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Cancer Research Center Nantes-Angers, Inserm, U892, Nantes, FranceCancer Research Center Nantes-Angers, CNRS, UMR6299, Nantes, FranceNantes University, Nantes, France
Cancer Research Center Nantes-Angers, Inserm, U892, Nantes, FranceCancer Research Center Nantes-Angers, CNRS, UMR6299, Nantes, FranceCancer Research Center Nantes-Angers, Inserm, U892, Angers, FranceCancer Research Center Nantes-Angers, CNRS, UMR6299, Angers, France
Cancer Research Center Nantes-Angers, Inserm, U892, Nantes, FranceCancer Research Center Nantes-Angers, CNRS, UMR6299, Nantes, FranceNantes University, Nantes, France
Cancer Research Center Nantes-Angers, Inserm, U892, Nantes, FranceCancer Research Center Nantes-Angers, CNRS, UMR6299, Nantes, FranceNantes University, Nantes, France
Cancer Research Center Nantes-Angers, Inserm, U892, Nantes, FranceCancer Research Center Nantes-Angers, CNRS, UMR6299, Nantes, FranceNantes University, Nantes, France
Cancer Research Center Nantes-Angers, Inserm, U892, Angers, FranceCancer Research Center Nantes-Angers, CNRS, UMR6299, Angers, FranceImmunology and Allergology Laboratory, University Hospital of Angers, Angers, France
Cancer Research Center Nantes-Angers, Inserm, U892, Nantes, FranceCancer Research Center Nantes-Angers, CNRS, UMR6299, Nantes, FranceNantes University, Nantes, France
Cancer Research Center Nantes-Angers, Inserm, U892, Nantes, FranceCancer Research Center Nantes-Angers, CNRS, UMR6299, Nantes, FranceNantes University, Nantes, France
Mesothelioma is a rare and aggressive cancer related to asbestos exposure. We recently showed that pleural effusions (PEs) from patients with mesothelioma contain high levels of the C-C motif chemokine ligand 2 (CCL2) inflammatory chemokine. In the present work, we studied the effect of CCL2 contained in mesothelioma samples, particularly on monocyte recruitment. Then, we studied the fate of these monocytes in malignant pleural mesothelioma (MPM) PEs and their impact on tumor cells' properties.
Methods
The implication of CCL2 in monocyte recruitment was evaluated using transmigration assays and a CCL2 blocking antibody. The phenotype of macrophages was determined by flow cytometry and enzyme-linked immunosorbent assay. Immunohistochemical analysis was used to support the results. Cocultures of macrophages with mesothelioma cells were performed to study cancer cell proliferation and resistance to treatment.
Results
We showed that CCL2 is a major factor of monocyte recruitment induced by MPM samples. Macrophages obtained in MPM samples were M2 macrophages (high CD14, high CD163, and interleukin-10 secretion after activation). The colony-stimulating factor 1 receptor/macrophage colony-stimulating factor (M-CSF) pathway is implicated in M2 polarization, and high levels of M-CSF were measured in MPM samples compared with benign PE (4.17 ± 2.75 ng/mL and 1.94 ± 1.47 ng/mL, respectively). Immunohistochemical analysis confirmed the presence of M2 macrophages in pleural and peritoneal mesothelioma. Finally, we showed that M2 macrophages increased mesothelioma cell proliferation and resistance to treatment.
Conclusions
These results demonstrate the implication of CCL2 in MPM pathogenesis and designate M-CSF as a new potential biomarker of MPM. This study also identifies CCL2 and colony-stimulating factor 1 receptor/M-CSF as interesting new targets to modulate pro-tumorigenic properties of the tumor microenvironment.
