Immunosuppressive M2 Macrophages Research Articles

Macrophages in Oral Carcinomas: Relationship with Cancer Stem Cell Markers and PD-L1 Expression.

Tumor-associated macrophages (TAMs) can be polarized into antitumoral M1 and protumoral and immunosuppressive M2 macrophages. This study investigated the clinical relevance of TAM infiltration in oral squamous cell carcinoma (OSCC), evaluating CD68 (M1 and M2 macrophage marker) and CD163 expression (M2 macrophage marker) in the tumor nests and surrounding stroma. Immunohistochemical analysis of both stromal/tumoral CD68+ and CD163+ TAMs was performed in paraffin-embedded tissue specimens from 125 OSCC patients, and correlated with clinical data. Potential relationships with the expression of cancer stem cell (CSC) markers and PD-L1 in the tumors were also assessed. Stromal CD163+ infiltration was significantly associated with the tumor location in the tongue, and stromal and tumoral CD68+ and CD163+-infiltrating TAMs were more abundant in nonsmokers and non-alcohol-drinkers. Strikingly, this study uncovers an inverse relationship between CD68+ and CD163+ TAMs and CSC marker expression (NANOG and SOX2) in OSCC. High infiltration of CD163+ TAMs in both tumor and stroma was strongly and significantly correlated with the absence of NANOG expression. Moreover, infiltration of both CD68+ and CD163+ TAMs was also significantly associated with high tumor expression of PD-L1. Our results suggest that there is a link between TAM infiltration and immune escape in OSCC.

Bioinformatic profiling of tumor immunity from patient biopsies to predict survival and response to immunotherapy.

e15198 Background: Immunotherapies have revolutionized cancer treatment, but responses are not universal and patients who initially respond to therapy develop resistance. The accurate quantification of tumor-infiltrating immune cells holds the promise to reveal the role of the immune system in human cancers and its involvement in tumor escape mechanisms and response to therapy. We present MIXTURE, a new algorithm for tumor immune cell-type proportions deconvolution from transcriptomic data that overcomes competitive methods and revealed novel associations of immune cell types with patient survival and immunotherapy response. Methods: We applied MIXTURE to transcriptomic data from BRCA (n = 1095), LUAD (n = 506), SKCM (n = 472), HNSC (n = 499) and COAD (n = 521) cohorts from TCGA and five published datasets of melanoma patients treated with anti-PD-1/anti-CLTA-4. Results: The analysis of TCGA breast cancer biopsies showed high proportions of M2-macrophages associated with poor patient survival (p < 0.001). In contrast, we found that proportions of follicular T helper cells were associated with better outcome (p < 0.001). We observed a differential immune composition in biopsies of lung adenocarcinoma patients with mutations in TP53 (WT; n = 247; Mut; n = 259) and EGFR (WT, n = 438; Mut, n = 68) related with their different response to immunotherapy (p < 0.05). We found correlations between immune cells proportions and current biomarkers of response to immunotherapy such as TMB, intratumoral heterogeneity, MSI and PD-L1 expression in the TCGA cohorts (p < 0.05). The meta-analysis of melanoma patients treated with immunotherapy showed a distinct immune infiltrate in patients who responded to anti-PD-1 with an increase of immune effector cells such as CD8, CD4 memory activated and gamma-delta T cells, and a decrease in immunosuppressive M2-macrophages (p < 0.05; R = 81; NR = 107). According with the latest findings, we observed higher presence of B cells in responders to anti-PD-1 on-treatment (p = 0.033; R = 31; NR = 23) and in baseline of responders to anti-CTLA-4 (p = 0.028; R = 14; NR = 26). Interestingly, patients that previously progressed to anti-CTLA-4 showed a differential immune profile that was associated with response to anti-PD-1 (p < 0.05; Ipi-Prog = 59; Ipi-Naïve = 102). Conclusions: We demonstrated the potential of MIXTURE to understand the tumor immune microenvironment and its relationship with patient survival and response to immunotherapies. MIXTURE is available for the wider scientific community as web application and as packages for R and Python.

PAM3 protects against DSS-induced colitis by altering the M2:M1 ratio

Inflammation of the gastrointestinal tract contributes to the development of inflammatory bowel disease (IBD). Human IBD is modeled by administering dextran sulfate sodium (DSS) to mice. In humans and mice, inflammatory M1 macrophages contribute to the progression of IBD whereas immunosuppressive M2 macrophages protect against colitis. The TLR2/1 agonist PAM3CSK4 (PAM3) induces human and murine monocytes to differentiate into immunosuppressive M2 macrophages, suggesting that PAM3 might be of benefit in the prevention/treatment of colitis. PAM3 was therefore administered to mice treated with DSS. As hypothesized, the number of M2 macrophages rose and disease severity decreased. The critical role of M2 macrophages in this process was established by transferring purified M2 macrophages from PAM3 treated control donors into DSS recipients and reducing colitis. These findings suggest that PAM3 may represent a novel approach to the treatment of human IBD.

