Abstract 1085: Glioma-infiltrating myeloid derived suppressor cells inhibit anti-tumor T cell responses

MDSCs represent a population of immature myeloid cells at various stages of differentiation that have the potential to inhibit anti-tumor T cell immunity. We demonstrate the accumulation of MDSCs in GL26 and M7-induced glioma (GBM) bearing mice. Absolute numbers of Ly-6G+ (Gr-1high) MDSCs showed a 200 fold increase within the tumor microenvironment (TME) 28 days post-tumor implantation. The numbers of Ly-6C+ (Gr-1low) MDSCs also showed a similar trend within the TME. While this massive influx of MDSCs was noted within intracranial tumors, MDSC levels did not increase in the dLNs, spleen or bone marrow (BM) of intracranial tumor bearing mice. MDSC numbers were significantly elevated in the blood of GL26 and M7 intracranial tumor bearing mice at 28 days. While both Gr-1high and Gr-1low MDSCs isolated from the TME of GL26 intracranial tumor bearing mice inhibited antigen-specific T cell proliferation, Gr-1low MDSC were found to be more efficient. Gr-1high or Gr-1low MDSCs from the bone marrow of intracranial tumor bearing mice failed to suppress antigen-specific T cell proliferation suggesting that TME derived factors may activate MDSCs to exert their immune-suppressive properties. In vivo, depletion of Gr-1+ cells enhanced the median survival of GBM bearing mice. Furthermore, when combined with Ad-TK + Ad-FLT3L immune-gene therapy, Gr-1+ depletion significantly enhanced the frequency of tumor-specific T cells within the TME and spleen and increased IFN-γ production by splenic T cells. Our data therefore indicates that inhibiting the accumulation of MDSCs with in the GBM TME promotes the generation of robust anti-tumor immunity. Preliminary experiments to determine the mechanism of MDSC trafficking to the TME point towards the receptor CXCR2 and its ligand CXCL1. Microarray analysis of glioma cell lines showed elevated levels of CXCL1 mRNA. Additionally a culture of primary mixed glial cells also produced CXCL1 when stimulated with GBM cell lysates. SB225002, a CXCR2 inhibitor suppressed the migration of MDSCs towards GBM cells in an in vitro migration assay. Overall, our data suggests that strategies that inhibit MDSC recruitment to the GBM TME and/or block their activity could enhance the T cell mediated tumor clearance and provide survival benefit. Work supported by grants from NIH-NINDS Citation Format: Neha Kamran, Youping Li, Mariela Moreno-Ayala, Hikmat Assi, Marianela Candolfi, Marta Dzaman, Pedro Lowenstein, Maria Castro. Depletion of glioma infiltrating myeloid derived suppressor cells promotes anti-tumor T cell responses. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 457. doi:10.1158/1538-7445.AM2015-457

