The ability of tumor cells to escape immune destruction and their acquired resistance to chemotherapy are major obstacles to effective cancer therapy. Although immune checkpoint therapies such as anti-PD-1 address these issues in part, clinical responses remain limited to a subpopulation of patients. In this report, we identified IL34 produced by cancer cells as a driver of chemoresistance. In particular, we found that IL34 modulated the functions of tumor-associated macrophages to enhance local immunosuppression and to promote the survival of chemoresistant cancer cells by activating AKT signaling. Targeting IL34 in chemoresistant tumors resulted in a remarkable inhibition of tumor growth when accompanied with chemotherapy. Our results define a pathogenic role for IL34 in mediating immunosuppression and chemoresistance and identify it as a tractable target for anticancer therapy. Cancer Res; 76(20); 6030–42. ©2016 AACR.

Although recent progress in cancer research has helped to develop new selective treatments based on increased understanding of cancer biology, chemotherapy is still one of the most common treatment options for many cancers (1). Chemotherapy is used to induce cell death in tumors and augment immune responses to cancer. However, many cancer patients experience recurrence and ultimately death because of chemoresistance and chemotherapy-induced immunosuppression. In the past few decades, several studies have underlined intrinsic mechanisms that occur inside cancer cells and contribute to chemoresistance such as the induction of antiapoptotic regulators, ABC transporters, aberrant NF-κB activities, and DNA damage machinery (2, 3). In addition, recent advances in cancer immunology have suggested that the therapeutic resistance of tumors also relies importantly on extrinsic mechanisms represented by the cross-talk between tumor cells and other cellular components of the tumor microenvironment (TME), in particular immune cells (4). Indeed, understanding the complex interaction between cancer and immune system has provided unique therapeutic opportunities to improve the clinical benefit of chemotherapy; for example, combining chemotherapy with antibodies that target immune-checkpoint molecules such as programmed cell death-1 (PD-1) in advanced non–small lung cancer patients (5). In spite of its promising results, clinical responses are still limited to a subpopulation of patients (6), and identifying new therapeutic targets is critically needed.

Tumor-associated macrophages (TAM) constitute the dominant myeloid cell population in many tumors and play critical roles in multiple aspects of TME, including therapeutic resistance (7–9). Importantly, increased frequency of protumorigenic TAMs following chemotherapy is a hallmark of developing chemoresistance and correlate with poor clinical outcomes (7–9). In particular, M2-polarized TAMs drive multiple protumorigenic processes, including immunosuppression, angiogenesis, metastasis, tumor survival, and cancer cell stemness (7–12). Consistent with this, targeting of macrophages by colony stimulating factor 1 receptor (CSF1R) inhibitors or blocking mAbs showed promising effects to improve chemotherapeutic responses (13, 14). However, considering the critical roles of macrophages in homeostasis and innate immunity, there are concerns about side effects that may arise from extended depletion of these cells at the systemic level. Alternatively, targeting factors induced by chemotherapy and affecting macrophage polarization may have the potential to improve the clinical outcome of cytotoxic chemotherapy with fewer side effects. Accordingly, further investigations are needed to identify mechanisms that drive macrophage polarization under chemotherapeutic conditions.

To identify such mechanisms, we developed a model of chemoresistance in lung cancer cells, the major leading cause of cancer-related deaths in the world and frequently accompanied with chemoresistance (15). Using this model, we identified IL34 as a novel factor that contributes to immunosuppression and survival of chemoresistant cancer cells in chemotherapy-treated TME. In this report, we described the role of IL34 as a molecular driver of chemoresistance, and discuss the significance of these basic findings on clinical applications in cancer patients.

Cell culture

All cell lines were obtained in 2013 as follows. Lung (A549, H1299), breast (MCF7, MDA-MB-231, MDA-MB-436, T47D), liver (Hep3B, HepG2), and colon (HCT116, SW480) cancer cell lines were obtained from the ATCC. KFr and TuoM-1 cell lines were provided by Prof. Yoshihiro Kikuchi (National Defense Medical College, Saitama, Japan) and Prof. Junzo Kigawa (Tottori University, School of Medicine), respectively. OVISE and OVTOKO cell lines were obtained from JCRB (Japanese Collection of Research Bio resources, Osaka, Japan). All cell lines were cultured at 37°C with 5% CO2 in an appropriate culture medium.

Generation of chemoresistant cells

Lung cancer cells were exposed to stepwise increasing concentrations of standard chemotherapies, including doxorubicin (0.01–1 μmol/L) or cisplatin (0.01–0.1 μmol/L). In each cycle, cells were treated with cytotoxic agent for 72 hours, and then drug was washed and cells were allowed to recover. After reaching maximal concentrations, chemoresistant cells were exposed to maximal toxic concentrations at regular intervals to maintain their drug resistance. In experiments that use supernatants of cell culture, chemoresistant cells were washed 5 times with sterilized PBS, and cultured in media without doxorubicin. The supernatants were harvested 72 hours later and passed through 0.2-μm filter.

Human monocyte culture

Purified human monocytes (Miltenyi Biotec 130-050-201) were stimulated with supernatants of chemosensitive or chemoresistant cell culture (50% of culture medium) on day 0, 3, and 6. For experiments using neutralizing antibodies, supernatants were pretreated with 10 μg/mL of α-M-CSF (Peprotech 500-P44), α-IL34 (R&D Systems 578416) or a matching isotype control (Biolegend 910801 or R&D Systems MAB002). For experiments using selective inhibitors, supernatants were pretreated with GW2580 (25 nmol/L, Abcam 142096), LY294002 (10 μmol/L, CST 9901), or DMSO.

Humanized mouse model

NOD-SCID-IL2rg−/− mice (females, 6 weeks old) were irradiated as described previously (2.5 G; refs. 16, 17) and transferred after 24 hours with 1 × 105 human bone marrow cells from one healthy donor (StemCell Technologies, catalog 70001.4, lot 626184005). No information regarding the HLA type of donor were available. Three weeks later, mice were inoculated subcutaneously with 1 × 106 of tumor cells, and treated with doxorubicin (intraperitoneal injection, 10 mg/kg/mice, 4 doses/1 dose per week) when tumors size reached 5 mm. Mice were maintained in a temperature-controlled, pathogen-free room at the Institute for Genetic Medicine, Hokkaido University, and treated with humane care according to animal procedures approved by Animal Care Committee of Hokkaido University (approval no. 14-0171).

Immunohistochemical analysis

Primary lung cancer samples were obtained earlier with informed consent from Kanagawa Cancer Center. Formalin-fixed and paraffin-embedded tissues were stained with α-IL34 (Abcam 101443) according to the manufacturer's protocol. Three independent investigators assessed IL34 positivity without prior knowledge of clinicopathologic data. IL34 staining was mostly homogenous in cytoplasm, and thus was semiquantitatively evaluated as absent, weak, or high. All clinical materials mentioned were approved by individual institutional ethics committees.

Statistical analysis

The significance of differences between two independent subjects' groups were determined by Student t test or two-sample t test with Welch's correction. Differences among three or more subjects were determined by one-way ANOVA. A P value of <0.05 was considered to be statistically significant.

