macrophages. E, The percentage of CD206 (+) cells was compared between the reference and variant group. Data were presented as means ± SEM. **, P < 0.01 by unpaired t test.

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We also purified mature macrophages by sorting to repeat the experiments of CSF-1R phosphorylation and endocytosis. The protocol of sorting mature macrophages was shown in Supplementary Fig. S5A. In brief, after macrophages were harvested, CD14 and CD206 were used to sort mature macrophages for functional study (Supplementary Fig. S5B and S5C). Consistent with the results shown in Fig. 3 and 4A, CSF-1 induced a rapid receptor endocytosis and remarkable phosphorylation in mature macrophages with the genotype of CSF1R c.1085 A_A.

CSF1R c.1085A>G genetic variant confers the sensitivity of macrophage survival to CSF-1R inhibitors

Small molecule inhibitors of CSF-1R offer an attractive strategy for reducing macrophage numbers associated with cancer as well as autoimmune and inflammatory disease. However, in the early phase clinical trials, the results of CSF-1R inhibitors were controversial, either positive or detrimental for cancer treatment. We here tested the hypothesis that the genetic alteration within the CSF1R locus could determine macrophage susceptibility to CSF-1R inhibitors. Three CSF-1R kinase inhibitors, including PLX3397, BLZ945, and GW2580, were used to study their effects on macrophage proliferation. In cellular assays of macrophages with CSF1R c.1085A>G genetic variant, these three CSF-1R inhibitors dose-dependently inhibited macrophage survival with IC50 in the 0.1 to 1 nmol/L range (Fig. 5A and B). By contrast, for macrophages with CSF1R genotype A_A, PLX3397, BLZ945, and GW2580 inhibited the macrophage survival with IC50 in the 10 to 100 nmol/L range. These results suggest that CSF1R genetic variant confers sensitivity to the treatment of CSF1R inhibitors.

Figure 5.

CSF1R c.1085A>G genetic variant confers the sensitivity of macrophage survival to CSF-1R inhibitors. Macrophages differentiated from peripheral blood mononuclear cells were incubated with various concentrations of three CSF-1R inhibitors, including PLX3397 (A), BLZ945 (B), and GW2580 (C) for 8 days. Percentage of cell survival was determined by CellTiter-Glo Luminescent Cell Viability Assay.

Figure 5.

CSF1R c.1085A>G genetic variant confers the sensitivity of macrophage survival to CSF-1R inhibitors. Macrophages differentiated from peripheral blood mononuclear cells were incubated with various concentrations of three CSF-1R inhibitors, including PLX3397 (A), BLZ945 (B), and GW2580 (C) for 8 days. Percentage of cell survival was determined by CellTiter-Glo Luminescent Cell Viability Assay.

Close modal

Here we identified the genetic alteration within CSF1R locus mediates macrophage function. This study highlights an important finding that macrophages with CSF1R c.1085A>G genetic variant display a refractory response to CSF-1 stimulation and susceptibility to CSF-1R inhibitors. This conclusion is supported by the following evidences. (i) In the reference group (CSF1R genotype A_A), CSF-1 induced a remarkable CSF-1R phosphorylation and a fast process of receptor endocytosis. However, macrophages with CSF1R c.1085A>G genetic variant showed refractory to CSF-1 stimulation, in terms of receptor phosphorylation and endocytosis. (ii) Significant CSF-1-mediated macrophage differentiation and M2 polarization were observed in the reference group. In contrast, macrophages with CSF1R c.1085A>G genetic variant demonstrated a poor response to CSF-1 stimulation while determined by the percentage of macrophage differentiation and expression of M2 marker. (iii) The genetic alteration within CSF1R locus confers the sensitivity to CSF-1R inhibitors. Compared to macrophages with CSF1R c.1085A>G genetic variant, the IC50 of CSF-1R inhibitors to inhibit macrophage survival was 10- to 100-fold higher in the reference group.