Malignant pleural mesothelioma (MPM) is a rare and aggressive cancer related to asbestos exposure, mainly during occupational activities. Clinical symptoms, including pleural effusion (PE), dyspnea, and chest pain, arise later in the development of the disease, 30 to 40 years after asbestos exposure, when the efficacy of therapeutic interventions is limited. Mesothelioma is described as a poorly immunogenic cancer. Immunosuppression in cancer may be related to, among other parameters, the coexistence of different immunosuppressive cells in the tumor microenvironment such as T-regulatory lymphocytes (Tregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs).
Macrophages are essential components of the innate immunity. Depending on their environment and their plasticity, macrophages can exhibit a M1 or M2 phenotype closely associated with the Th1-Th2 polarization of lymphocytes.
This subtle cooperation between M1 and M2 macrophages is altered in diseases such as cancer. Indeed, the presence and the density of CD163-positive M2 macrophages are associated with bad prognosis.
Macrophages can promote tumor development by secreting a large number of factors, including cytokines, growth factors, matrix remodeling enzymes, and proteases. Moreover, M2 macrophages are known to participate in immunosuppression and are characterized by the secretion of interleukin (IL)-10, an immunoregulatory cytokine. Previous studies have demonstrated that mesothelioma tumors are infiltrated by M2 macrophages.
Thus, the level of M2 macrophages could be a prognostic factor for patients with epithelioid mesothelioma and for local tumor outgrowth at the intervention site.
Intratumoral macrophage phenotype and CD8(+) T lymphocytes as potential tools to predict local tumor outgrowth at the intervention site in malignant pleural mesothelioma.
C-C motif chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1, belongs to the CC chemokine superfamily and is a chemokine that is well known for its chemoattractive properties toward monocytes.
This inflammatory chemokine is produced by tumor cells and cells from the microenvironment. CCL2 leads to the recruitment of several players implicated in tumor development (macrophages, subsets of lymphocytes [including Tregs], and endothelial cells) and participates in tumor promotion by acting through its receptor, C-C motif chemokine receptor 2 (CCR2).
The aim of this study was to analyze the function of CCL2 in the pathogenesis of mesothelioma and especially in the recruitment of macrophages to the tumor microenvironment. We first investigated the implication of the CCL2 contained in PE or in MPM cell culture supernatants (SNs) on the recruitment of monocytes. Then, we determined the fate of these monocytes in the tumor microenvironment, mainly their macrophage polarization. Finally, we studied the effects of these macrophages on MPM cells' proliferation and resistance to therapy.
Materials and Methods
Patient Samples
PEs from patients with MPM or with a nonmalignant disease were collected aseptically at the Nantes Hospital. All patients had signed informed consent. These samples were centrifuged at 800 g for 20 minutes at 4°C and SNs were aliquoted and stored at –80°C.
Cell Lines
The mesothelioma cell lines were established from the pleural fluids of patients with mesothelioma.
All cell lines were maintained in Roswell Park Memorial Institute-1640 (RPMI-1640) medium (Gibco, Carlsbad, CA) supplemented with 2 mM L-glutamine (Gibco), 100 IU/mL penicillin (Gibco), 0.1 mg/mL streptomycin (Gibco) and 10% heat-inactivated fetal calf serum (HyClone [GE Healthcare, Pittsburgh, PA]).
Preparation and Collection of the MPM Cell Culture SNs
MPM cells were seeded in six-well plates (Falcon [Thermo Fisher Scientific, Waltham, MA) at 2 × 106 cells per well. After 24 hours, the medium was replaced by 2 mL of fresh medium containing 2% fetal calf serum for an additional 24 hours. Then, SNs were collected, centrifuged for 5 minutes at 800 g, and used immediately for migration or monocyte differentiation experiments or aliquoted and stored at –80°C.
ELISA Assays
CCL2 levels were determined using the Human Monocyte Chemoattractant Protein-1 ELISA (Enzyme-Linked Immunosorbent Assay) Kit (PromoKine, Heidelberg, Germany). M-CSF levels were measured using Human M-CSF Immunoassay (Quantikine [R&D Systems, Minneapolis, MN]). IL-10 and IL-12 levels were determined using Human DuoSet ELISA kits (R&D Systems).