Abstract A14: Irreversible electroporation is an “in situ vaccine” and induces antitumor immune responses in pancreatic cancer

Abstract The pancreatic cancer (PC) microenvironment is notoriously immunosuppressive and responds poorly to conventional checkpoint blockade immunotherapy, necessitating a need to overcome this challenge. Irreversible electroporation (IRE) is a nonthermal method of inducing cell death that is currently being used clinically for selected patients with locally advanced PC. Using a robust syngeneic mouse model of PC using cells established from a tumor arising in an LSL-KrasG12D/+; LSL-Trp53R172H/+; PDX1Cre/+; LSL-ROSA26 Luc/+ mouse (KPC), we have demonstrated that IRE alone can serve as an “in situ vaccine” and induce systemic immune responses that prevent secondary tumor growth (prophylactic immunity). We have furthermore demonstrated that these effects are augmented by combination with innate immune activators and PD-1 checkpoint blockade. We observed a significant decrease in immunosuppressive myeloid-derived suppressor cells (MDSCs) (CD11b+, CD11c-, GR1+) at one week in the IRE and drug alone groups with an additive effect in the combination groups. We also saw a profound shift in the ratio of immunostimulatory M1 (MHC II+, CD206 lo): immunosuppressive M2 (MHC II -, CD206 hi) macrophages in the combination groups and a modest increase in CD8+ T cells with IRE alone that was almost doubled in the combination groups with a concomitant decrease in T regulatory cells. Using exome and RNA sequencing of the KPC tumors expressed, nonsynonymous mutations in these tumors were identified and 101 long peptides (20 mer) were generated harboring these mutations at positions 9 and 15. When the CD8+ T-cells post-IRE were incubated along with these mutant peptides and bone marrow-derived dendritic cells, we were able to observe that these T cells were reactive (Interferon gamma secretion) to these neoantigenic peptide pools. Further, we were able to identify specific mutant peptides against which T-cell response was stimulated post IRE that were similar or even better than vaccinating the mice with irradiated KPC cells, suggesting that IRE induced tumor neoantigen-specific prophylactic immune response. Specifically, local delivery of toll-like receptor 7 (TLR7) agonist (1V270) and agonistic CD40 antibody resulted in complete regression of treated primary tumors in 7/10 mice in the experimental group. Increased presence of CD8+ dendritic cells (CD45+ CD11C+) in the draining lymph node suggested that the agonistic CD40 antibody could have triggered increased dendritic cell maturation and cross-presentation. Moreover, it resulted in the complete elimination of the untreated secondary tumors in the group, which IRE as a monotherapy failed to achieve, showing that with appropriate combination strategies, successful therapeutic systemic antitumor immunity can also be achieved with IRE to be used for the management of metastatic PC in the near future. Citation Format: Jayanth S. Shankara Narayanan, Aaron Miller, Tomoko Hayashi, Partha Ray, Stephen Schoenberger, Dennis Carson, Rebekah White. Irreversible electroporation is an “in situ vaccine” and induces antitumor immune responses in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A14.

Elevated expression of DOK3 indicates high suppressive immune cell infiltration and unfavorable prognosis of gliomas

Docking protein 3 has been implicated in immune response, including interferon-β production in macrophage and plasma cell differentiation. And its importance in lung adenocarcinoma has been reported. However, studies about its role in gliomas are rare. In this study, we explored the clinical and prognostic characteristics of DOK3 expression in 921 glioma samples. Kaplan-Meier survival analysis and Cox regression analysis verified the independent unfavorable prognostic value and high prognostic accuracy of DOK3 expression for overall survival. Functional analysis with Database for Annotation, Visualization and Integrated Discovery (DAVID) and Gene Set Enrichment Analysis (GSEA) implied the involvement of DOK3 in immune related responses. Immune cell infiltration analysis with online tools, CIBERSORT and EPIC, showed that samples with higher DOK3 expression were infiltrated with much more macrophages. DOK3 was also found to be strongly positively correlated with marker genes of tumor-associated macrophages and M2 macrophages, not M1. Results of immunohistochemical staining also demonstrated that samples with higher DOK3 expression level were infiltrated with more microglia/macrophages and immunosuppressive M2 macrophages. In summary, our results demonstrated the correlation between high DOK3 expression level and malignant progression of gliomas, and the possible involvement of DOK3 in immunosuppressive responses in gliomas.