Carcinogen-associated oral cavity cancers are a heterogeneous group of aggressive cancers with a high recurrence rate after definitive treatment and a poor 5-year survival. The genetic alteration rate of these cancers tends to be high, and many oral cancers express immune checkpoint molecules in the tumor microenvironment with potential to respond to checkpoint blocking immunotherapy. Local immunosuppression mediated by both the tumor cells and other infiltrating immune cells are likely a major mechanism of resistance to immunotherapy. Here, we investigated the role of myeloid derived suppressor cells (MDSCs) in a highly aggressive but poorly immunogenic syngeneic model of carcinogen-induced oral cavity cancer (MOC2). Performing a time course analysis of immune infiltrates, MOC2 tumors demonstrated robust recruitment of CXCR2+CSFR1+CCR2− MDSCs that peaked at 15 days following tumor transplantation. This intratumoral accumulation of MDSCs preceded draining lymph node and splenic MDSC accumulation by 6 and 9 days respectively. The accumulation of MDSCs corresponded to a sharp decrease in the presence of tumor infiltrating CD8+ T-lymphocytes (TIL), CD4+ TIL, FoxP3+CD4+ Tregs and CD3−NK1.1+ NK cells. As CD8+ TIL numbers decreased, cell surface expression of the lymphocyte activation markers CD69, CD44 and ICOS decreased with the checkpoint molecule CTLA-4 increased. Phenotypically, the great majority of tumor-infiltrating MDSCs were granulocytic MDSCs (Ly6GhiLy6Cint), while few were monocytic MDSCs (Ly6GloLy6Chi). In a CFSE-based functional T-lymphocyte suppression assay, sorted peripheral and tumor-infiltrating MDSCs strongly suppressed T-lymphocyte proliferation at MDSC:T-lymphocyte ratios as low as 1:32. Hypothesizing that decreased tumor MDSCs would impact primary tumor growth, MOC2 tumors were allowed to engraft to 100mm3 and depletion of granulocytic MDSCs was performed with a rat anti-mouse Ly6G depleting antibody (clone RB6-8C5). This depletion resulted in a statistically significant delay in MOC2 primary tumor growth. Flow cytometric analysis of tumor tissues revealed that MDSC depletion was transient with rapid rebound of MDSC tumor infiltration within days of depleting antibody administration. This data highlights the critical role that MDSCs play in local immunosuppression and suggest that the syngeneic MOC model represents a powerful tool to study MDSC pathobiology. Functional inhibition or elimination of MDSCs from the tumor microenvironment represents an exciting adjuvant therapy that may enhance the response to checkpoint inhibition in patients with oral cavity cancer. Citation Format: Paul E. Clavijo, Ruth Davis, Zhong Chen, Carter Van Waes, Clint T. Allen. MDSC depletion delays primary tumor growth in syngeneic models of oral cavity cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1448.

Introduction: Pancreatic adenocarcinoma (PA) is a highly aggressive and chemo-resistant malignancy with a median survival of only 6 months. Myeloid derived suppressor cells (MDSC) have been implicated in the immune suppression and growth of several malignancies. MDSC are highly prevalent in the tumor microenvironment, and represent a potential therapeutic target. Here, we assess the tumor-promoting ability of MDSC in a model of PA and explore MDSC targeting strategies using ZA, a drug known to inhibit MDSC. Methods: C57BL/6 mice were inoculated in the right flank with 100,000 or 50,000 of the syngeneic PA line, Pan02. Tumor volume was determined by standard calculation using caliper measurements. Mice were treated with Gemcitabine 20mg/kg Qwk IP and/or ZA 1ug QOD SQ once tumors were palpable. Analysis for MDSC accumulation was determined in the tumor, spleen and bone marrow of mice at sacrifice using flow cytometry for standard markers of MDSC (CD45, CD11b, Gr1, Ly6G, and Ly6C). MDSC prevalence is expressed as a percentage of total leukocytes (CD45+). Statistical analysis was performed using non-parametric student's t-test. Results: MDSC are up regulated in the spleen and bone marrow of mice with flank injected Pan02 compared to non-tumor bearing mice. MDSC accumulate over time and increase in number until 4 weeks post-inoculation (CD45 + 11b+Gr1+, Bone Marrow: 26.06% 42.04%, spleen 1.07% 7.18%, p=0.0021). Treatment of tumor bearing mice with ZA decreases tumor associated MDSC (CD45+11b+Gr1+: 50.36 29.38; CD45+11b+Ly6G+: 49.45 30.47) and tumor volume (2.45 1.71 p=0.034). ZA also works in concert with Gemcitabine to decrease MDSC (Gr1 +: 50.36 39.38; Ly6G+: 49.45 37.28;) and tumor volume (2.45 1.02, p=0.0011). Conclusion: MDSC play an integral role in the development of PA. Depletion of MDSC by ZA results in significantly smaller tumors and can be added to the standard chemotherapeutic regimen (Gemcitabine) successfully. The addition of ZA to Gemcitabine represents an attractive and innovative regimen to improve the treatment of this deadly malignancy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-148.