Monocyte Transmigration Assay
Monocytes and human serum albumin (HSA) were obtained from the clinical transfer platform of Nantes Hospital. First, 600 μL of PE, SN, or RPMI supplemented with 2% HSA and containing or not recombinant CCL2 (Abcam, Cambridge, U.K.) were added to 12-well plates (Falcon). Then, 5-μm transwell filters (Corning, Corning, NY) were inserted into the wells and loaded with 100 μL of RPMI 2% HSA (Vialebex, French Laboratory of Fractionation and Biotechnology, Les Ulis, France) containing 1 × 105 monocytes. After 3 hours of incubation at 37°C and 5% CO2, liquids in the 12-well plates were collected and centrifuged for 5 minutes at 800 g. Pellets were resuspended in 300 μL of phosphate-buffered saline containing acridine orange (Chemometec, Copenhagen, Denmark) at 2 μg/mL. The use of acridine orange made it possible to eliminate inert particles present in PE and to count only monocytes. The number of monocytes was determined by flow cytometry (FACS Calibur [BD Biosciences, San Jose, CA]) as the number of events counted in the FL2 channel during 90 seconds of acquisition at maximal speed. Results were analyzed using Flowjo software (BD Biosciences). To avoid variations between donors, monocyte migration was normalized according to the spontaneous monocytes migration in response to RPMI 2% HSA.
For the neutralizing experiments, anti-CCL2 antibodies (Abcam) or IgG1κ isotype control (Abcam) were incubated with the samples at 10 μg/mL for 30 minutes at room temperature before the migration experiments.
Monocyte Differentiation
Monocytes were seeded in six-well plates at a density of 5 × 106 cells in 5 mL of culture medium. To obtain M1 macrophages, the medium was supplemented with granulocyte-macrophage colony-stimulating factor at 20 ng/mL (Cellgenix, Freiburg im Breisgau, Germany) and to obtain M2 macrophages, the medium was supplemented with M-CSF at 50 ng/mL (Isokine, Kopavogur, Iceland). PEs were diluted 10 times in culture medium and SN were used undiluted. After 3 days of incubation with or without colony-stimulating factor 1 receptor (CSF1R) inhibitor GW2580 (1 μM) (LC Laboratories, Woburn, MA), cells were collected and labeled with anti–CD14–fluorescein isothiocyanate (FITC) (Beckman Coulter, Fullerton, CA) and anti–CD163- allophycocyanin (APC) (R&D Systems) antibodies or IgG2a-FITC (Beckman Coulter) and IgG1-APC (R&D Systems) isotype controls. Analysis of the cells was performed using flow cytometry. For lipopolysaccharide (LPS) stimulation, macrophages were seeded in 96-well plates at a density of 2 × 105 cells in 200 μL of culture medium. Cells were treated with 200 ng/mL of LPS (Sigma-Aldrich, St. Louis, MO) and the SNs were collected after 24 hours for IL-12 measurement or 48 hours for IL-10 measurement.
Immunohistochemical Analysis
Immunohistochemical analysis was performed on mesothelioma tissue microarrays (TMAs) (US BIOMAX, Inc., Rockville, MD) using standard techniques. The anti-CD163 antibody (Bioss, Inc., Woburn, MA) was used at 5 μg/mL.