Infiltration of CD163-positive macrophages in glioma tissues after treatment with anti-PD-L1 antibody and role of PI3Kγ inhibitor as a combination therapy with anti-PD-L1 antibody in in vivo model using temozolomide-resistant murine glioma-initiating cells

Although chemoimmunotherapy often lengthens glioblastoma (GBM) survival, early relapses remain problematic as immunosuppressive M2 macrophages (Mϕ) that function via inhibitory cytokine and PD-L1 production cause immunotherapy resistance. Here, we detail anti-PD-L1 antibody effects on the tumor microenvironment, including Mϕ infiltration, using a temozolomide (TMZ)-treated glioma model. In addition, we tested combinations of anti-PD-L1 antibody and the M2Mϕ inhibitor IPI-549 on tumor growth. We simulated late TMZ treatment or relapse stage, persistent GBM cells by generating TMZ-resistant TS (TMZRTS) cells. M2Mϕ-associated cytokine production and PD-L1 expression in these cells were investigated. TMZRTS cells were then subcutaneously implanted into C57BL/6 mice to determine the effectiveness of an anti-PD-L1 antibody and/or IPI-549 treatment on infiltration of CD163-positive Mϕ, usually considered as an M2Mϕ marker into tumor tissues. CD163 expression in samples from human GBM patients were also evaluated. CD163-positive Mϕ heavily infiltrated TMZRS tumor tissues after in vivo anti-PD-L1 antibody treatment. Tumor growth was strongly inhibited by anti-PD-L1 antibody and IPI-549 combination therapy. Anti-PD-L1 antibody treatment significantly reduced infiltration of CD163-positive Mϕ into tumors, while combined PD-L1 antibody and IPI-549 therapy remarkably inhibited tumor growth. These therapies may be useful for recurrent or chronic GBM after TMZ treatment, but clinical safety and effectiveness studies are needed.

Exosomal communication by metastatic osteosarcoma cells modulates alveolar macrophages to an M2 tumor-promoting phenotype and inhibits tumoricidal functions

ABSTRACT Osteosarcoma metastasizes to the lung, and there is a link between the predominance of tumor-promoting immunosuppressive M2 macrophages in the metastases and poor patient survival. By contrast, M1 macrophage predominance correlates with longer survival. M2 macrophages can be induced by various stimuli in the tumor microenvironment, including exosomes, which are 40- to 150-nm vesicles that are involved in intercellular communication and contribute to tumor progression and immune evasion. Recognizing that tumor cells can influence the tumor microenvironment to make it more permissive and because of the link between M2 dominance and curtailed patient survival, we evaluated the effect of exosomes from non-metastatic K7 and Dunn osteosarcoma cells and the metastatic sublines K7M3 and DLM8 on macrophage phenotype and function. Incubating MHS mouse alveolar macrophages with K7M3 and DLM8 exosomes induced expression of IL10, TGFB2, and CCL22 mRNA (markers of M2 macrophages) and decreased phagocytosis, efferocytosis, and macrophage-mediated tumor cell killing. In contrast, exosomes from non-metastatic K7 or Dunn cells did not inhibit phagocytosis, efferocytosis, and macrophage-mediated cytotoxicity or induce increased expression of IL10, TGFB2 or CCL22 mRNA. In addition, metastatic osteosarcoma cell exosomes significantly increased the secretion of TGFB2, a key signaling pathway associated with tumor- mediated immune suppression. Finally, the inhibition of TGFB2 reversed the suppressive activity of alveolar macrophages exposed to metastatic osteosarcoma cell exosomes. Our data suggest that the exosomes from metastatic osteosarcoma cells can modulate cellular signaling of tumor-associated macrophages, thereby promoting the M2 phenotype and creating an immunosuppressive, tumor-promoting microenvironment through the production of TGFB2.

Mesenchymal Stem Cell-Derived Exosomes and Other Extracellular Vesicles as New Remedies in the Therapy of Inflammatory Diseases.

There is growing evidence that mesenchymal stem cell (MSC)-based immunosuppression was mainly attributed to the effects of MSC-derived extracellular vesicles (MSC-EVs). MSC-EVs are enriched with MSC-sourced bioactive molecules (messenger RNA (mRNA), microRNAs (miRNAs), cytokines, chemokines, immunomodulatory factors) that regulate phenotype, function and homing of immune cells. In this review article we emphasized current knowledge regarding molecular mechanisms responsible for the therapeutic effects of MSC-EVs in attenuation of autoimmune and inflammatory diseases. We described the disease-specific cellular targets of MSC-EVs and defined MSC-sourced molecules, which were responsible for MSC-EV-based immunosuppression. Results obtained in a large number of experimental studies revealed that both local and systemic administration of MSC-EVs efficiently suppressed detrimental immune response in inflamed tissues and promoted survival and regeneration of injured parenchymal cells. MSC-EVs-based anti-inflammatory effects were relied on the delivery of immunoregulatory miRNAs and immunomodulatory proteins in inflammatory immune cells (M1 macrophages, dendritic cells (DCs), CD4+Th1 and Th17 cells), enabling their phenotypic conversion into immunosuppressive M2 macrophages, tolerogenic DCs and T regulatory cells. Additionally, through the delivery of mRNAs and miRNAs, MSC-EVs activated autophagy and/or inhibited apoptosis, necrosis and oxidative stress in injured hepatocytes, neurons, retinal cells, lung, gut and renal epithelial cells, promoting their survival and regeneration.