In terms of molecular and structural aspects, we propose two possible mechanisms to explain the great impact of c.1085A_G allele on macrophage function. First, the CSF1R c.1085 A>G genetic variant results in a change of amino acid from histidine to arginine within the immunoglobulin-like domain 4 which is essential for the formation of receptor dimerization. Although arginine and histidine are both basic amino acids, histidine has an imidazole side chain that would lose one proton at a pH above its pK of 6.0 leading to the change from positive charge to a neutral status (16). The different side chain of amino acid and the change of positive charge in dimerization domain of CSF-1R caused by the substitution of histidine by arginine might affect the process of CSF-1-induced dimerization, subsequent phosphorylation and endocytosis of CSF-1R. Second, the substitution of amino acid could change the protein stability and alter the level of protein expression. Zhao and colleagues reported arginine-to-histidine mutations of voltage-gated potassium channel also had an impact on the stability of the protein and reduced the protein expression on the cell surface (17). The substitution of histidine by arginine caused CSF1R c.1085 A>G genetic variant might also have an effect on CSF-1R expression. Different levels of CSF-1R expression may explain the different responses to CSF-1 stimulation observed in macrophages with different CSF1R genotypes. Further study is needed to confirm these hypotheses.

Our clinical study showed that CSF1R c.1085 genetic variant was associated with clinical outcomes. M2-like macrophage has been reported to be associated with a poor outcome in different types of cancers (3). Here we found that tumor M1/M2 ratio was significantly higher in the surgical specimens of CSF1R c.1085A>G, compared with that of reference group. In addition, M1/M2 ratio of tumor-associated macrophages inversely correlated with VEGF expression in tumor tissues. It seems that CSF1R c.1085A>G genetic variant regulated tumor immunity through differential recruitment of tumor-associated macrophages and related cytokines. Importantly, when analyzing the effect of CSF1R genetic variant on clinical outcome in patients with stage III colorectal cancer, the reference group demonstrated a poorer disease-free survival than patients with CSF1R c.1085 genetic variant. Endometrial cancer patients were CSF1R c.1085 genotype A_A also had a poor overall survival than those with CSF1R c.1085 A_G. For patients with ovarian cancer, there was also a trend showing a shorter survival in the group of CSF1R c.1085 genotype A_A than genotype A_G. These results imply that CSF1R genetic variant has an impact on tumor immunity associated with clinical outcomes.

CSF-1 induced a strong CSF-1R signaling in macrophages with CSF1R c.1085 genotype A_A, leading to more macrophage differentiation and M2 polarization. Importantly, we observed higher concentration of CSF-1R inhibitors was required to inhibit CSF-1-induced receptor phosphorylation in macrophages with CSF1R c.1085 genotype A_A. Because CSF-1R singling is important for survival, proliferation, and differentiation of macrophages (18), it is not surprised that higher concentration of CSF-1R inhibitors was required to inhibit the survival of macrophages with CSF1R genotype A_A. We observed the IC50 of CSF-1R inhibitors was 10- to 100-fold lower in macrophages with CSF1R c.1085 A_G than A_A. These results imply that CSF-1R inhibitors should be used with caution and highlight the importance of testing CSF1R genotypes before prescribing CSF-1R inhibitors clinically. In patients with CSF1R genotype A_G, targeting CSF-1R signaling by CSF-1R inhibitors may cause excess deaths of macrophages and the beneficial M1 macrophages may also be depleted. The excess death of macrophages would jeopardize innate immunity which would put the host in the risk of infections. And, the depletion of beneficial M1 macrophages may reduce the efficacy of CSF-1R inhibitors in patients with CSF1R c.1085 genotype A_G.

The allele frequency of CSF1R c.1085A>G varies in population. This genetic variant has a high incidence in Eastern Asian population with the allele frequency of 42.86%. However, only 7% to 11% of this genetic variant was observed in Africa, America, south Asia, and European. In the era of precision medicine, using genetic markers to predict the benefit of treatment or avoid potential toxicities is getting more and more important. A good example to show the importance of varied frequency of genetic variants among different ethnic groups is the efficacy of EGFR inhibition in phase III trial of lung cancer, the INTEREST study (19). The Asian lung adenocarcinoma had the high epidermal growth factor receptor (EGFR) mutation frequency (∼50%) and the low EGFR mutation frequency occurred in west population, around 10%. The 20% of Asian population in INTEREST study led to failure of demonstrating better efficacy of EGFR inhibitor than traditional chemotherapy and withdrawal of gefitinib from the US market. A recent study tried to show the different effects of CSF-1R monoclonal antibody on macrophage survival (10). Pradel and colleagues (10) investigated the impact of SNP rs10079250 on the depletion of macrophages by RG7155, a humanized anti-CSF-1R monoclonal antibody blocking the receptor dimerization. The results showed a trend toward a reduced response to RG7155 in donors carrying the variant allele. The SNP rs10079250 is the genetic variant, CSF1R c.1085A>G investigated in our study. The different frequency of CSF1R c.1085 genetic variant among ethnic groups associated with the different response to CSF-1R inhibition raises an important issue that CSF1R genetic variant might be a potential predictive biomarker in targeting CSF-1R signaling clinically.