Coculture of MPM Cells with M2 Macrophages
M2 macrophages were obtained as already described by incubating monocytes with M-CSF for 3 days. Macrophages were labeled with anti–CD14-FITC (Beckman Coulter) and anti–CD163-APC (R&D Systems) antibodies or IgG2a-FITC (Beckman Coulter) and IgG1-APC (R&D Systems) isotype controls, and analyzed by flow cytometry to confirm their M2 phenotype. MPM cells were seeded at a density of 5 × 103 cells in 96-well plates (Nunclon Delta Surface, Thermo Scientific, Waltham, MA). At the same time, 0, 10, or 100 M2 macrophages were added to the MPM cells. After 24 hours, cisplatin (Sigma-Aldrich) at 0.8 mg/mL and/or pemetrexed (Sigma-Aldrich) at 80 μM were added for an additional 72 hours. Uptiblue cell counting reagent (Interchim, Montluçon, France) (5%, v/v) was then added to the culture medium for 2 hours at 37°C. Fluorescence was measured at 605 nm after a green epi-illumination excitation using a ChemiDoc MP imaging system (Bio-Rad, Hercules, CA). Quantification was performed using ImageJ 1.41o software.
Statistical Analysis
Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA). Data were expressed as means plus or minus the standard error of the mean of at least three independent experiments. Statistical comparisons were made using the nonparametric Mann-Whitney u-test. Correlation analyzes were performed using the Pearson’s test.
Results
Implication of the CCL2 Contained in PEs and in MPM Cell Culture SNs in the Recruitment of Monocytes
To study the implication of the CCL2 contained in MPM samples on monocyte migration, we performed monocyte migration assays using transwell filters. To this aim, first, monocyte migration was evaluated in response to PE and SN with previously determined CCL2 concentrations.
In a first set of experiments, no correlation was observed between monocyte migration and CCL2 concentrations in PE and SN (Fig. 1A and 1B). In a second set of experiments, to determine the real impact of CCL2 in monocyte migration, we used an anti-CCL2 neutralizing antibody. The efficacy and the specificity of the antibody was confirmed in a monocyte migration assay in response to RPMI medium containing 2% HSA and 10 ng/mL of recombinant CCL2 (Fig. 1C and Supplementary Fig. 1). In the presence of the neutralizing antibody (10 μg/mL), monocyte migration was reduced by 95%. In response to PE and SN, monocyte migration was also drastically reduced by the anti-CCL2 blocking antibody (by 82.5 ± 5.6% and 76.5 ± 12.8%, respectively). To confirm these results, we used a CCR2 antagonist (Supplementary Fig. 2). We showed that the use of a CCR2 antagonist reduced migration induced by recombinant CCL2 and PE by 90% and 75%, respectively (Supplementary Fig. 2A). The effect is specific regarding the absence of toxicity of the CCR2 antagonist on monocyte viability (Supplementary Fig. 2B) and the absence of modification of monocyte migration in the presence of DMSO (solvent of CCR2 antagonist) (Supplementary Fig. 2A). All these results demonstrate that whereas there is no direct correlation between monocyte migration and CCL2 concentrations in PE and SN, this chemokine plays a major role in monocyte recruitment by these samples.
Figure 1Effect of C-C motif chemokine ligand 2 (CCL2) present in mesothelioma microenvironment on monocyte migration. Migration of monocytes through transwell filters was evaluated in the presence of pleural effusions (PEs) from patients with mesothelioma (A) or mesothelioma cell culture supernatants (SN) (B) containing various CCL2 concentrations. Using a blocking antibody directed against CCL2 (10 μg/mL), we determined the implication of this chemokine in monocyte migration. (C) Validation of the blocking antibody in standard conditions (2% human serum albumin [HSA]; 10 ng/mL of CCL2 [n = 5]). The effect of the antibody was studied on monocyte migration induced by PEs from patients with MPM (n = 9) (D) or by mesothelioma cell culture SNs (n = 8) (E). r = Spearman’s r; ***p < 0.001.