Myeloid-derived suppressor cells (MDSC) accumulate in patients and animals with cancer where they mediate systemic immune suppression and obstruct immune-based cancer therapies. We have previously demonstrated that inflammation, which frequently accompanies tumor onset and progression, increases the rate of accumulation and the suppressive potency of MDSC. To determine how inflammation enhances MDSC levels and activity we used mass spectrometry to identify proteins produced by MDSC induced in highly inflammatory settings. Proteomic pathway analysis identified the Fas pathway and caspase network proteins, leading us to hypothesize that inflammation enhances MDSC accumulation by increasing MDSC resistance to Fas-mediated apoptosis. The MS findings were validated and extended by biological studies. Using activated caspase 3 and caspase 8 as indicators of apoptosis, flow cytometry, confocal microscopy, and Western blot analyses demonstrated that inflammation-induced MDSC treated with a Fas agonist contain lower levels of activated caspases, suggesting that inflammation enhances resistance to Fas-mediated apoptosis. Resistance to Fas-mediated apoptosis was confirmed by viability studies of MDSC treated with a Fas agonist. These results suggest that an inflammatory environment, which is frequently present in tumor-bearing individuals, protects MDSC against extrinsic-induced apoptosis resulting in MDSC with a longer in vivo half-life, and may explain why MDSC accumulate more rapidly and to higher levels in inflammatory settings.

Purpose: Myeloid derived suppressor cells (MDSCs) are a major component of the immunosuppressive environment responsible for immune tolerance in cancer. These cells facilitate carcinogenesis and tumor progression by impairing T and NK cells activation and contributing to regulation of angiogenesis. In current study, we evaluated whether accumulation of MDSCs occur in two murine prostate cancer models: a castrate resistant, syngenic transplantable tumor (CR Myc-Cap) and the Hi-myc transgenic tumor models. Tasquinimod is an anti-angiogenic agent tested in prostate cancer and has been found to bind to an inflammatory protein S100A9 and therefore have the potential to affect accumulation and function of MDSCs. We investigated whether tasquinimod affects the induction and function of MDSCs and facilitate vaccine therapy in these castrate resistant prostate cancer models. Experimental design: Splenocytes and blood cells were isolated from naïve and CR Myc-Cap tumor bearing mice. Blood samples were taken from Hi-myc transgenic mice and age-matched littermates at different ages. All these splenocytes and blood samples were subjected to staining with MDSCs markers, Gr1 and CD11b, and to flow cytometry analysis. CR Myc-Cap tumor bearing mice were treated with either vehicle, tasquinimod (1mg/kg), modified survivin vaccine peptide SurVaxM, or combination. Tumor growth, MDSCs accumulation and function upon treatments were monitored. Results: The peripheral MDSCs (in blood and spleen) significantly increased in tumor bearing mice. The MDSCs number increase correlated with disease progression in the transgenic model. Low doses of tasquinimod (1-5mg/kg) did not change the CD11b+Gr1high MDSCs number, but CD11b+Gr1high, CD11b+Gr1int, CD11b+Gr1− cells were differently accumulated in vehicle or tasquinimod treated mice. Tasquinimod in vivo treatment reduced peripheral CD11b+Gr1int, CD11b+Gr1− cells number. Low doses of tasquinimod have minimal effect on CD11b+Gr1high MDSCs function and did not enhance the SurVaxM vaccine therapy in CR Myc-Cap model.Conclusions: In summary, our study demonstrates MDSCs accumulation in two prostate cancer models and suggests that MDSCs may contribute to immune tolerance in prostate cancer. The study also suggests low dose tasquinimod affect maturation of MDSCs into other immunosuppressive cell types (CD11b+Gr1int, CD11b+Gr1−). Higher doses of tasquinimod (≥10mg/kg) are being tested for their potential to affect MDSCs accumulation and function in prostate cancer models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1551. doi:1538-7445.AM2012-1551