Supernatants of chemoresistant cells enhance monocyte differentiation into immunosuppressive M2-polarized macrophages

Monocytes are recruited into tumors by factors secreted in TME and serve as the cellular resource of TAMs (18). Increased frequencies of monocytes and its differentiation into protumorigenic M2-polarized TAMs have been considered as a hallmark of enhanced chemoresistance in tumors (7–9). Consistent with this, we found that frequencies of monocytes, but not other myeloid subsets, in total peripheral blood leukocytes (PBL) of healthy donors were increased when stimulated with supernatants of doxorubicin-resistant A549 (A549-DR) and cisplatin-resistant H1299 (H1299-CR) cells compared with chemosensitive counterparts (A549-DS and H1299-CS; Fig. 1A and Supplementary Figs. S1 and S2A). Next, to evaluate the effects of chemoresistant cell–derived factors on monocytes differentiation, we stimulated purified CD14+ monocytes with supernatants of chemosensitive or chemoresistant cell culture. In both cases, monocytes differentiated into spindle-adherent cells (Fig. 1B) that exhibit M2-polarized MΦ characteristics as evidenced by high expression of the pan-macrophage marker CD68 (Fig. 1C and Supplementary Fig. S2B) and CD163, which serves as a biomarker of M2-polarized macrophages activated toward tumor-promoting and immunosuppressive phenotype (Fig. 1D and Supplementary Fig. S2B; ref. 19). Importantly, CD68 and CD163 expression levels were remarkably higher in monocytes stimulated with supernatants of A549-DR (A549-DR-MΦ) compared with A549-DS (A549-DS-MΦ; Fig. 1C and D and Supplementary Fig. S2B). Furthermore, A549-DR-MΦ exhibit decreased levels of MHC (class I and II) and costimulatory molecules such as CD80 and CD86, and enhanced levels of the T-cell–inhibitory molecule B7-H1 (PD-L1) compared with A549-DS-M (Supplementary Fig. S2C and S2D), indicating an enhanced immunosuppressive phenotype of macrophages when stimulated with supernatants of chemoresistant cells. Consistent with this enhancement, A549-DR-MΦ showed increased expression levels of immunosuppressive factors such as IL10 and TGFβ (Fig. 1E) and potent capability to suppress T-cell response compared with A549-DS-MΦ (Fig. 1F). Together, these results suggest that soluble factors in the supernatants of chemoresistant A549 and H1299 cells mediate monocyte differentiation into M2-polarized macrophages with an enhanced immunosuppressive phenotype.

Figure 1

Monocytes differentiate into M2-polarized macrophages with enhanced immunosuppressive phenotype when stimulated with supernatants of chemoresistant cells. A frequencies of CD14+CD11b+ fraction in PBLs treated for 7 days with media or supernatants of chemosensitive (A549-DS) or chemoresistant (A549-DR) cells as evaluated by FACS (left) and graph (right). Forward scatter (FSC) and side scatter (SSC) parameters were used to detect cell size distribution and perform initial gating to remove debris. B representative photomicrographs of macrophages differentiated from monocytes cultured for 7 days in the presence of A549-DS or A549-DR supernatants. C and D FACS analysis for CD68 (C) or CD163 (D) expression in purified CD14+ monocytes cultured in media or in the presence of A549-DS or A549-DR supernatants for 7 days. Large cells were gated on the basis of forward scatter and side scatter. Mean fluorescence intensity (MFI) of monocytes was considered as 1. E qRT-PCR analysis of various cytokines and chemokines expression in A549-DS-MΦ or A549-DR-MΦ. Data from purified CD14+ monocytes were set as 1. F evaluation of the immunosuppressive function of macrophages. A549-DS-MΦ or A549-DR-MΦ were cocultured with autologous CD4 or CD8 T cells stimulated with anti-CD3/28 for 72 hours (ratio 1:5) and IFNγ production was determined by ELISA. Data, as mean ± SEM; *, P < 0.05.

Figure 1

Monocytes differentiate into M2-polarized macrophages with enhanced immunosuppressive phenotype when stimulated with supernatants of chemoresistant cells. A frequencies of CD14+CD11b+ fraction in PBLs treated for 7 days with media or supernatants of chemosensitive (A549-DS) or chemoresistant (A549-DR) cells as evaluated by FACS (left) and graph (right). Forward scatter (FSC) and side scatter (SSC) parameters were used to detect cell size distribution and perform initial gating to remove debris. B representative photomicrographs of macrophages differentiated from monocytes cultured for 7 days in the presence of A549-DS or A549-DR supernatants. C and D FACS analysis for CD68 (C) or CD163 (D) expression in purified CD14+ monocytes cultured in media or in the presence of A549-DS or A549-DR supernatants for 7 days. Large cells were gated on the basis of forward scatter and side scatter. Mean fluorescence intensity (MFI) of monocytes was considered as 1. E qRT-PCR analysis of various cytokines and chemokines expression in A549-DS-MΦ or A549-DR-MΦ. Data from purified CD14+ monocytes were set as 1. F evaluation of the immunosuppressive function of macrophages. A549-DS-MΦ or A549-DR-MΦ were cocultured with autologous CD4 or CD8 T cells stimulated with anti-CD3/28 for 72 hours (ratio 1:5) and IFNγ production was determined by ELISA. Data, as mean ± SEM; *, P < 0.05.

Close modal

Chemoresistant cell–derived IL34 enhances monocyte differentiation into M2-polarized macrophages

Macrophages acquire their phenotype in response to various signals present within individual microenvironments (20). In tumors, chemotherapy induces inflammatory responses that enrich TME with factors that repolarize myeloid cells into different phenotype (21). Consistent with this, we found that chemoresistant cells showed enhanced levels of several cytokines and chemokines compared with chemosensitive counterparts (Supplementary Fig. S2E). To clarify the importance of these factors on TAMs phenotype, we first started with the neutralization of M-CSF, because CSF1R-mediated signaling is critical for the recruitment and differentiation of TAMs (13, 14). Unexpectedly, M-CSF blockade did not efficiently decrease expression levels of CD68 and CD163 induced by A549-DR and H1299-CR supernatants (Fig. 2A and B and Supplementary Fig. S2G). On the other hand, a specific inhibitor for CSF1R (GW2580) was effective to abolish these effects (data not shown), suggesting that chemoresistant cells produce a factor that can bind to CSF1R and affect monocyte differentiation rather than M-CSF. The existence of a second ligand for CSF1R has been discovered recently by functional screening of the extracellular proteome and designated as IL34 (22). IL34 supports survival of monocytes and macrophages, and induces monocytes differentiation into M2-polarized macrophages with potent immunosuppressive properties (23). Interestingly, ELISA measurement showed that IL34 was produced by chemoresistant A549 and H1299 cells but not their chemosensitive counterparts (Fig. 2C and Supplementary Fig. S2F). Furthermore, IL34 blockade was effective to reduce expression levels of CD68 and CD163 induced in monocytes by supernatants of chemoresistant A549 and H1299 cells (Fig. 2D and E and Supplementary Fig. S2G), indicating that effects of these supernatants depend largely on IL34. In addition, pretreatment of A549-DR supernatants with α-IL34 significantly decreased IL10 and TGFβ expression levels in A549-DR-MΦ (Fig. 2F), and impaired the immunosuppressive capabilities of A549-DR-MΦ to suppress T-cell response (Fig. 2G) compared with α-M-CSF. Together, these results suggest that IL34 produced by chemoresistant cells is involved in the enhancement of the immunosuppressive phenotype of TAMs. Thus, we focused in next experiments on IL34 as a modulator of TME under chemotherapeutic conditions.

Figure 2

Chemoresistant cell–derived IL34 mediates monocytes differentiation into M2-polarized macrophages with enhanced immunosuppressive properties. A and B purified CD14+ monocytes were cultured in media or A549-DR supernatants pretreated with α-M-CSF or control Ig (rabbit polyclonal) for 7 days. The expression of CD68 (A) or CD163 (B) was evaluated on day 7 by FACS (left) and graph (right). Mean fluorescence intensity (MFI) of monocytes was considered as 1. Large cells were gated on the basis of forward scatter and side scatter. C ELISA measurement of IL34 in the supernatants of chemosensitive or chemoresistant cells as measured in undiluted 72 hours culture. D and E purified CD14+ monocytes were cultured in media or A549-DR supernatants pretreated with α-M-CSF, α-IL34 (mouse monoclonal IgG1), or control Ig (rabbit polyclonal and mouse IgG1) for 7 days. CD68 (D) or CD163 (E) expression was evaluated on day 7 by FACS (left) and graph (right). Mean fluorescence intensity of monocytes was considered as 1. Large cells were gated on the basis of forward scatter and side scatter. F qRT-PCR analysis of IL10 and TGFβ expression in A549-DR-MΦ. Data from purified CD14+ monocytes were set as 1. G ELISA measurement of IFNγ production by CD3/CD28-stimulated CD4 or CD8 T cells cocultured for 72 hours with A549-DR-MΦ (5:1) as indicated. Data, as mean ± SEM; *, P < 0.05; N.D., not detected.