Taken together, this is the first study to demonstrate CSF1R genetic variant regulates the CSF-1R signaling and sensitivity to CSF-1R inhibitors (summarized in Table 1). Our work showed CSF-1 induced remarkable phosphorylation and endocytosis of CSF-1R in macrophages with CSF1R c.1085 genotype A_A. The prominent activation and dynamics of CSF-1R were accompanied by the significant CSF-1-mediated macrophage differentiation and M2 polarization. M2 predominant distribution of tumor-associated macrophages was also observed in clinical specimens with CSF1R genotype A_A and colorectal cancer patients with CSF1R genotype A_A had a poorer disease-free survival. Different susceptibility to CSF-1R inhibitors in macrophages with different CSF1R genotypes suggests this genetic variant is worth being tested as a predictive marker when targeting CSF-1R signaling by small molecular inhibitors, especially in the population in which the prevalence of CSF1R c.1085A>G genetic variant is high.

Table 1.

The genetic alteration within CSF1R locus mediates macrophage function

Genetic variantCSF1R c.1085 A_ACSF1R c.1085 A_G
Global distribution ∼90% in African, American, European, and South Asian ∼40% in East Asian 
Response to CSF-1 
 CSF-1R phosphorylation Strong Weak 
 CSF-1R endocytosis Rapid Slow 
 M2 polarization Prominent Less prominent 
Sensitivity to CSF-1R inhibitors 
 IC50 10–100 nmol/L 0.1–1 nmol/L 
Potential clinical impact Poor outcome Favorable outcome 
Genetic variantCSF1R c.1085 A_ACSF1R c.1085 A_G
Global distribution ∼90% in African, American, European, and South Asian ∼40% in East Asian 
Response to CSF-1 
 CSF-1R phosphorylation Strong Weak 
 CSF-1R endocytosis Rapid Slow 
 M2 polarization Prominent Less prominent 
Sensitivity to CSF-1R inhibitors 
 IC50 10–100 nmol/L 0.1–1 nmol/L 
Potential clinical impact Poor outcome Favorable outcome 

No potential conflicts of interest were disclosed.

Conception and design: Y.-M. Yeh, S.-J. Hsu, P.-C. Lin, M.-R. Shen

Development of methodology: Y.-M. Yeh, S.-J. Hsu

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y.-M. Yeh, S.-J. Hsu, P.-C. Lin, K.-F. Hsu, P.-Y. Wu, W.-C. Su

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y.-M. Yeh, S.-J. Hsu, P.-C. Lin, K.-F. Hsu, P.-Y. Wu, J.-Y. Chang

Writing, review, and/or revision of the manuscript: Y.-M. Yeh, M.-R. Shen

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y.-M. Yeh, S.-J. Hsu, M.-R. Shen