Increasing evidence indicates that myeloid-derived suppressor cells (MDSCs) negatively regulate immune responses during tumor progression, inflammation and infection. However, the underlying molecular mechanisms of their development and mobilization remain to be fully delineated. Kruppel-like factor KLF4 is a transcription factor that has an oncogenic function in breast cancer development, but its function in tumor microenvironment, a critical component for tumorigenesis, has not been examined. By using a spontaneously metastatic 4T1 breast cancer mouse model and an immunodeficient NOD/SCID mouse model, we demonstrated that KLF4 knockdown delayed tumor development and inhibited pulmonary metastasis, which was accompanied by decreased accumulation of MDSCs in bone marrow, spleens and primary tumors. Mechanistically, we found that KLF4 knockdown resulted in a significant decrease of circulating GM-CSF, an important cytokine for MDSC biology. Consistently, recombinant GM-CSF restored the frequency of MDSCs in purified bone marrow cells incubated with conditioned medium from KLF4 deficient cells. In addition, we identified CXCL5 as a critical mediator to enhance the expression and function of GM-CSF. Reduced CXCL5 expression by KLF4 knockdown in primary tumors and breast cancer cells was correlated with a decreased GM-CSF expression in our mouse models. Finally, we found that CXCL5/CXCR2 axis facilitated MDSC migration and that anti-GM-CSF antibodies neutralized CXCL5-induced accumulation of MDSCs. Taken together, our data suggest that KLF4 modulates maintenance of MDSCs in bone marrow by inducing GM-CSF production via CXCL5 and regulates recruitment of MDSCs into the primary tumors through the CXCL5/CXCR2 axis, both of which contribute to KLF4-mediated mammary tumor development. Citation Format: Fang Yu, Daping Fan, Walden Ai. Deficiency of Kruppel-like factor KLF4 in mammary tumor cells resulted in inhibited tumor growth and pulmonary metastasis accompanied by compromised recruitment of myeloid-derived suppressor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-323. doi:10.1158/1538-7445.AM2013-LB-323

Accumulation of myeloid-derived suppressor cells (MDSCs) is a hallmark of cancer. However, the underlying mechanism of MDSC accumulation in the tumor microenvironment (TME) remain incompletely understood. We report that MDSC accumulation is regulated by the TNFα-RIP1-mediated necroptosis. We determined that inhibition of DNMTs with Decitabine (DAC) abolished MDSC accumulation and increased activation of antigen-specific cytotoxic T lymphocytes (CTLs) in tumor-bearing mice. DAC-induced decrease of MDSC accumulation is correlated with increased IRF8 expression in MDSCs. However, DAC also abolished MDSC-like cell accumulation in IRF8 KO mice, indicating that DNA methylation does not regulate MDSC lineage differentiation but mediates MDSC accumulation at post differentiation stage. We determined that DAC decreased MDSC accumulation through increasing cell death and identified RIP1-dependent necroptosis as target of DNA methylation in MDSCs. Genome-wide DNA bisulfite sequencing revealed that the Tnf promoter is hypermethylated in tumor-induced MDSCs in vivo. Consequently, DAC dramatically increased TNFα level in MDSCs and neutralizing TNFα significantly decreased MDSC cell death. Furthermore, recombinant TNFα induced MDSC cell death in a does- and RIP1-dependent manner. IL6 which is expressed in MDSCs in tumor-bearing mice and human colorectal cancer patients. Our data shows that the autocrine IL6 activates the STAT3-DNMT axis to epigenetically silence the TNFα-RIP1 necroptosis pathway to sustain MDSC survival and accumulation in cancer. Targeting the TNFα-RIP1 necroptosis is potentially an effective approach to supress MDSCs to activate tumor-reactive CTLs in the TME.