Figure 2

Chemoresistant cell–derived IL34 mediates monocytes differentiation into M2-polarized macrophages with enhanced immunosuppressive properties. A and B purified CD14+ monocytes were cultured in media or A549-DR supernatants pretreated with α-M-CSF or control Ig (rabbit polyclonal) for 7 days. The expression of CD68 (A) or CD163 (B) was evaluated on day 7 by FACS (left) and graph (right). Mean fluorescence intensity (MFI) of monocytes was considered as 1. Large cells were gated on the basis of forward scatter and side scatter. C ELISA measurement of IL34 in the supernatants of chemosensitive or chemoresistant cells as measured in undiluted 72 hours culture. D and E purified CD14+ monocytes were cultured in media or A549-DR supernatants pretreated with α-M-CSF, α-IL34 (mouse monoclonal IgG1), or control Ig (rabbit polyclonal and mouse IgG1) for 7 days. CD68 (D) or CD163 (E) expression was evaluated on day 7 by FACS (left) and graph (right). Mean fluorescence intensity of monocytes was considered as 1. Large cells were gated on the basis of forward scatter and side scatter. F qRT-PCR analysis of IL10 and TGFβ expression in A549-DR-MΦ. Data from purified CD14+ monocytes were set as 1. G ELISA measurement of IFNγ production by CD3/CD28-stimulated CD4 or CD8 T cells cocultured for 72 hours with A549-DR-MΦ (5:1) as indicated. Data, as mean ± SEM; *, P < 0.05; N.D., not detected.

Close modal

IL34 production is controlled by NF-κB and mediates an autocrine pathway in chemoresistant cells

First, we asked about the mechanism that controls IL34 production in chemoresistant cells. Previous studies have suggested the involvement of transcription factor NF-κB in IL34 production (24, 25). Thus, we hypothesized that NF-κB activation by chemotherapy may contribute to IL34 production in chemoresistant cells (26). As expected, NF-κB activation was significantly stronger in A549-DR than A549-DS cells (Fig. 3A and B). Furthermore, A549-DR cells but not their chemosensitive counterparts showed active NF-κB under steady state (Fig. 3A and B). Following stimulation with chemotherapy, NF-κB activities in A549-DS cells increased in a time-dependent manner but remained almost constant in A549-DR cells at the time course of treatment (Fig. 3A and B). Next, we used a specific inhibitor of NF-κB and examined its effect on IL34 production. We found that treatment of A549-DR cells with NF-κB inhibitor resulted in decreased IL34 mRNA levels in a dose-dependent manner (Fig. 3C), suggesting that constitutive activation of NF-κB is required for IL34 production in chemoresistant cells. In addition, survival of doxorubicin-treated A549-DR cells was also decreased in correlation with IL34 expression (data not shown). Thus, we asked whether IL34 has an autocrine effect, which in turn requires the expression of CSF1R in cancer cells. Previous studies have shown that CSF1R expression can be detected in lung and breast cancer cell lines in addition to tumor cells from patients (27, 28). Consistent with this, we found that CSF1R is weakly expressed at mRNA but not protein level in A549-DS cells (Fig. 3D and E). On the other hand, CSF1R expression is increased at mRNA level and can be detected at protein level in A549-DR cells (Fig. 3D and E). Furthermore, IL34 blockade resulted in decreased survival of doxorubicin-treated A549-DR cells (Fig. 3F), suggesting that IL34 mediates an autocrine signaling pathway that enhances survival of chemoresistant cells.

Figure 3

NF-κB controls IL34 production in chemoresistant cells. A immunoblots for phospho-IKKβ, total-IKKβ, phospho-p65, total-p65, and actin on lysates from A549-DS or A549-DR cells after exposure to doxorubicin (1 μmol/L) at the indicated times. B quantification densitometry of immunoblots described in A. C PCR analysis of IL34 mRNA expression (normalized to actin) in A549-DR cells 12 hours after treatment with BAY11-7082 at the indicated doses. D and E CD115 and actin expression was confirmed by qRT-PCR (D) or Western blot (E) in chemosensitive or chemoresistant A549 cells compared with purified CD14+ monocytes as a positive control. F cell survival assay of A549-DR cells pretreated with α-IL34 or control Ig (mouse IgG1) 1 hour before stimulation with doxorubicin (1 μmol/L). Data, mean ± SEM; *, P < 0.05.

Figure 3

NF-κB controls IL34 production in chemoresistant cells. A immunoblots for phospho-IKKβ, total-IKKβ, phospho-p65, total-p65, and actin on lysates from A549-DS or A549-DR cells after exposure to doxorubicin (1 μmol/L) at the indicated times. B quantification densitometry of immunoblots described in A. C PCR analysis of IL34 mRNA expression (normalized to actin) in A549-DR cells 12 hours after treatment with BAY11-7082 at the indicated doses. D and E CD115 and actin expression was confirmed by qRT-PCR (D) or Western blot (E) in chemosensitive or chemoresistant A549 cells compared with purified CD14+ monocytes as a positive control. F cell survival assay of A549-DR cells pretreated with α-IL34 or control Ig (mouse IgG1) 1 hour before stimulation with doxorubicin (1 μmol/L). Data, mean ± SEM; *, P < 0.05.

Close modal

IL34 enhances survival of chemoresistant cells via CSF1R-mediated AKT signaling

Next, we investigated the molecular mechanism by which IL34 enhances survival of chemoresistant cells. Previous report suggested the ability of IL34 to bind CSF1R with high affinity and induces strong phosphorylation of CSF1R and downstream mediators (29). Transphosphorylation of tyrosine residues in the cytoplasmic domain of CSF1R could be observed in A549-DRMock cells (Fig. 4A). On the other hand, CSF1R phosphorylation was significantly reduced in A549-DR cells deficient for IL34 (Fig. 4A), which suggests a functional relevance for IL34 in this autocrine loop. Next, we compared A549-DRMock cell survival when pretreated with various kinases inhibitors that act downstream of CSF1R (30). Among these inhibitors, LY294002 (an inhibitor for PI3K/AKT) was capable to significantly reduce survival of doxorubicin-treated A549-DRMock cells, indicating that survival of these cells is AKT-dependent (Supplementary Fig. S3). AKT acts as a key signaling molecule that links between oncogenic receptors and many essential prosurvival cellular functions (31). Consistent with this, A549-DRMock cells showed increased levels of phospho-AKT compared with A549-DS cells (Fig. 4B). On the other hand, AKT phosphorylation was significantly reduced in A549-DRΔIL34 and comparable with A549-DS cells (Fig. 4B). Furthermore, AKT phosphorylation was decreased in A549-DRMock cells when treated with α-IL34 in a dose-dependent manner (Fig. 4C), which consequently sensitized A549-DRMock cells to doxorubicin treatment (Fig. 4E). On the other hand, stimulating A549-DRΔIL34 cells with recombinant IL34 (rIL34) enhanced AKT phosphorylation in a dose-dependent manner (Fig. 4D), and increased viability of doxorubicin-treated A549-DRΔIL34 cells (Fig. 4F). Finally, we compared the ability of M-CSF and IL34 to induce AKT activation in chemoresistant cells. Levels of phospho-AKT increased gradually in M-CSF–stimulated A549-DRΔIL34 cells in a time-dependent manner to reach a peak 60 minutes after stimulation and then start to drop (Fig. 4G and H). On the other hand, IL34 potently induced AKT phosphorylation, reaching its peak 30 to 60 minutes after stimulation, and peak duration was significantly elongated compared with M-CSF (Fig. 4G and H). Together, these results suggest that IL34 enhances survival of chemoresistant cells by modulating AKT signal downstream of CSF1R.