MDM2 inhibitor APG-115 synergizes with PD-1 blockade through enhancing antitumor immunity in the tumor microenvironment

BackgroundProgrammed death-1 (PD-1) immune checkpoint blockade has achieved clinical successes in cancer therapy. However, the response rate of anti-PD-1 agents remains low. Additionally, a subpopulation of patients developed hyperprogressive disease upon PD-1 blockade therapy. Combination therapy with targeted agents may improve immunotherapy. Recent studies show that p53 activation in the myeloid linage suppresses alternative (M2) macrophage polarization, and attenuates tumor development and invasion, leading to the hypothesis that p53 activation may augment antitumor immunity elicited by anti-PD-1 therapy.MethodUsing APG-115 that is a MDM2 antagonist in clinical development as a pharmacological p53 activator, we investigated the role of p53 in immune modulation and combination therapy with PD-1 blockade.ResultsIn vitro treatment of bone marrow-derived macrophages with APG-115 resulted in activation of p53 and p21, and a decrease in immunosuppressive M2 macrophage population through downregulation of c-Myc and c-Maf. Increased proinflammatory M1 macrophage polarization was observed in the spleen from mice treated with APG-115. Additionally, APG-115 has co-stimulatory activity in T cells and increases PD-L1 expression in tumor cells. In vivo, APG-115 plus anti-PD-1 combination therapy resulted in enhanced antitumor activity in Trp53wt, Trp53mut, and Trp53-deficient (Trp53−/−) syngeneic tumor models. Importantly, such enhanced activity was abolished in a syngeneic tumor model established in Trp53 knockout mice. Despite differential changes in tumor-infiltrating leukocytes (TILs), including the increases in infiltrated cytotoxic CD8+ T cells in Trp53wt tumors and M1 macrophages in Trp53mut tumors, a decrease in the proportion of M2 macrophages consistently occurred in both Trp53wt and Trp53mut tumors upon combination treatment.ConclusionOur results demonstrate that p53 activation mediated by APG-115 promotes antitumor immunity in the tumor microenvironment (TME) regardless of the Trp53 status of tumors per se. Instead, such an effect depends on p53 activation in Trp53 wild-type immune cells in the TME. Based on the data, a phase 1b clinical trial has been launched for the evaluation of APG-115 in combination with pembrolizumab in solid tumor patients including those with TP53mut tumors.

IMMU-19. QUINOLINATE-INDUCED IMMUNE SUPPRESSION IN GLIOBLASTOMA

Abstract Glioblastoma continues to be an invariably fatal malignancy with limited treatment options. We performed integrative cross-platform analyses coupling global metabolomic profiling with genomics in patient-derived tumors to provide insight into metabolic programs that may be driving the aggressive phenotype of this malignancy. A persistent theme that emerged was the interplay between metabolic remodeling and immune response. Most notable was a 64-fold accumulation of quinolinate, which represents a downstream intermediary of tryptophan metabolism, in glioblastoma, when compared to low-grade astrocytoma. We discovered a dynamic interaction between cells within the tumor microenvironment that contributes to the generation of quinolinate. Specifically, neither tumor cells nor immune cells demonstrated the capacity to completely metabolize tryptophan. Tumor cells were required to metabolize tryptophan to kynurenine, while only cells of monocyte lineage, including M1/M2 macrophages and microglial cells, demonstrated the capacity to generate quinolinate from tumor-generated kynurenine. We next sought to determine if the observed accumulation of quinolinate in glioblastoma had immune consequences. We discovered a novel role quinolinate plays in increasing (~50%) immunosuppressive M2 macrophage (CD45+CD11b+F4/80+ and CD206+) polarization and promoting a suppressive molecular phenotype (IL4Rα +). These findings were functionally recapitulated, with quinolinate-induced M2 macrophages demonstrating potent inhibition of CD8+T-cell proliferation. Intriguingly, quinolinate also skewed M1 polarization towards an “M2-like” phenotype both molecularly (CD45+CD11b+F4/80+CD206+) and functionally, by inhibiting CD8+T-cell proliferation and decreasing phagocytosis efficiency. Targeting quinolinate’s upstream enzyme kynurenine-3-monooxygenase (KMO) inhibited M2 polarization in vitro and deceased M2 macrophage levels in vivo by 60%. This was unique to KMO, as targeting tryptophan metabolism by inhibiting the more established, upstream target IDO1 did not influence M2 macrophage levels. As emerging studies suggest M2 macrophages play a central role in contributing towards the potent immunosuppressive microenvironment in glioblastoma, therapeutic strategies designed to target quinolinate metabolism may serve as a novel immuno-metabolic checkpoint with direct clinical implications in this malignancy.