Recent studies indicate that sunitinib, a tyrosine kinase inhibitor, could decease accumulation of myeloid-derived suppressor cells (MDSC) in tumors and also in peripheral blood. This study aims to investigate the behavior and dynamic profile of myeloid-derived suppressor cells in tumor tissues and peripheral blood of sunitinib-treated mice bearing prostate adenocarcinoma TRAMP-C1 tumors. To this end, 3×106 TRAMP-C1 tumor cells were inoculated at shank muscle of C57BL6/J mouse and sunitinib was administered by intraperitoneal injection of a daily dose of 20mg/kg into mice bearing 4 mm diameter tumor. Comparing to tumors in untreated group, sunitinib administration slightly delayed tumor growth delay for 2 days, but significantly decreased micro-vascular density and induced chronic hypoxia in tumors, resulting in the specific accumulation of CD11b+Gr-1+ MDSCs at the tumor necrotic region within CA-IX positive chronic hypoxic area. Flow cytometry was used to analyze the change of CD11b+ myeloid cells within tumor tissues and peripheral blood. Results showed that sunitinib treatment decreased the percentage of CD11b+Ly6G-LyC- tumor-associated macrophages (TAMs), but increased the percentage of CD11b+Ly6G-Ly6C+ monocytic MDSCs (M-MDSCs) while had no effect on CD11b+Ly6G+Ly6C+ neutrophilic MDSCs (N-MDSCs) within tumor tissues. In contrast, both the percentage of N-MDSCs and M-MDSC were expanded in peripheral blood of sunitinib-treated mice. Multiplex immunoassay showed significant increase of CCL2, CCL3, CCL5, CXCL5, IL-17a, GM-CSF, G-CSF and VEGF-A in tumor tissues, but only CXCL5, IL-á, IL-6 and G-CSF in the peripheral blood of sunitinib-treated mice were affected. In conclusion, this study shows that sunitinib has anti-vascular effect and could disrupt the recruitment of CD11b+Ly6G-Ly6C- TAMs into tumors. We also propose that sunitinib might exert different effects on tumor microenvironment and peripheral blood. Citation Format: Fang-Hsin Chen, Sheng-Yung Fu, Chun-Chieh Wang, Chi-Shiun Chiang, Ji-Hong Hong. Sunitinib has opposite roles to regulate the myeloid-derived suppressor cells in tumors and peripheral blood. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 759.

The abnormal differentiation of myeloid cells is one of the key features of altered immune response in cancer. Myeloid derived suppressor cells (MDSC) are a major component of immune suppressive network. MDSC accumulate in large numbers in peripheral lymphoid organs and inside tumors. Previous data indicated that the fate of MDSC depends on the localization of these cells. In contrast to peripheral lymphoid organs such as spleen, inside tumors, MDSC rapidly differentiate to tumor associated macrophages with potent immune suppressive activity. We have demonstrated that this phenomenon is mediated by dramatic down-regulation of STAT3 activity in MDSC at tumor site. In cancer patients, MDSC at tumor site also had substantially lower level of pSTAT3 than MDSC in peripheral blood. These results were unexpected, since high level of STAT3 activity is a hallmark of MDSC present in blood and lymphoid organs. The mice with constitutively active STAT3 had shown the dramatic expansion of MDSC and down-regulation of macrophage population in tumor microenvironment, emphasizing the crucial role of STAT3 in myeloid cell differentiation. Hypoxia is an intricate part of tumor microenvironment. We have found that hypoxia caused the down-regulation of STAT3 activity in MDSC. However, the effect of hypoxia on STAT3 activity was not observed in tumor cells. We further demonstrate that the up-regulation of CD45 tyrosine phosphatase activity in MDSC exposed to hypoxia in tumor site was responsible for down-regulation of STAT3 activity. Using KO model we have shown that MDSC devoid of CD45 failed to experience the effect of hypoxia on pSTAT3 down-regulation as well as their differentiation to macrophage at tumor site. Furthermore, the positive regulation of CD45 activity in hypoxia was mediated by the disruption of CD45 protein dimerization via sialylation. The treatment of sialidase abrogated the effect of hypoxia on STAT3 activity in MDSC and differentiation of MDSC to macrophages. This novel mechanism of regulation of myeloid cell differentiation by STAT3 may provide rationale for re-evaluation of therapeutic strategies in cancer. Citation Format: Vinit Kumar, Pingyan Cheng, Thomas Condamine, Dmitry Gabrilovich. Tumor microenvironment regulates the fate of myeloid cells by controlling STAT3 activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2343. doi:10.1158/1538-7445.AM2015-2343