Study of the Macrophage Phenotype Obtained from Monocytes Incubated in PE from Patients with MPM
When recruited in the tumor microenvironment, monocytes differentiate in macrophages. These cells polarize into M1 macrophages, which are equipped with beneficial capacities such as tumor cell killing, or into M2 macrophages, which acquire deleterious properties increasing immune suppression and tumor promotion. To define the phenotype of the macrophages obtained from monocytes incubated in PE, cells were labeled with CD14-FITC and CD163-APC antibodies and analyzed by flow cytometry. As controls, monocytes were differentiated with granulocyte-macrophage colony-stimulating factor (20 ng/mL) to obtain M1 macrophages (CD14low/CD163 low) or with M-CSF (50 ng/mL) to obtain M2 macrophages (CD14mild/CD163high) (Fig. 2A). In the presence of PE, monocytes differentiated into macrophages with mild CD14 and high CD163 expression like M2 macrophages.
Figure 2Effect of pleural effusions (PEs) from patients with malignant pleural mesothelioma (MPM) and MPM cell culture supernatants (SNs) on macrophage phenotype. (A) Monocytes were incubated with granulocyte-macrophage colony-stimulating factor (GM-CSF) (20 ng/mL), macrophage colony-stimulating factor (M-CSF) (50 ng/mL), or PEs from patients with MPM (n = 8). Cells were labeled with an anti–CD14–fluorescein isothiocyanate and an anti–CD163-allophycocyanin and analyzed by flow cytometry. (B) Macrophages obtained in (A) were stimulated with lipopolysaccharide (200 ng/mL). Then, interleukin-12 (IL-12) and interleukin-10 levels were measured in SNs using enzyme-linked immunosorbent assay. (C) M-CSF levels in benign PE (BPE) (n = 11) and in PEs from patients with MPM (n = 22). ∗p < 0.05. (D) Monocytes were incubated for 3 days with M-CSF (50 ng/mL) or PE (n = 8) in the presence or absence of 1 μM GW2580. ∗∗p < 0.01. (E) Monocytes were incubated with GM-CSF (20 ng/mL), M-CSF (50 ng/mL), or MPM cell culture SNs (n = 8). Cells were labeled with an anti–CD14–fluorescein isothiocyanate and an anti–CD163- allophycocyanin and analyzed by flow cytometry. (F) M-CSF levels in MPM cell culture SNs (n = 14). RFMI, relative mean fluorescence intensity.
It was previously reported that after LPS stimulation, M1 macrophages can produce mainly IL-12 and low levels of IL-10 whereas M2 macrophages produce high amounts of IL-10 but no IL-12. To confirm the M2 phenotype of the cells obtained in PE, macrophages were stimulated with LPS for 24 hours, and IL-10 and IL-12 levels were measured in the cell culture SNs. Activated M1 macrophages produced IL-10 and high levels of IL-12, whereas activated M2 macrophages produced only IL-10. Activated macrophages derived from monocytes incubated in PE produced only IL-10, confirming the M2 phenotype of these cells (Fig. 2B). Then, we measured the level of the chemokine M-CSF, which is known to induce the differentiation of monocytes into M2 macrophages, in these samples (Table 1). Interestingly, M-CSF was detected in all the tested PEs (Fig. 2C). Moreover, we observed that the M-CSF level was significantly higher in PE from patients with MPM than in benign PE (BPE) (4.17 ± 2.75 ng/mL and 1.94 ± 1.47 ng/mL, respectively). No difference in M-CSF level was observed between MPM subtypes (Supplementary Fig. 3).
Table 1Description of Pleural Effusion Groups and Demographic Characteristics of Recruited Patients
We also measured M-CSF in serum from BPE and patients with MPM. The observed concentrations were low, and we did not see any significant difference between BPE and MPM (Supplementary Fig. 4 and Supplementary Table 1).
M-CSF acts through its binding to CSF1R. To demonstrate the implication of the CSF1-R pathway in the differentiation of monocytes into M2 macrophages, we used a CSF1R inhibitor, GW 2580 (Fig. 2D). This molecule inhibits CSF1R intracellular signaling after binding of ligand. When monocytes were incubated with GW2580, the relative mean fluorescence intensities (RMFIs) of CD14 and CD163 were strongly decreased compared with those of monocytes incubated with M-CSF (70% and 90% of inhibition for CD14 and CD163 RMFI, respectively) or PE (40% and 50% of inhibition for CD14 and CD163 RMFI, respectively).