Combination Treatment of the Oral CHK1 Inhibitor, SRA737, and Low-Dose Gemcitabine Enhances the Effect of Programmed Death Ligand 1 Blockade by Modulating the Immune Microenvironment in SCLC

IntroductionDespite the enthusiasm surrounding cancer immunotherapy, most SCLC patients show very modest response to immune checkpoint inhibitor monotherapy treatment. Therefore, there is growing interest in combining immune checkpoint blockade with chemotherapy and other treatments to enhance immune checkpoint blockade efficacy. Based on favorable clinical trial results, chemotherapy and immunotherapy combinations have been recently approved by the U.S. Food and Drug Administration for frontline treatment for SCLC. Methods and ResultsHere, we show that combined treatment of SRA737, an oral CHK1 inhibitor, and anti–programmed death ligand 1 (PD-L1) leads to an antitumor response in multiple cancer models, including SCLC. We further show that combining low, non-cytotoxic doses of gemcitabine with SRA737 + anti–PD-L1/anti–PD-1 significantly increased antitumorigenic CD8+ cytotoxic T cells, dendritic cells, and M1 macrophage populations in an SCLC model. This regimen also led to a significant decrease in immunosuppressive M2 macrophage and myeloid-derived suppressor cell populations, as well as an increase in the expression of the type I interferon beta 1 gene, IFNβ, and chemokines, CCL5 and CXCL10. ConclusionsGiven that anti–PD-L1/anti–PD-1 drugs have recently been approved as monotherapy and in combination with chemotherapy for the treatment of SCLC, and that the SRA737 + low dose gemcitabine regimen is currently in clinical trials for SCLC and other malignancies, our preclinical data provide a strong rational for combining this regimen with inhibitors of the PD-L1/PD-1 pathway.

Abstract 3192: Activation of p53 in the tumor microenvironment by MDM2 inhibitor APG-115 synergizes with PD-1 blockade independently of p53 status of tumor cells

Abstract Blockade of the checkpoint inhibitor programmed death 1 (PD-1) has gained big success in cancer therapy. However, the response rate of anti-PD1 agents remains low. Molecularly targeted agents offer selectivity and high tumor response rates, but patients develop resistance to these drugs inevitably. Combinations of targeted agents and immunotherapy provide new opportunities to improve cancer treatments. Recent studies found that p53 activation in the myeloid linage suppressed alternative (M2) macrophage polarization and attenuates tumor development and invasion, leading to the hypothesis that p53 activation may further augment antitumor immunity elicited by anti-PD-1 therapy. APG-115 is an orally active, selective, small molecule inhibitor of the MDM2-p53 protein-protein interaction. APG-115 acts as an antitumor agent by activating the p53 tumor suppressor in p53 wild-type tumors. However, its role in regulating immune responses remained unknown. In this study, we investigated the role of APG-115 in immune modulation both in vitro and in vivo. Enhanced antitumor activity was first demonstrated in p53 wild-type MH-22A, p53 mutant MC38, and p53 knockout (p53-/-) MH-22A syngeneic tumor models after the combination treatment of APG-115 and anti-PD-1 antibody. Despite differential changes in tumor-infiltrating leukocytes, including an increase in cytotoxic CD8+ T cells in the p53 wild-type tumors and an increase in proinflammatory M1 macrophages in the p53 mutant tumors, the combination treatment consistently reduced immunosuppressive M2 macrophages in the tumor microenvironment regardless of p53 status of tumor cells. In addition, in vitro, the treatment of bone marrow-derived macrophages with APG-115 resulted in activation of p53 and p21 gene expression, as well as a decrease in M2 macrophages population and reduction of c-MYC and M2-related gene expression. Moreover, enhanced M1 macrophage polarization in the spleen was also observed in naïve mice treated with APG-115. Furthermore, APG-115 increased production of multiple proinflammatory cytokines, including IFN-γ, TNF-α, IL-2 and IL-6, in stimulated T cells. Collectively, for the first time, our findings suggest that p53 activation by a pharmacological MDM2 inhibitor enables reversal of immunosuppressive tumor microenvironment and enhance antitumor immunity independently of p53 status of tumors. Specifically, in complementary to PD-1 blockade that predominantly activates cytotoxic CD8+ T cell populations, APG-115 primarily targets tumor-associated macrophages. Collectively, our data provide a rationale for applying the combination of APG-115 plus PD-1 blockade to a broader patient population with p53 mutant tumors. Accordingly, a clinical trial of APG-115 in combination with pembrolizumab in metastatic melanoma patients regardless of p53 status has been initiated in the USA. Citation Format: Douglas D. Fang, Qiuqiong Tang, Yanhui Kong, Qixin Wang, Jiaxing Gu, Xu Fang, Peng Zou, Tao Rong, Jingwen Wang, Dajun Yang, Yifan Zhai. Activation of p53 in the tumor microenvironment by MDM2 inhibitor APG-115 synergizes with PD-1 blockade independently of p53 status of tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3192.