Figure 4

IL34 enhances survival of chemoresistant cells via AKT-mediated mechanism. A Western blot analysis of whole-cell lysates from A549-DS, A549-DRMock, or A549-DRΔIL34 cells stained with α-CSF1R (CD115), α-phospho-CSF1R (P-Tyr708 or P-Tyr723), and actin. Quantification densitometry of detected bands is demonstrated in the top panel. B phosphorylation levels of AKT compared between A549-DS, A549-DRMock, or A549-DRΔIL34 cells by immunoblots and densitometric quantification. C phosphorylation levels of AKT in A549-DRMock pretreated with indicated doses of α-IL34 as estimated by immunoblots and densitometric quantification. D phosphorylation levels of AKT in A549-DRΔIL34 stimulated with indicated doses of rIL34 as estimated by immunoblots and densitometric quantification. E cell survival assay of A549-DRMock cells pretreated with indicated doses of α-IL34 for 1 hour before stimulation with doxorubicin (1 μmol/L). F cell survival assay of A549-DRΔIL34 cells stimulated with indicated doses of rIL34 for 1 hour before stimulation with doxorubicin (1 μmol/L). G time-course of AKT phosphorylation in A549-DRΔIL34 stimulated with rM-CSF or rIL34 (1 ng/mL) as estimated by Western blot analysis. H dynamism of AKT phosphorylation induced by rM-CSF or rIL34 in A549-DRΔIL34 cells as estimated by densitometric quantification of immunoblots shown in G. Data, mean ± SEM; *, P < 0.05; N.D., not detected.

Figure 4

IL34 enhances survival of chemoresistant cells via AKT-mediated mechanism. A Western blot analysis of whole-cell lysates from A549-DS, A549-DRMock, or A549-DRΔIL34 cells stained with α-CSF1R (CD115), α-phospho-CSF1R (P-Tyr708 or P-Tyr723), and actin. Quantification densitometry of detected bands is demonstrated in the top panel. B phosphorylation levels of AKT compared between A549-DS, A549-DRMock, or A549-DRΔIL34 cells by immunoblots and densitometric quantification. C phosphorylation levels of AKT in A549-DRMock pretreated with indicated doses of α-IL34 as estimated by immunoblots and densitometric quantification. D phosphorylation levels of AKT in A549-DRΔIL34 stimulated with indicated doses of rIL34 as estimated by immunoblots and densitometric quantification. E cell survival assay of A549-DRMock cells pretreated with indicated doses of α-IL34 for 1 hour before stimulation with doxorubicin (1 μmol/L). F cell survival assay of A549-DRΔIL34 cells stimulated with indicated doses of rIL34 for 1 hour before stimulation with doxorubicin (1 μmol/L). G time-course of AKT phosphorylation in A549-DRΔIL34 stimulated with rM-CSF or rIL34 (1 ng/mL) as estimated by Western blot analysis. H dynamism of AKT phosphorylation induced by rM-CSF or rIL34 in A549-DRΔIL34 cells as estimated by densitometric quantification of immunoblots shown in G. Data, mean ± SEM; *, P < 0.05; N.D., not detected.

Close modal

IL34 enhances the immunosuppressive function of TAMs through C/EBPβ-mediated mechanism

Depending on various signals present within individual microenvironments, marked changes in the activity and gene expression programs in macrophages can occur and determine macrophage phenotype and function (32). TAMs are often considered to be synonymous with M2-phenotype characterized with unique transcriptional profile (32, 33). Consistent with this, stimulation of monocytes with supernatants of lung cancer cell culture induced the expression of transcriptional factors (TF) responsible for M2-polarization such as interferon regulating factor 4 (IRF4), STAT6, and CCAAT/enhancer-binding protein β (C/EBPβ; Supplementary Fig. S4A). As suggested above, A549-DR-MΦ showed enhanced immunosuppressive phenotype in an IL34-dependent manner. However, because A549-DS-MΦ and A549-DR-MΦ showed comparable expression levels of M2 phenotype-related TFs, we hypothesized that IL34-mediated signaling may affect TAM function by modulating the activation of certain TFs downstream of CSF1R. To confirm this hypothesis, we first compared the phosphorylation levels of CSF1R in monocytes stimulated with supernatants of A549-DS, A549-DRMock or A549-DRΔIL34 cells. We found that CSF1R phosphorylation was stronger in macrophages stimulated with supernatants of IL34-producing A549-DRMock compared with nonproducing A549-DS or A549-DRΔIL34 cells (Fig. 5A). The enhancement of CSF1R phosphorylation in A549-DRMock-MΦ has a consequent effect on increased activation of AKT signaling (Fig. 5B), which interestingly correlated with a remarkable increase in phosphorylation levels of C/EBPβ compared with other TFs (Fig. 5C and Supplementary Fig. S4B). C/EBPβ, a hallmark of M2-polarized macrophages, belongs to bZIP family of TFs that play essential roles in myeloid development and macrophage activation (32). In particular, C/EBPβ has been shown to be essential for the expression of immunosuppressive cytokines such as IL10 and Arginase-1 (ARG1) in M2-polarized macrophages (34). Consistent with this, we found that C/EBPβ activation is accompanied with increased expression levels of IL10 and ARG1 in A549-DRMock-MΦ (Fig. 5D). C/EBPβ specifically regulates M2-associated genes under the control of another bZIP family transcription factor, known as cAMP-responsive element-binding protein (CREB; ref. 35). Furthermore, CREB activities are enhanced upon the activation of PI3K/AKT signaling pathway (36). From these backgrounds, we focused on C/EBPβ as a possible candidate that acts downstream of CSF1R and contributes to IL34 effects on TAMs function. First, we examined the phosphorylation status of CSF1R, AKT, and C/EBPβ in monocytes stimulated with A549-DRMock supernatants when pretreated with α-IL34 and/or a selective pharmacologic inhibitor of PI3K/AKT (LY294002). As expected, α-IL34 has a similar effect to LY294002 on reducing AKT phosphorylation, which in turn has impact on C/EBPβ phosphorylation in A549-DRMock-MΦ (Fig. 5E). Importantly, α-IL34 or LY294002 has similar effects on reducing IL10 and ARG1 expression levels in A549-DRMock-MΦ in correlation with decreased C/EBPβ phosphorylation (Fig. 5F). On the other hand, adding rIL34 to A549-DRΔIL34 supernatants resulted in a remarkable enhancement of AKT and C/EBPβ phosphorylation (Fig. 5G), and a consequent increase in IL10 and ARG1 expression levels (Fig. 5H). Importantly, these effects were abolished by LY294002 (Fig. 5G and H), demonstrating the importance of IL34-induced activation of AKT signaling on the regulation of C/EBPβ function. Collectively, these results suggest a possible mechanism by which IL34 enhances the immunosuppressive phenotype of TAMs by activating M2-regulating factors such as C/EBPβ.