Study of the Macrophage Phenotype Obtained from Monocytes Incubated in SN
To evaluate the role of MPM cells in the differentiation of monocytes into M2 macrophages, monocytes were incubated with SN from MPM cell lines. The phenotype of the macrophages was defined as previously, by labeling cells with anti–CD14-FITC and anti–CD163-APC antibodies and flow cytometry analysis. As observed for macrophages obtained from monocytes incubated in PE, macrophages obtained from monocytes incubated in SN expressed mild levels of CD14 and high levels of CD163 as the control M2 macrophages (Fig. 2E). We then measured the levels of M-CSF in SN using ELISA assay (Fig. 2F). M-CSF was detected in all the tested SNs, thus demonstrating that MPM cells can secrete M-CSF in their environment and could be responsible for or participate in the production of M-CSF in PE.
CD163-Positive Macrophages Infiltrate Pleural and Peritoneal Mesothelioma Tumors
To confirm our results obtained in vitro, we labeled TMAs, including pleural and peritoneal mesothelioma, with an anti-CD163 antibody. Immunohistochemical analysis of the TMAs revealed the presence of CD163-positive macrophages in all pleural (n = 9) and peritoneal mesotheliomas (n = 7) (Fig. 3). This confirms that the mesothelioma microenvironment allows the differentiation of monocytes into M2 macrophages.
Figure 3Presence of M2 macrophages in human malignant mesothelioma tumors. Representative results of immunohistological labeling of CD163 in two malignant pleural mesotheliomas (MPMs) and two peritoneal mesotheliomas on a tissue microarray. Arrows indicate CD163-labeled macrophages.
Impact of the Presence of M2 Macrophages on Proliferation and Resistance of MPM Cells to Chemotherapeutic Agents
In the tumor microenvironment, by secreting a large number of factors, M2 macrophages can change the oncogenic properties of tumor cells. Here, we cocultivated MPM cell lines with M2 macrophages. Then, we measured MPM cell proliferation and also analyzed resistance to the first-line regimen used to treat patients with MPM, a combination of cisplatin and pemetrexed. In the presence of M2 macrophages, the proliferation rate of MPM cells was significantly increased (Fig. 4A). Sensitivity of MPM cells to cisplatin or pemetrexed, used alone or in combination, was drastically reduced in the presence of M2 macrophages (Fig. 4B). Toxic effects of cisplatin and pemetrexed used alone were drastically inhibited in the presence of M2 macrophages. Likewise, the ability of the cisplatin-pemetrexed combination to decrease MPM cell viability was reduced in the presence of M2 macrophages. Indeed, the viability of MPM cells treated with the cisplatin-pemetrexed combination was 28.46 % ± 4.35, whereas in the presence of M2 macrophages the viability of MPM cells was higher (45.67% ± 6.05 in the presence of 100 M2 macrophages per 5 × 103 MPM cells and 51.34% ± 10.07 in the presence of 10 M2 macrophages per 5 × 103 MPM cells).
Figure 4Effect of M2 macrophages on malignant pleural mesothelioma (MPM) cell proliferation and chemotherapy sensitivity. Monocytes were incubated with macrophage colony-stimulating factor (50 ng/mL) to obtain M2 macrophages. MPM cells were cocultured with increasing amounts of M2 macrophages (10 or 100 M2 macrophages for 5 × 103 MPM cells). (A) MPM cell growth was evaluated using Uptiblue cell counting reagent. Results are expressed as the percentage of MPM cell growth measured in the absence of M2 macrophages (100%). (B) Sensitivity of MPM cells to cisplatin (Cis) (0.8 mg/L) and/or pemetrexed (80 μM). Cell growth was measured after 72 hours of treatment using Uptiblue cell counting reagent. Results are expressed as the percentage of MPM cell growth measured in the absence of treatment (100%). Results are the means plus standard error of the mean of experiments performed on three MPM cell lines. *p < 0.05, ***p < 0.001.