Immunological and Clinicopathological Significance of MFG-E8 Expression in Patients with Oral Squamous Cell Carcinoma.

Milk fat globule-epidermal growth factor 8 (MFG-E8) is a glycoprotein secreted by the activated macrophages and acts as a bridge between apoptotic cells and phagocytes. Aside from macrophages, a variety of malignant cells also express MFG-E8. The objective of this study is to elucidate the clinical relevance and significance of MFG-E8 in the tumor microenvironment (TME) of patients with oral squamous cell carcinoma (OSCC). We investigated MFG-E8 expression in 74 patients with OSCC by immunohistochemistry and evaluated the relationship between MFG-E8 expression and various clinicopathological factors including immune cell infiltration. MFG-E8 expression was detected in 34 of 74 (45.9%) patients with OSCC and a significant correlation was observed with levels of infiltrating T cells, macrophages, and immunosuppressive M2 macrophages. Furthermore, MFG-E8 expression was also associated with clinical stage, lymphatic/vascular invasion, and Ki-67+ tumor cells but not with survival. Our results suggest that MFG-E8 may play an important role in shaping the immune suppressive network in TME as well as tumor progression.

HCC-Derived Exosomes: Critical Player and Target for Cancer Immune Escape.

Hepatocellular carcinoma (HCC) is a primary malignancy of the liver, and currently the second most common cause of cancer-related deaths worldwide with increasing incidence and poor prognosis. Exosomes are now considered as important mediators of host anti-tumor immune response as well as tumor cell immune escape. HCC-derived exosomes have been shown to attenuate the cytotoxicity of T-cells and NK cells, and promote the immuno-suppressive M2 macrophages, N2 neutrophils, and Bregs. These exosomes harbor several immune-related non-coding RNAs and proteins that drive immune-escape and tumor progression, and thus may serve as potential diagnostic biomarkers and therapeutic targets for HCC. In a previous study, we identified miR146a as an exosomal factor that promotes M2-polarization and suppresses the anti-HCC function of T-cells. In this review, we summarized the role of tumor-derived exosomes and their key components in mediating tumor immune escape during HCC development.

α-Mannosyl-Functionalized Cationic Nanohydrogel Particles for Targeted Gene Knockdown in Immunosuppressive Macrophages.

Immunosuppressive M2 macrophages govern the immunophathogenic micromilieu in many severe diseases including cancer or fibrosis, thus, their re-polarization through RNA interference is a promising concept to support combinatorial therapies. For targeted siRNA delivery, however, safe and stable carriers are required that manage cell specific transport to M2 macrophages. Here, siRNA-loaded cationic nanogels are reported with α-mannosyl decorated surfaces that target and modify M2 macrophages selectively. Via amphiphilic precursor block copolymers bearing one single α-mannosyl moiety at their chain end mannosylated cationic nanohydrogel particles (ManNP) were obtained of 20 nm diameter determined by dynamic light scattering and cryogenic electron transmission microscopy. α-Mannosyl surface modification is confirmed by agglutination with concanavalin A. SiRNA-loaded ManNP preferentially targets the overexpressed mannose receptor CD206 on M2 macrophages, as shown by in vitro cell uptake studies in M2 polarized primary macrophages. This specificity is confirmed, since ManNP uptake could be reduced by blocking of CD206 with mannan. Effective ManNP-guided siRNA delivery is confirmed by sequence-specific gene knockdown of CSF-1R in M2-type macrophages exclusively, while the expression levels in M1-polarized macrophages is not affected. In conclusion, α-mannosyl-functionalized ManNPs are promising universal siRNA carriers for targeted immunomodulatory treatment of immunosuppressive macrophages.

Microenvironmental signals govern the cellular identity of testicular macrophages.