Figure 5

IL34 modifies TAMs function through C/EBPβ-mediated mechanism. A phosphorylation levels of CSF1R (P-Tyr708 or P-Tyr723) in A549-DS-MΦ (blue bars), A549-DRMock-MΦ (red bars), or A549-DRΔIL34-MΦ (green bars) at day 7 of culture as estimated by immunoblots (left) and densitometric quantification (right). B phosphorylation or total levels of AKT in macrophages described in A as estimated by immunoblots (left) and densitometric quantification (right). C phosphorylation levels of C/EBPβ in macrophages described in A as estimated by immunoblots (left) and densitometric quantification (right). D qRT-PCR analysis of IL10 and ARG1 expression in macrophages described in A at day 7 of culture. Data from purified CD14+ monocytes were set as 1. E phosphorylation levels of AKT or C/EBPβ in A549-DRMock-MΦ as estimated by immunoblots (left) and densitometric quantification (right). Monocytes were cultured in the presence of A549-DRMock supernatants pretreated with α-IL34 and/or LY294002 for 7 days. F qRT-PCR analysis of IL10 and ARG1 expression in macrophages described in E. Data from purified CD14+ monocytes were set as 1. G phosphorylation levels of AKT or C/EBPβ in A549-DRΔIL34-MΦ as estimated by immunoblots (left) and densitometric quantification (right). Monocytes were cultured in the presence of A549-DRΔIL34 supernatants after the addition of rIL34 and/or LY294002 for 7 days. H qRT-PCR analysis of IL10 and ARG1 expression in macrophages described in G. Data from purified CD14+ monocytes were set as 1. Data, mean ± SEM; *, P < 0.05.

Figure 5

IL34 modifies TAMs function through C/EBPβ-mediated mechanism. A phosphorylation levels of CSF1R (P-Tyr708 or P-Tyr723) in A549-DS-MΦ (blue bars), A549-DRMock-MΦ (red bars), or A549-DRΔIL34-MΦ (green bars) at day 7 of culture as estimated by immunoblots (left) and densitometric quantification (right). B phosphorylation or total levels of AKT in macrophages described in A as estimated by immunoblots (left) and densitometric quantification (right). C phosphorylation levels of C/EBPβ in macrophages described in A as estimated by immunoblots (left) and densitometric quantification (right). D qRT-PCR analysis of IL10 and ARG1 expression in macrophages described in A at day 7 of culture. Data from purified CD14+ monocytes were set as 1. E phosphorylation levels of AKT or C/EBPβ in A549-DRMock-MΦ as estimated by immunoblots (left) and densitometric quantification (right). Monocytes were cultured in the presence of A549-DRMock supernatants pretreated with α-IL34 and/or LY294002 for 7 days. F qRT-PCR analysis of IL10 and ARG1 expression in macrophages described in E. Data from purified CD14+ monocytes were set as 1. G phosphorylation levels of AKT or C/EBPβ in A549-DRΔIL34-MΦ as estimated by immunoblots (left) and densitometric quantification (right). Monocytes were cultured in the presence of A549-DRΔIL34 supernatants after the addition of rIL34 and/or LY294002 for 7 days. H qRT-PCR analysis of IL10 and ARG1 expression in macrophages described in G. Data from purified CD14+ monocytes were set as 1. Data, mean ± SEM; *, P < 0.05.

Close modal

IL34 enhances immunosuppression by TAMs and attenuates antitumor effects of chemotherapy

Next, we evaluated the significance of IL34 on the attenuation of chemotherapeutic effects of doxorubicin in a humanized NOD-SCID-IL2rg−/− mouse model (16, 17). In vivo treatment with doxorubicin had little impact on A549-DRMock tumor growth compared with A549-DS (Fig. 6A and B). Notably, tumor growth of A549-DRΔIL34 tumors was significantly suppressed compared with IL34-producing A549-DRMock tumors (Fig. 6A and B). Increased frequencies of M2-polarized protumorigenic TAMs have been considered as a hallmark of enhanced chemoresistance in tumors (7–9). Consistent with this, A549-DRMock tumors were highly infiltrated with CD68+CD163+ M2-polarized TAMs (Fig. 6C and D), which correlate with suppression of cytotoxic CD8+ T-cell response (Fig. 6E and F and Supplementary Fig. S5A and S5B), in addition to increased Treg to Th1 cells ratio compared with IL34-nonproducing tumors (Supplementary Fig. S5C–S5E), which indicates the importance of IL34-modified TAMs in regulating immune tolerance within TME. In addition, we have also compared tumor growth in normal (nonhumanized) NOD-SCID-IL2rg−/− mice. As shown in Supplementary Fig. S5F, doxorubicin had suppressed growth of A549-DS and A549-DRΔIL34 tumors, which can be explained by the direct effect of doxorubicin on A549 cells. On the other hand, doxorubicin had little impact on A549-DRMock tumor growth due to the resistant nature of these tumors (as enhanced by autocrine signaling of IL34). Adding lymphocytes in humanized NOD-SCID-IL2rg−/− mice resulted in more suppression of tumor growth in A549-DS and A549-DRΔIL34 tumors but not in A549-DRMock tumors due to the enhanced immunosuppression in these tumors, which suggests that suppression of tumor growth in this model is a collective action of the direct effect of doxorubicin on A549 cells in addition to antitumor immune response. It is worth to be mentioned here that the HLA-type matching between tumor and immune cells was not confirmed in this model due to the lack of information regarding the HLA type of donor. Thus, it is unknown whether interactions and phenotypes are antigen/MHC-dependent. A recent report has suggested a potent role of IL34 in suppressing allogeneic immune responses against transplants (37). Thus, IL34 produced by chemoresistant tumors in this model may similarly suppress antitumor T-cell responses within TME.

Figure 6

High infiltration of immunosuppressive TAMs in IL34-producing chemoresistant tumors. A tumor growth in humanized NOD-SCID-IL2rg−/− mice challenged with A549-DS, A549-DRMock or A549-DRΔIL34 cells and treated with doxorubicin (DOX) or PBS (n = 5/group). Error bars were removed for better view and are shown in B. B comparison of tumor size at day 25 between different groups described in A. C and D frequencies of CD68+CD163 or CD68+CD163+ TAMs populations as evaluated by FACS (C) or graph (D). E and F frequencies of CD8+CD3+ cells in tumor-infiltrating lymphocytes as evaluated by FACS (E) or graph (F). Forward scatter and side scatter parameters were used to detect cell size distribution and to perform initial gating to remove debris, and isotype control antibody staining was used to set gates. G qRT-PCR analysis of various factors expressed in CD68+CD163+ TAMs isolated from A549-DS, A549-DRMock or A549-DRΔIL34 tumors. Data, mean ± SEM; *, P < 0.05. n.s., nonsignificant.

Figure 6

High infiltration of immunosuppressive TAMs in IL34-producing chemoresistant tumors. A tumor growth in humanized NOD-SCID-IL2rg−/− mice challenged with A549-DS, A549-DRMock or A549-DRΔIL34 cells and treated with doxorubicin (DOX) or PBS (n = 5/group). Error bars were removed for better view and are shown in B. B comparison of tumor size at day 25 between different groups described in A. C and D frequencies of CD68+CD163 or CD68+CD163+ TAMs populations as evaluated by FACS (C) or graph (D). E and F frequencies of CD8+CD3+ cells in tumor-infiltrating lymphocytes as evaluated by FACS (E) or graph (F). Forward scatter and side scatter parameters were used to detect cell size distribution and to perform initial gating to remove debris, and isotype control antibody staining was used to set gates. G qRT-PCR analysis of various factors expressed in CD68+CD163+ TAMs isolated from A549-DS, A549-DRMock or A549-DRΔIL34 tumors. Data, mean ± SEM; *, P < 0.05. n.s., nonsignificant.

Close modal

In addition to immunosuppression, TAMs release various factors that contribute to tumor progression and chemoresistance (7–9). Consistent with this, we found that CD68+CD163+ TAMs isolated from A549-DRMock tumors showed enhanced expression levels of immunosuppressive cytokines such as IL10 and TGFβ, factors that provide critical survival signals to cancer cells such as IL6, TNFα, and EGF, in addition to factors that enhance angiogenesis such as VEGF and modulate matrix components of TME to facilitate tumor progression and invasion such as MMP9 and MMP13 (Fig. 6G). Importantly, this enhancement was abolished in CD68+CD163+ TAMs isolated from A549-DRΔIL34 tumors (Fig. 6G). Collectively, these results suggest that IL34 has significant impact on enhancing the protumorigenic properties of TAMs as characterized by increased ability to potently suppress antitumor immune response and provide cancer cells with various factors necessary to survive under chemotherapeutic conditions.