Study supervision: M.-R. Shen

This work was supplied by a grant from the Ministry of Science and Technology, Ministry of Health and Welfare (MOHW106-TDU-B-211-113003), and National Cheng Kung University Hospital.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Wynn
TA
,
Chawla
A
,
Pollard
JW
. 
Macrophage biology in development, homeostasis and disease
.
Nature
2013
;
496
:
445
55
.
2.
Van Overmeire
E
,
Stijlemans
B
,
Heymann
F
,
Keirsse
J
,
Morias
Y
,
Elkrim
Y
, et al
M-CSF and GM-CSF receptor signaling differentially regulate monocyte maturation and macrophage polarization in the tumor microenvironment
.
Cancer Res
2016
;
76
:
35
42
.
3.
Mantovani
A
,
Sozzani
S
,
Locati
M
,
Allavena
P
,
Sica
A
. 
Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes
.
Trends Immunol
2002
;
23
:
549
55
.
4.
Pyonteck
SM
,
Akkari
L
,
Schuhmacher
AJ
,
Bowman
RL
,
Sevenich
L
,
Quail
DF
, et al
CSF-1R inhibition alters macrophage polarization and blocks glioma progression
.
Nat Med
2013
;
19
:
1264
72
.
5.
DeNardo
DG
,
Brennan
DJ
,
Rexhepaj
E
,
Ruffell
B
,
Shiao
SL
,
Madden
SF
, et al
Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy
.
Cancer Discov
2011
;
1
:
54
67
.
6.
Zhu
Y
,
Knolhoff
BL
,
Meyer
MA
,
Nywening
TM
,
West
BL
,
Luo
J
, et al
CSF1/CSF1R blockade reprograms tumor-infiltrating macrophages and improves response to T-cell checkpoint immunotherapy in pancreatic cancer models
.
Cancer Res
2014
;
74
:
5057
69
.
8.
Rademakers
R
,
Baker
M
,
Nicholson
AM
,
Rutherford
NJ
,
Finch
N
,
Soto-Ortolaza
A
, et al
Mutations in the colony stimulating factor 1 receptor (CSF1R) gene cause hereditary diffuse leukoencephalopathy with spheroids
.
Nat Genet
2011
;
44
:
200
5
.
9.
Kang
HG
,
Lee
SY
,
Jeon
HS
,
Choi
YY
,
Kim
S
,
Lee
WK
, et al
A functional polymorphism in CSF1R gene is a novel susceptibility marker for lung cancer among never-smoking females
.
J Thorac Oncol
2014
;
9
:
1647
55
.
10.
Pradel
LP
,
Ooi
CH
,
Romagnoli
S
,
Cannarile
MA
,
Sade
H
,
Ruttinger
D
, et al
Macrophage susceptibility to emactuzumab (RG7155) treatment
.
Mol Cancer Ther
2016
;
15
:
3077
86
.
12.
Fan
CT
,
Lin
JC
,
Lee
CH
. 
Taiwan Biobank: a project aiming to aid Taiwan's transition into a biomedical island
.
Pharmacogenomics
2008
;
9
:
235
46
.
13.
Genomes Project
C
,
Auton
A
,
Brooks
LD
,
Durbin
RM
,
Garrison
EP
,
Kang
HM
, et al
A global reference for human genetic variation
.
Nature
2015
;
526
:
68
74
.
14.
Li
W
,
Stanley
ER
. 
Role of dimerization and modification of the CSF-1 receptor in its activation and internalization during the CSF-1 response
.
EMBO J
1991
;
10
:
277
88
.
15.
Lou
J
,
Low-Nam
ST
,
Kerkvliet
JG
,
Hoppe
AD
. 
Delivery of CSF-1R to the lumen of macropinosomes promotes its destruction in macrophages
.
J Cell Sci
2014
;
127
:
5228
39
.
16.
Tu
Z
,
Young
A
,
Murphy
C
,
Liang
JF
. 
The pH sensitivity of histidine-containing lytic peptides
.
J Pept Sci
2009
;
15
:
790
5
.
17.
Zhao
J
,
Zhu
J
,
Thornhill
WB
. 
Spinocerebellar ataxia-13 Kv3.3 potassium channels: arginine-to-histidine mutations affect both functional and protein expression on the cell surface
.
Biochem J
2013
;
454
:
259
65
.
18.
Pixley
FJ
,
Stanley
ER
. 
CSF-1 regulation of the wandering macrophage: complexity in action
.
Trends Cell Biol
2004
;
14
:
628
38
.
19.
Kim
ES
,
Hirsh
V
,
Mok
T
,
Socinski
MA
,
Gervais
R
,
Wu
YL
, et al
Gefitinib versus docetaxel in previously treated non-small-cell lung cancer (INTEREST): a randomised phase III trial
.
Lancet
2008
;
372
:
1809
18
.