In conclusion, these data show that M2 macrophages decrease the efficacy of cisplatin-pemetrexed treatment in malignant mesothelioma.
Discussion
Cellular composition of the tumor microenvironment conditions disease progression. Tumor infiltration by Tregs, MDSCs, or TAMs is associated with bad prognosis. Inflammatory processes related to tumor development, cells in the tumor microenvironment, and tumor cells themselves are all responsible for the recruitment of these immunosuppressive cells by modifying the chemokine landscape. We previously reported that the chemokine CCL2 is expressed at high levels in PEs from patients with mesothelioma and that this chemokine can be produced directly by the tumor cells.
and to study the implication of this chemokine in mesothelioma, we evaluated the effect of PE and SN on monocyte recruitment. We showed, by using an anti-CCL2 neutralizing antibody, that CCL2 is a major chemokine involved in the monocyte recruitment by MPM samples. Then, we determined the phenotype of macrophages obtained from monocytes incubated in the presence of PE or SN. We demonstrated that monocytes undergo M2 polarization, probably driven by the presence of M-CSF in the environment as shown by ELISA. Infiltration of mesothelioma tumors by M2 macrophages was also confirmed by immunohistochemical analysis using an anti-CD163 antibody. Finally, we demonstrated that M2 macrophages increased proliferation and resistance of mesothelioma cells to cisplatin and pemetrexed treatments, alone or in combination.
CCL2 is an immunomodulatory chemokine present in the microenvironment of numerous tumors, including mesothelioma.
According to its described function, inhibition of CCL2 or CCR2 decreases monocyte migration induced by PE and SN from mesothelioma samples. Our results demonstrate that CCL2 is a major player in monocyte recruitment induced by mesothelioma samples. However, the complex composition of PE and SN, which includes additional chemoattractant factors and probably inhibitors of migration, may be responsible for the absence of direct correlation between CCL2 concentrations and monocyte migration.
PE and SN capacity to induce M2 polarization of macrophages is associated with the presence of M-CSF, a well-known chemokine implicated in this process. The differentiation of monocytes into M2 macrophages induced by M-CSF or PE is decreased by the use of GW2580, an inhibitor of CSF1R signaling, thus demonstrating the crucial implication of the CSF1R pathway in this process. M-CSF is a major factor given that all PE samples used for the differentiation experiments do not contain the second CSF1R ligand, IL-34. Indeed, the PE samples contained M-CSF in concentrations ranging from 0.85 to 8.68 ng/mL, whereas IL-34 was not detected (IL-34 concentrations less than 100 pg/mL or null according to ELISA kit sensitivity, Human IL-34 DuoSet ELISA, [R&D Systems]). The difference in activity of GW2580 between M-CSF and PE conditions (approximately 30%) raises the question of the implication of other cytokines, such as IL-4 or IL-13 for example, in PE.
The ability of the MPM tumor environment to drive M2 differentiation is confirmed by immunohistochemical analysis, as shown by the presence of CD163-positive cells in MPM tumors, as previously described.
However, for the first time, we describe the presence of M2 macrophages in peritoneal mesothelioma tumors, suggesting high similarities between the pleural and peritoneal mesothelioma microenvironments.
The presence of M-CSF in PEs appears as a potent soluble biomarker to differentiate BPE from mesothelioma PE. This observation constitutes the first description of potent diagnostic properties for M-CSF in mesothelioma. M-CSF measurements performed on SN from MPM cells showed that tumor cells participate in the production of the M-CSF present in PE, which confirmed recent results obtained by Cioce et al.