IL34 expression in primary human lung cancers correlates with poor prognosis

Finally, we asked whether IL34 expression could be detected in primary human lung cancers. Recent protocols suggest the application of chemotherapy as an adjuvant treatment after surgical resection of tumors. Because the majority of lung cancer patients in our institute are treated with chemotherapy after the surgical removal of tumors, it was extremely difficult to obtain samples from lung cancer patients before and after chemotherapy. Thus, we chose to evaluate IL34 expression at mRNA and protein levels in primary lung cancer tissues removed by surgical procedures. This cohort comprises data from lung cancer patients without any preselection. 45% of samples were obtained from Japanese women over 60-year-old without smoking history, and 77.4% of cases were diagnosed with stage I of non–small lung cancers with 5 years of follow-up period (Supplementary Table S1 and S2). qRT-PCR analysis showed detectable levels of IL34 mRNA in primary lung cancer tissues compared with normal lung tissues (Fig. 7A). More importantly, immunohistochemical staining revealed that IL34 could be also detected at protein levels in cancer tissues but not in adjacent normal tissues (Fig. 7B), and high expression of IL34 significantly correlates with poor prognosis (Fig. 7C and Supplementary Fig. S6A), suggesting that IL34 expression may serve as an important prognostic biomarker in lung cancer patients. In addition, IL34 and CSF1R expression, which can be detected at low levels in primary lung adenocarcinoma cells, were induced after exposure to doxorubicin (Fig. 7D) or cisplatin (Fig. 7E). Thus, IL34 may also serve as a biomarker to monitor the development and progression of chemoresistance in cancer patients treated with chemotherapy.

Figure 7

IL34 is expressed in primary lung cancer tissues and correlates to poor prognosis. A qRT-PCR analysis of IL34 expression in primary lung cancer tissues from patients diagnosed with adenocarcinoma (ADC), squamous cell carcinoma (SCC), or small-cell lung carcinoma (SCLC), as normalized to actin. B immunohistochemical staining of IL34 in cancer or normal tissues from lung cancer patients. IL34 staining in cerebral cortex or skin is showed in the bottom as positive controls. C Kaplan–Meier analysis of overall survival in 332 lung cancer patients stratified as high (green line, n = 83) or weak/absent (blue line, n = 249). D and E qRT-PCR analysis of IL34 and CSF1R expression in primary lung adenocarcinoma cells after exposure to doxorubicin (D) or cisplatin (E) at the indicated doses. Data, mean ± SEM; *, P < 0.05.

Figure 7

IL34 is expressed in primary lung cancer tissues and correlates to poor prognosis. A qRT-PCR analysis of IL34 expression in primary lung cancer tissues from patients diagnosed with adenocarcinoma (ADC), squamous cell carcinoma (SCC), or small-cell lung carcinoma (SCLC), as normalized to actin. B immunohistochemical staining of IL34 in cancer or normal tissues from lung cancer patients. IL34 staining in cerebral cortex or skin is showed in the bottom as positive controls. C Kaplan–Meier analysis of overall survival in 332 lung cancer patients stratified as high (green line, n = 83) or weak/absent (blue line, n = 249). D and E qRT-PCR analysis of IL34 and CSF1R expression in primary lung adenocarcinoma cells after exposure to doxorubicin (D) or cisplatin (E) at the indicated doses. Data, mean ± SEM; *, P < 0.05.

Close modal

Finally, we examined IL34 and CSF1R expression in various cancer cell lines including breast, liver, colon, and ovary cancers. Quantitative PCR analysis showed detectable levels of IL34 and CSF1R mRNA in different cancer cells with some variations (Supplementary Fig. S6B and S6C). Importantly, IL34 and CSF1R expression levels were higher in a cisplatin-resistant ovarian cancer cell line compared with its chemosensitive counterpart (Supplementary Fig. S6D). Together, these observations suggest that IL34 and CSF1R may characterize a population of chemoresistant cancer cells in various types of cancer.

Recent progress in understanding the relation between macrophage function and therapeutic resistance has helped to improve new therapeutic strategies based on the characteristics of TME (7–9). In this report, we provide evidence that chemotherapy-treated TME adopt a novel strategy to develop chemoresistance and suppress antitumor immunosurveillance by triggering IL34 production in tumor cells (Supplementary Fig. S6E).

IL34 is a newly discovered cytokine, which shares a common receptor (CSF1R) with M-CSF (22). Although both cytokines mediate monocytes/macrophages survival and proliferation, IL34 binds to CSF-1R with higher affinity and induces stronger tyrosine phosphorylation of CSF-1R and downstream mediators (29). IL34 plays important roles in the pathogenicity of diseases associated with chronic inflammation such as viral infections and inflammatory bowel disease (38, 39). In cancer, IL34 was found to promote tumor progression and metastatic process of osteosarcoma via promotion of angiogenesis and macrophage recruitment (40). Emerging evidence indicates that chemotherapy treatment increases the proportion of M2-polarized protumorigenic TAMs, which in turn limit the efficacy of chemotherapy (7–9). In addition, chemotherapy induces NF-κB activation, which plays critical roles in the development and progression of cancer chemoresistance via intrinsic mechanisms such as induction of antiapoptotic genes expression, and extrinsic mechanisms such as the expression of several cytokines and chemokines that modulate the interaction between tumor cells and other cellular components of TME (41, 42). Importantly, NF-κB activation is linked to IL34 production following stimulation with proinflammatory cytokines such as TNFα and IL1β (43). As shown in this study, chronic exposure of lung cancer cells to cytotoxic agents results in enhanced NF-κB activation, which in turn induced IL34 expression in chemoresistant cells. Thus, we expected that IL34 play critical roles in the modulation of TME under chemotherapeutic conditions.

First, we identified a paracrine effect of IL34 represented by recruiting high frequencies of M2-polarized TAMs and enhancing its immunosuppressive phenotype (Supplementary Fig. S6E). Chemoresistance is significantly enhanced when chemotherapy increases the proportion of M2-polarized protumorigenic TAMs, which in turn limit the efficacy of chemotherapy (12). Indeed, IL34 secreted by chemoresistant cells enhanced monocytes differentiation into M2-polarized macrophages in vitro. In addition, in a humanized mouse model, IL34-producing chemoresistant tumors were infiltrated with increased frequencies of M2-polarized TAMs, which was negatively correlated with the frequencies of tumor-infiltrating cytotoxic CD8+ T cells, indicating the importance of IL34 in recruiting M2-polarized TAMs with potent abilities to suppress antitumor immune response under chemotherapeutic condition. Furthermore, TAMs showed enhanced expression levels of immunosuppressive and chemoprotective factors in IL34-producing chemoresistant tumors. Previous reports have suggested that cancer cells produce various factors that affect differentiation and immunosuppressive features of tumor-infiltrating myeloid cells (44). In our experiments, we found that macrophages showed comparable expression levels of factors responsible for transcription control in macrophages such as IRF4, STAT6, and C/EBPβ when stimulated with supernatants of chemosensitive or chemoresistant cells. Even then, macrophages showed enhanced phosphorylation level of C/EBPβ when stimulated with supernatants of chemoresistant cells, which was mediated by IL34-induced activation of the CSF1R/AKT signaling pathway. C/EBPβ plays critical roles in the regulation of the immunosuppressive phenotype of myeloid cells, including M2 macrophages and myeloid-derived suppressor cells (MDSC; ref. 32). The activation of C/EBPβ cascade induces M2 macrophage–specific gene expression and promotes muscle injury repair (35). Similarly, IL34-induced C/EBPβ activation may help to enhance the immunosuppressive and protumorigenic functions of TAMs to protect tumors from immune attack and maintain homeostasis of TME under chemotherapeutic conditions.