Cancer immunotherapy aims to harness the power of the immune system to fight advanced stages of cancer. A main hindrance to cancer immunotherapy is tumor-induced immune suppression. Within the tumor microenvironment are a plethora of immune suppressive cells including Myeloid-Derived Suppressor Cells (MDSC). MDSC are present in nearly all types of cancer blocking T cell activation and shifting immunity towards a type 2 tumor-promoting phenotype through cross-talk with macrophages that enhances the secretion of pro-tumor molecules such as IL-10, and simultaneously drops IL-12 production by macrophages. MDSC accumulation is induced by pro-inflammatory cytokines including IL-1β, IL-6 and VEGF as well as alarmins such as S100A8/9. Because the alarmin HMGB1 is increased in many cancers, we are determining if HMGB1 drives MDSC. Inhibition of HMGB1 in vitro by chemical inhibitors Glycyrrhizin and Ethyl Pyruvate decreased cross-talk-induced IL-10 production by MDSC and IL-6 production by macrophages. Ethyl Pyruvate also reduced MDSC suppressive capacity in transgenic T cell suppression assays, suggesting that HMGB1 regulates multiple aspects of MDSC function. Treatment of BALB/c mice with 4T1 mammary carcinoma with HMGB1 inhibitors Glycyrrhizin and Ethyl Pyruvate reduced lung metastases and did not impact primary tumor progression. Treatment of C57BL/6 mice with MC38 colon carcinoma with an HMGB1 neutralizing antibody (2G7) reduced MDSC levels in the blood, spleen, and tumor. HMGB1 consists of two domains, the pro-inflammatory B box domain and an anti-inflammatory A box domain. Treatment of C57BL/6 mice with MC38 tumor with A box significantly reduced primary tumor growth and decreased MDSC levels in the blood. These findings indicate that full-length HMGB1 drives inflammation and tumor progression, while the anti-inflammatory A box domain counteracts full-length HMGB1 and reduces tumor growth and MDSC. These results suggest that HMGB1 drives MDSC accumulation and tumor progression and that A box can be used to target MDSC-mediated tumor-induced immune suppression. (Research supported by NIH RO1CA115880 and RO1CA84232) Citation Format: Katherine Parker, Suzanne Rosenberg, Pratima Sinha, Huan Yang, Kevin Tracey, Jianhua Li. Inhibition of HMGB1 delays tumor progression, reduces MDSC-mediated immune suppression, and diminishes MDSC-macrophage cross-talk interaction. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 461. doi:10.1158/1538-7445.AM2013-461

β2-AR Signaling Enhances MDSC Survival through Metabolic Reprograming and Impairs Therapeutic Efficacy in Hematologic Malignancies