Testicular macrophages (TM) comprise the largest immune cell population in the mammalian testis. They are characterized by a subdued proinflammatory response upon adequate stimulation, and a polarization toward the immunoregulatory and immunotolerant M2 phenotype. This enables them to play a relevant role in supporting the archetypical functions of the testis, namely spermatogenesis and steroidogenesis. During infection, the characteristic blunted immune response of TM reflects the need for a delicate balance between a sufficiently strong reaction to counteract invading pathogens, and the prevention of excessive proinflammatory cytokine levels with the potential to disturb or destroy spermatogenesis. Local microenvironmental factors that determine the special phenotype of TM have just begun to be unraveled, and are discussed in this review.

Abstract 1751: MSC-1 is a first-in-class humanized monoclonal antibody that modulates the tumor microenvironment by inhibiting a novel cancer immunotherapy target, LIF

Abstract Leukemia Inhibitory Factor (LIF) is a member of the IL-6 family of cytokines and is involved in many physiological and pathological processes including the promotion of an immunosuppressive environment to support embryo implantation, down-regulation of autoimmune processes and the regulation of stem cell homeostasis and differentiation. In cancer, LIF is hypothesized to have a complex role tumor development and progression, creating an immunosuppressive tumor microenvironment as well as promoting the activity of cancer initiating cells (CICs). LIF is highly expressed in a subset of tumors across multiple tumor types, e.g. glioblastoma multiforme (GBM), non-small cell lung cancer (NSCLC), colon, ovarian, prostate, and pancreatic cancer, and correlates with poor prognosis. Given the pleiotropic role LIF is hypothesized to play in cancer, inhibition of LIF represents an exciting new therapeutic concept sitting at the intersection of two key therapeutic approaches in oncology: blockade of tumor evasion of the immune system and blockade of tumor growth via inhibition of CICs. We have identified and developed MSC-1, a first-in-class humanized IgG1 monoclonal antibody that is a potent and selective inhibitor of LIF. MSC-1 cross reacts with mouse and non-human primate LIF and inhibits LIF signaling by blocking the recruitment of gp130 to the LIF-LIFR-gp130 signaling complex. The efficacy of MSC-1 was evaluated in multiple mouse tumor models and the mechanism of action investigated. LIF inhibition with MSC-1 or shRNAs reduced tumor growth in multiple syngeneic tumor models (NSCLC, ovarian and colon), and clear target engagement was shown for MSC-1. Investigations into the mechanism of action identified that inhibition of LIF with MSC-1 reprogrammed the tumor microenvironment by decreasing immunosuppressive M2 macrophages and increasing the number of intratumoral NK cells and total/activated T cells. MSC-1 also decreased immunosuppressive M2 macrophages in an orthotopic GBM xenograft model and human GBM organotypic tumor slices in an ex vivo model. Similarly, immunosuppressive macrophage genes were decreased when monocytes were co-cultured with supernatants from a GBM cell line in which LIF expression had been knocked-down. Given the effects of MSC-1 on intratumoral immune cells, we hypothesized that MSC-1 could be effectively combined with checkpoint inhibitors and we are currently evaluating MSC-1/checkpoint inhibitor combination therapy. Taken together, these findings form the basis of a robust therapeutic hypothesis, whereby MSC-1 treatment will lead to clinical activity in multiple cancer indications. A Phase I dose-escalation and expansion study of MSC-1 is planned to initiate early 2018 in patients with advanced solid tumors that will incorporate target engagement and PD biomarkers, as well as safety and efficacy endpoints. Citation Format: Angus Sinclair, Robin Hallett, Patricia Giblin, Isabel Huber-Ruano, Judit Anido, Naimish Pandya, Kimberly Hoffman, Ada Sala, Monica Pascual, Vanessa Chigancas, Swetha Raman, Johan Fransson, Jean-Philippe Julien, Robert Wasserman, Jeanne Magram, Joan Seoane. MSC-1 is a first-in-class humanized monoclonal antibody that modulates the tumor microenvironment by inhibiting a novel cancer immunotherapy target, LIF [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1751.

Tumor-Associated Macrophages: Therapeutic Targets for Skin Cancer.

Tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) are significant components of the microenvironment of solid tumors in the majority of cancers. TAMs sequentially develop from monocytes into functional macrophages. In each differentiation stage, TAMs obtain various immunosuppressive functions to maintain the tumor microenvironment (e.g., expression of immune checkpoint molecules, production of Treg-related chemokines and cytokines, production of arginase I). Although the main population of TAMs is immunosuppressive M2 macrophages, TAMs can be modulated into M1-type macrophages in each differential stage, leading to the suppression of tumor growth. Because the administration of certain drugs or stromal factors can stimulate TAMs to produce specific chemokines, leading to the recruitment of various tumor-infiltrating lymphocytes, TAMs can serve as targets for cancer immunotherapy. In this review, we discuss the differentiation, activation, and immunosuppressive function of TAMs, as well as their benefits in cancer immunotherapy.