The second role of IL34 in chemoresistance is the unexpected autocrine effect on activating AKT signal pathway downstream of CSF1R in chemoresistant cells (Supplementary Fig. S6E). AKT plays critical roles in chemoresistance to cytotoxic agents such as paclitaxel and cisplatin in human cancers (45–47). AKT also acts downstream of CSF1R to transduce signals from M-CSF and IL34 (48–50). In addition to myeloid cells, recent reports have suggested that CSF1R mRNA can be detected in some cancers like lung and breast cancer cells (27, 28). In our experiments, we found that CSF1R expression is upregulated and can be detected at protein levels in chemoresistant cells. Upon binding to IL34, CSF1R enhances AKT activation, providing a critical signal that help cancer cells to survive under chemotherapeutic conditions.

In summary, we identify a novel role for IL34 produced by cancer cells following chemotherapy treatment in the formation of chemoprotective niche in a paracrine manner through the recruitment of M2-polarized TAMs with enhanced protumorigenic phenotype in addition to autocrine effects by enhancing AKT-mediated survival signal downstream of CSF1R, and thus help to maintain chemoresistance in cancer cells, suggesting IL34 as a promising target to overcome chemoresistance in future therapeutic strategies.

No potential conflicts of interest were disclosed.

Conception and design: M. Baghdadi, K.-i. Seino

Development of methodology: M. Baghdadi, Y. Daigo

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Baghdadi, H. Wada, S. Nakanishi, H. Abe, N. Han, W.E. Putra, D. Endo, H. Watari, N. Sakuragi, Y. Hida, K. Kaga, Y. Miyagi, T. Yokose, A. Takano, Y. Daigo

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Baghdadi, H. Wada, S. Nakanishi, H. Abe, N. Han, W.E. Putra, Y. Miyagi, Y. Daigo, K.-i. Seino

Writing, review, and/or revision of the manuscript: M. Baghdadi, K. Kaga, Y. Daigo, K.-i. Seino

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Baghdadi, S. Nakanishi, Y. Miyagi

Study supervision: M. Baghdadi, K.-i. Seino

The authors thank Ms. Rei Okabe, Ms. Chie Kusama, Ms. Ayano Yamauchi, and Ms. Sena Kudo for academic and technical assistance.

This study was supported in part by JSPS/KAKENHI (M. Baghdadi, H. Wada, and K.-i. Seino), Kanae Foundation for the Promotion of Medical Science (M. Baghdadi), grant in aid of Uehara Memorial Foundation (M. Baghdadi), Takeda Science Foundation (M. Baghdadi), and The Cell Science Research Foundation (M. Baghdadi).