Serum levels of M-CSF were not discriminating between patients with BPE and patients with mesothelioma PE (Supplementary Fig. 4). However, it was previously shown that serum levels of M-CSF could be a prognostic factor in mesothelioma
Pretreatment serum levels of cytokines and cytokine receptors in patients with non-small cell lung cancer, and correlations with clinicopathological features and prognosis. M-CSF—an independent prognostic factor.
All these observations suggest that M-CSF could be an interesting biomarker for mesothelioma, but additional studies on a larger cohort of samples are required to confirm this hypothesis.
In coculture experiments, we showed that M2 macrophages induce MPM cell proliferation and resistance to cisplatin and pemetrexed used alone or in combination. This suggests that M2 macrophages could participate in tumor promotion and resistance to treatment in mesothelioma, as previously described for other cancers.
In addition, we demonstrated that M2 macrophages, derived from monocytes incubated in the presence of PE, secreted the immunosuppressive chemokine IL-10 after LPS stimulation. These observations could explain the association of a high level of M2 macrophages in tumors with a worst prognosis in mesothelioma.
Intratumoral macrophage phenotype and CD8(+) T lymphocytes as potential tools to predict local tumor outgrowth at the intervention site in malignant pleural mesothelioma.
All of our results highlight the cooperation between the chemokines CCL2, which participates in the recruitment of monocytes, and M-CSF, which drives M2 polarization, to promote mesothelioma development. However, no correlation was observed between CCL2 and M-CSF concentrations in PE and SN (Supplementary Fig. 5). Additional functions were described for these chemokines in cancer, which designate them as interesting therapeutic targets. Indeed, CCL2 could be involved in the recruitment of other immunosuppressive cells such as Tregs and MDSC
CCL2 inhibition using blocking antibodies was already demonstrated to induce tumor growth reduction in animal cancer models with an improvement of the immune response.
In a model of lung adenocarcinoma, it was demonstrated that this effect was produced by a change in TAM polarization. Moreover, cytotoxic CD8-positive T cells were more activated. In addition, SCID mice or CD8 T-cell–depleted mice lost the benefit of CCL2 blockade. These data indicate that CCL2 blockade can inhibit tumor growth by altering TAM phenotype and activating CTLs.
However, the efficacy of these strategies remains to be confirmed in clinical practice. M-CSF/CSF1R signaling was also described as an autocrine pathway that promotes chemoresistance of mesothelioma cells.
Anti–M-CSF/CSF1R therapies were developed, mainly inhibitors (drugs or antibodies) of CSF1R. The blocking of CSF-1R has shown interesting effects against tumor growth, metastasis spreading, and resistance to chemotherapeutic agents.
In conclusion, our work designates CCL2/CCR2 and M-CSF/CSF1R pathways as potential targets to reduce M2 macrophage levels in the mesothelioma microenvironment and in this way to improve the efficacy of chemotherapeutic and immunotherapeutic strategies. Additionally, as we previously suggested for CCL2,
M-CSF levels in PE could have a diagnostic value to discriminate patients with BPE and patients with MPM. Studies using larger cohorts of samples are required to confirm this hypothesis.
Acknowledgments
This work was supported by the Research Institute Respiratory Pays de la Loire en santé respiratoire des Pays de la Loire, the French Ligue Against Cancer (committees of Morbihan, Sarthe, Vendée et Loire-Atlantique), ARSMESO44, Nantes University Hospital, and Région Pays de la Loire for TB grant. The authors thank the Cytocell core facility for the flow cytometry experiments and the clinical transfer platform (CIC-biothérapies Nantes) for monocyte purification.
Intratumoral macrophage phenotype and CD8(+) T lymphocytes as potential tools to predict local tumor outgrowth at the intervention site in malignant pleural mesothelioma.
Pretreatment serum levels of cytokines and cytokine receptors in patients with non-small cell lung cancer, and correlations with clinicopathological features and prognosis. M-CSF—an independent prognostic factor.
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