IL-4 blockade alters the tumor microenvironment and augments the response to cancer immunotherapy in a mouse model.

Recent findings show that immune cells constitute a large fraction of the tumor microenvironment and that they modulate tumor progression. Clinical data indicate that chronic inflammation is present at tumor sites and that IL-4, in particular, is upregulated. Thus, we tested whether IL-4 neutralization would affect tumor immunity. Current results demonstrate that the administration of a neutralizing antibody against IL-4 enhances anti-tumor immunity and delays tumor progression. IL-4 blockade also alters inflammation in the tumor microenvironment, reducing the generation of both immunosuppressive M2 macrophages and myeloid-derived suppressor cells, and enhancing tumor-specific cytotoxic T lymphocytes. In addition, IL-4 blockade improves the response to anti-OX40 Ab or CpG oligodeoxynucleotide immunotherapies. These findings suggest that IL-4 affects anti-tumor immunity and constitutes an attractive therapeutic target to reduce immune suppression in the tumor microenvironment, thus enhancing the efficacy of cancer therapy.

Abstract PR01: Blocking Colony Stimulating Factor 1 Receptor (CSF-1R) and Tropomyosin Receptor Kinase (Trk) improves antitumor efficacy of immunotherapy

Abstract The tumor microenvironment can provide a strong limitation to the antitumor activity of immunotherapy. Established tumors can escape immune responses by creating an inflammatory milieu that supports and recruits tumor-infiltrating myeloid cells (TIMs) with immune suppressive functions. Colony stimulating factor-1 (CSF-1) is a cytokine that is secreted by various cancer types including melanoma. It binds to colony stimulating factor-1 receptor (CSF-1R) on the myeloid cells. It stimulates the proliferation and recruitment of immunosuppressive M2-macrophages and myeloid derived suppressor cells (MDSC) into the tumor microenvironement. In addition, neurotrophins are a family of growth factors that includes nerve growth factor (NGF). NGF is the ligand for a family of tyrosine kinase receptors (Trks) that includes Trk-A, -B, and -C. Signal transduction through the Trk receptors regulates tumor growth. Trk receptors have been shown to be overexpressed in different cancer types. For example, Trk-C is overexpressed in 60% of patients with melanoma at various stages. PLX7486 is a novel orally bioavailable small molecule Trk and CSF-1R dual-inhibitor that is now being studied in Phase I clinical trials. PLX7486 can target Trks directly on cancer cells and block myeloid cell recruitment to the tumor microenvironement through CSF-1R. We hypothesize that PLX7486 would synergize with immunotherapies to result in a greater antitumor effect by inhibiting the recruitment of immunosuppressive macrophages. In order to block the Trk receptors on cancer cells with PLX7486, we first confirmed the expression of Trk receptors in murine cancer cell lines, such as MC38, SA1N, MB49, 4T1, B16F10, and 3LL. These cancer cell lines have also been exposed to PLX7486 with a broad range of concentrations (0.1 to 10 μM). We have shown that PLX7486 has a direct cytotoxic effect with IC50 of 5-8μM on most of the cancer cell lines at a 72 hour time point. Futher, the exposure of MC38 to PLX7486 resulted in the expected effects of inhibiting downstream AKT pathway signaling. In addition, PLX7486 has also been shown to have cytotoxic direct effect on bone marrow-derived macrophages and murine macrophage cell line RAW264.7 with IC50<1μM, including inhibition of the MAPK pathway. In order to test whether PLX7486 is toxic to T-cells, pmel-1/Thy1.1+ T-cells were adoptively transferred into mice, which then received daily oral gavage with PLX7486 (20mg/kg) for 5 days. Interestingly, the amount of pmel-1/Thy1.1+ T-cells increased in the spleen (vehicle: 1.76±0.2%, PLX7486: 3.52±0.4%). We then tested the combination of PLX7486 and immunotherapy reagents such as anti-CTLA-4 and anti-PD-1 in vivo in MC38 and B16F10 tumor models. We have observed a superior antitumor effect in the combined therapy group compared with either single treatment group alone. In conclusion, our promising data derived from in vivo models combining Trk/CSF-1R blockade and immunotherapy supports the rationale for further testing of such combination in patients with cancer. This abstract is also being presented as Poster A06. Citation Format: Stephen Mok, Colm Duffy, Reginald Du, James P. Allison. Blocking Colony Stimulating Factor 1 Receptor (CSF-1R) and Tropomyosin Receptor Kinase (Trk) improves antitumor efficacy of immunotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr PR01.