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.
Urruticoechea
A
,
Alemany
R
,
Balart
J
,
Villanueva
A
,
Viñals
F
,
Capellá
G
. 
Recent advances in cancer therapy: an overview
.
Curr Pharm Des
2010
;
16
:
3
10
.
2.
Wilson
TR
,
Longley
DB
,
Johnston
PG
. 
Chemoresistance in solid tumours
.
Ann Oncol
2006
;
17
Suppl 1
:
x315
24
.
3.
Holohan
C
,
Van Schaeybroeck
S
,
Longley
DB
,
Johnston
PG
. 
Cancer drug resistance: an evolving paradigm
.
Nat Rev Cancer
2013
;
13
:
714
26
.
4.
Pardoll
DM
. 
The blockade of immune checkpoints in cancer immunotherapy
.
Nat Rev Cancer
2012
;
12
:
252
64
.
5.
Kanda
S
,
Goto
H
,
Shiraishi
H
,
Kubo
E
,
Tanaka
A
,
Utsumi
H
, et al
Phase I study of anti-PD-1 ONO-4538 and chemotherapy in patients with advanced non–small cell lung cancer
.
Ann Oncol
2014
;
25
Suppl 4
:
iv466
.
6.
Topalian
SL
,
Hodi
FS
,
Brahmer
JR
,
Gettinger
SN
,
Smith
DC
,
McDermott
DF
, et al
Safety, activity, and immune correlates of anti-PD-1 antibody in cancer
.
N Engl J Med
2012
;
366
:
2443
54
.
7.
Noy
R
,
Pollard
JW
. 
Tumor-associated macrophages: from mechanisms to therapy
.
Immunity
2014
;
41
:
49
61
.
8.
De Palma
M
,
Lewis
CE
. 
Macrophage regulation of tumor responses to anticancer therapies
.
Cancer Cell
2013
;
23
:
277
86
.
9.
Ruffell
B
,
Coussens
LM
. 
Macrophages and therapeutic resistance in cancer
.
Cancer Cell
2015
;
27
:
462
72
.
10.
Curiel
TJ
,
Coukos
G
,
Zou
L
,
Alvarez
X
,
Cheng
P
,
Mottram
P
, et al
Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival
.
Nat Med
2004
;
10
:
942
9
.
11.
Ruffell
B
,
Chang-Strachan
D
,
Chan
V
,
Rosenbusch
A
,
Ho
CMT
,
Pryer
N
, et al
Macrophage IL-10 blocks CD8+ T cell-dependent responses to chemotherapy by suppressing IL-12 expression in intratumoral dendritic cells
.
Cancer Cell
2014
;
26
:
623
37
.
12.
De Palma
M
,
Lewis
CE
. 
Cancer: macrophages limit chemotherapy
.
Nature
2011
;
472
:
303
4
.
13.
Mitchem
JB
,
Brennan
DJ
,
Knolhoff
BL
,
Belt
BA
,
Zhu
Y
,
Sanford
DE
, et al
Targeting tumor-infiltrating macrophages decreases tumor-initiating cells, relieves immunosuppression, and improves chemotherapeutic responses
.
Cancer Res
2013
;
73
:
1128
41
.
14.
Ries
CH
,
Cannarile
MA
,
Hoves
S
,
Benz
J
,
Wartha
K
,
Runza
V
, et al
Targeting tumor-associated macrophages with anti-CSF-1R antibody reveals a strategy for cancer therapy
.
Cancer Cell
2014
;
25
:
846
59
.
15.
Sève
P
,
Dumontet
C
. 
Chemoresistance in non–small cell lung cancer
.
Curr Med Chem Anticancer Agents
2005
;
5
:
73
88
.
16.
Katano
I
,
Ito
R
,
Kamisako
T
,
Eto
T
,
Ogura
T
,
Kawai
K
, et al
NOD-Rag2null IL-2Rγnull mice: an alternative to NOG mice for generation of humanized mice
.
Exp Anim
2014
;
63
:
321
30
.
17.
Zhou
Q
,
Facciponte
J
,
Jin
M
,
Shen
Q
,
Lin
Q
. 
Humanized NOD-SCID IL2rg−/− mice as a preclinical model for cancer research and its potential use for individualized cancer therapies
.
Cancer Lett
2014
;
344
:
13
9
.
18.
Cortez-Retamozo
V
,
Etzrodt
M
,
Newton
A
,
Rauch
PJ
,
Chudnovskiy
A
,
Berger
C
, et al
Origins of tumor-associated macrophages and neutrophils
.
Proc Natl Acad Sci U S A
2012
;
109
:
2491
6
.
19.
Komohara
Y
,
Jinushi
M
,
Takeya
M
. 
Clinical significance of macrophage heterogeneity in human malignant tumors
.
Cancer Sci
2014
;
105
:
1
8
.
20.
Wynn
TA
,
Chawla
A
,
Pollard
JW
. 
Macrophage biology in development, homeostasis and disease
.
Nature
2013
;
496
:
445
55
.
21.
Takeuchi
S
,
Baghdadi
M
,
Tsuchikawa
T
,
Wada
H
,
Nakamura
T
,
Abe
H
, et al
Chemotherapy-derived inflammatory responses accelerate the formation of immunosuppressive myeloid cells in the tissue microenvironment of human pancreatic cancer
.
Cancer Res
2015
;
75
:
2629
40
.
22.
Lin
H
,
Lee
E
,
Hestir
K
,
Leo
C
,
Huang
M
,
Bosch
E
, et al
Discovery of a cytokine and its receptor by functional screening of the extracellular proteome
.
Science
2008
;
320
:
807
11
.
23.
Foucher
ED
,
Blanchard
S
,
Preisser
L
,
Garo
E
,
Ifrah
N
,
Guardiola
P
, et al
IL-34 induces the differentiation of human monocytes into immunosuppressive macrophages. antagonistic effects of GM-CSF and IFNγ
.
PLoS ONE
2013
;
8
:
e56045
.
24.
Boström
EA
,
Lundberg
P
. 
The newly discovered cytokine IL-34 is expressed in gingival fibroblasts, shows enhanced expression by pro-inflammatory cytokines, and stimulates osteoclast differentiation
.
PLoS ONE
2013
;
8
:
e81665
.
25.
Yu
Y
,
Yang
D
,
Qiu
L
,
Okamura
H
,
Guo
J
,
Haneji
T
. 
Tumor necrosis factor-α induces interleukin-34 expression through nuclear factor-κB activation in MC3T3-E1 osteoblastic cells
.
Mol Med Rep
2014
;
10
:
1371
6
.
26.
Godwin
P
,
Baird
AM
,
Heavey
S
,
Barr
MP
,
O'Byrne
KJ
,
Gately
K
. 
Targeting nuclear factor-kappa B to overcome resistance to chemotherapy
.
Front Oncol
2013
;
3
:
120
.
27.
Hung
JY
,
Horn
D
,
Woodruff
K
,
Prihoda
T
,
LeSaux
C
,
Peters
J
, et al
Colony-stimulating factor 1 potentiates lung cancer bone metastasis
.
Lab Invest
2014
;
94
:
371
81
.
28.
Patsialou
A
,
Wang
Y
,
Pignatelli
J
,
Chen
X
,
Entenberg
D
,
Oktay
M
, et al
Autocrine CSF1R signaling mediates switching between invasion and proliferation downstream of TGFβ in claudin-low breast tumor cells
.
Oncogene
2015
;
34
:
2721
31
.
29.
Chihara
T
,
Suzu
S
,
Hassan
R
,
Chutiwitoonchai
N
,
Hiyoshi
M
,
Motoyoshi
K
, et al
IL-34 and M-CSF share the receptor Fms but are not identical in biological activity and signal activation
.
Cell Death Differ
2010
;
17
:
1917
27
.
30.
Pixley
FJ
,
Stanley
ER
. 
CSF-1 regulation of the wandering macrophage: complexity in action
.
Trends Cell Biol
2004
;
14
:
628
38
.
31.
Martini
M
,
De Santis
MC
,
Braccini
L
,
Gulluni
F
,
Hirsch
E
. 
PI3K/AKT signaling pathway and cancer: an updated review
.
Ann Med
2014
;
46
:
372
83
.
32.
Lawrence
T
,
Natoli
G
. 
Transcriptional regulation of macrophage polarization: enabling diversity with identity
.
Nat Rev Immunol
2011
;
11
:
750
61
.
33.
Martinez
FO
,
Gordon
S
,
Locati
M
,
Mantovani
A
. 
Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: new molecules and patterns of gene expression
.
J Immunol
2006
;
177
:
7303
11
.
34.
El Kasmi
KC
,
Qualls
JE
,
Pesce
JT
,
Smith
AM
,
Thompson
RW
,
Henao-Tamayo
M
, et al
Toll-like receptor-induced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens
.
Nat Immunol
2008
;
9
:
1399
406
.
35.
Ruffell
D
,
Mourkioti
F
,
Gambardella
A
,
Kirstetter
P
,
Lopez
RG
,
Rosenthal
N
, et al
A CREB-C/EBPbeta cascade induces M2 macrophage-specific gene expression and promotes muscle injury repair
.
Proc Natl Acad Sci U S A
2009
;
106
:
17475
80
.
36.
Caravatta
L
,
Sancilio
S
,
di Giacomo
V
,
Rana
R
,
Cataldi
A
,
Di Pietro
R
. 
PI3-K/Akt-dependent activation of cAMP-response element-binding (CREB) protein in Jurkat T leukemia cells treated with TRAIL
.
J Cell Physiol
2008
;
214
:
192
200
.
37.
Bézie
S
,
Picarda
E
,
Ossart
J
,
Tesson
L
,
Usal
C
,
Renaudin
K
, et al
IL-34 is a Treg-specific cytokine and mediates transplant tolerance
.
J Clin Invest
2015
;
1
:
3952
64
.
38.
Yu
G
,
Bing
Y
,
Zhu
S
,
Li
W
,
Xia
L
,
Li
Y
, et al
Activation of the interleukin-34 inflammatory pathway in response to influenza A virus infection
.
Am J Med Sci
2015
;
349
:
145
50
.
39.
Zwicker
S
,
Martinez
GL
,
Bosma
M
,
Gerling
M
,
Clark
R
,
Majster
M
, et al
Interleukin 34: a new modulator of human and experimental inflammatory bowel disease
.
Clin Sci
2015
;
129
:
281
90
.
40.
Ségaliny
AI
,
Mohamadi
A
,
Dizier
B
,
Lokajczyk
A
,
Brion
R
,
Lanel
R
, et al
Interleukin-34 promotes tumor progression and metastatic process in osteosarcoma through induction of angiogenesis and macrophage recruitment
.
Int J Cancer
2015
;
137
:
73
85
.
41.
Castells
M
,
Thibault
B
,
Delord
J-P
,
Couderc
B
. 
Implication of tumor microenvironment in chemoresistance: tumor-associated stromal cells protect tumor cells from cell death
.
Int J Mol Sci
2012
;
13
:
9545
71
.
42.
Karin
M
,
Cao
Y
,
Greten
FR
,
Li
Z-W
. 
NF-kappaB in cancer: from innocent bystander to major culprit
.
Nat Rev Cancer
2002
;
2
:
301
10
.
43.
Eda
H
,
Shimada
H
,
Beidler
DR
,
Monahan
JB
. 
Proinflammatory cytokines, IL-1β and TNF-α, induce expression of interleukin-34 mRNA via JNK- and p44/42 MAPK-NF-κB pathway but not p38 pathway in osteoblasts
.
Rheumatol Int
2011
;
31
:
1525
30
.
44.
Kusmartsev
S
,
Gabrilovich
DI
. 
Effect of tumor-derived cytokines and growth factors on differentiation and immune suppressive features of myeloid cells in cancer
.
Cancer Metastasis Rev
2006
;
25
:
323
31
.
45.
Manning
BD
,
Cantley
LC
. 
AKT/PKB signaling: navigating downstream
.
Cell
2007
;
129
:
1261
74
.
46.
Gagnon
V
,
Mathieu
I
,
Sexton
E
,
Leblanc
K
,
Asselin
E
. 
AKT involvement in cisplatin chemoresistance of human uterine cancer cells
.
Gynecol Oncol
2004
;
94
:
785
95
.
47.
Kim
S-H
,
Juhnn
Y-S
,
Song
Y-S
. 
Akt involvement in paclitaxel chemoresistance of human ovarian cancer cells
.
Ann N Y Acad Sci
2007
;
1095
:
82
9
.
48.
Droin
N
,
Solary
E
. 
Editorial: CSF1R, CSF-1, and IL-34, a “menage a trois” conserved across vertebrates
.
J Leukoc Biol
2010
;
87
:
745
7
.
49.
Nakamichi
Y
,
Udagawa
N
,
Takahashi
N
. 
IL-34 and CSF-1: similarities and differences
.
J Bone Miner Metab
2013
;
31
:
486
95
.
50.
Cioce
M
,
Canino
C
,
Goparaju
C
,
Yang
H
,
Carbone
M
,
Pass
HI
. 
Autocrine CSF-1R signaling drives mesothelioma chemoresistance via AKT activation
.
Cell Death Dis
2014
;
5
:
e1167
.