The abnormal differentiation of myeloid cells is the key element of altered immune response in cancer. Myeloid derived suppressor cells (MDSC) are a major component of immune suppressive network. MDSC accumulate in large numbers in peripheral lymphoid organs and inside tumors. Recent data indicated that the fate of MDSC depends on the localization of these cells. In contrast to peripheral lymphoid organs, inside tumors, MDSC rapidly differentiate to tumor associated macrophages with potent immune suppressive activity. Hypoxia is intricate part of tumor microenvironment. We have found that hypoxia plays a major role in regulation of MDSC differentiation to macrophages in tumor site. Hypoxia caused dramatic reduction in phosphorylated STAT3 in MDSC. These results were unexpected, since high level of STAT3 activity is a hallmark of MDSC present in blood and lymphoid organs. In our experiments spleen MDSC in tumor-bearing mice had much higher level of STAT3 phosphorylation than their counterparts in tumor site from the same mice. In cancer patients MDSC in tumor site also had substantially lower level of pSTAT3 than MDSC in peripheral blood. In vitro culture of spleen MDSC in hypoxia led to the suppression of STAT3 activation. Furthermore, mice with constitutively active STAT3 had shown the dramatic expansion of MDSC and down-regulation of macrophage population in tumor microenvironment, suggesting that STAT3 is a downstream target for hypoxia driven myeloid cells differentiation in cancer. We found that CD45 protein tyrosine phosphatase was a molecule that regulated STAT3 activation in hypoxia. The activity but not expression of CD45 phosphatase was up-regulated in tumor MDSC as compared to MDSC in spleens. The exposure of spleen MDSC to hypoxia led to the significant increase in CD45 activity. The siRNA mediated silencing of CD45 abrogated the effect of hypoxia on the down-regulation of STAT3 activity in MDSC. This suggests that hypoxia driven up-regulation of CD45 phosphatase activity suppresses STAT3 activation, which in turn plays a key role in differentiation of myeloid cells in tumor site. These unexpected findings may provide rationale for re-evaluation of therapeutic strategies targeting myeloid cells in cancer. Citation Format: Vinit Kumar. Hypoxia determines the fate of myeloid cell differentiation by controlling STAT3 activation in tumor site. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3616. doi:10.1158/1538-7445.AM2014-3616

Elevated levels of circulating monocytic myeloid derived suppressor cells in splenectomised β-thalassaemia/HbE patients.

Introduction: The phosphatidylserine (PS)-targeting antibody, bavituximab, is currently in Phase IIb clinical trials in patients with lung cancer. Bavituximab, and its murine counterpart, 2aG4, induce the attack of monocytes and macrophages on PS-expressing tumor vascular endothelium and tumor cells and inhibit the immunosuppressive effects of PS in the tumor microenvironment. Myeloid-derived suppressor cells (MDSC) are one of the major cells responsible for the immunosuppressed state in tumors. In this study, we tested the influence of 2aG4 on the differentiation of MDSC into M1-like tumor associated macrophages (TAMs). Methods: MDSC were isolated from 4T1 tumor bearing mice with anti-Gr1-coated magnetic beads. The purified MDSC were then cultured for 5 days in the presence of 2aG4 or control C44 antibody. For in vivo studies, PC3 tumor-bearing mice were treated with 2aG4 for 30 days and MDSC and TAMs in tumors and spleens were analyzed by FACS and immunohistochemistry. Results: 2aG4 treatment of purified MDSC switched their cytokine production from an immunosuppressive IL-10-dominated response to a pro-inflammatory IL-12- and TNFα -dominated response. The percentage of Gr1+ cells decreased to 8% in the 2aG4-treated cultures (P<0.0001) but only to 50 – 57% in the PBS and C44-treated cultures. Treatment with 2aG4 induced the differentiation of MDSC into M1-like macrophages that expressed lower CD206 and produced more NO than control cultures. Treatment of mice bearing PC3 prostate tumors with 2aG4 decreased the percentage of MDSC from 7% to 4% (P<0.001) in the tumors and from 28% to 20% (P<0.001) in the spleens. The antibody treatment also increased the ratio of M1 to M2 TAMs in PC3 tumors from 0.7% to 1.4% (P<0.001). Conclusion: Taken together, our results suggest that 2aG4 causes the differentiation of MDSCs into macrophages having an M1-like phenotype. 2aG4 treatment decreased IL-10 production and increased IL-12 and TNFα-production. These results suggest that 2aG4 treatment reactivates innate immunity in tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3651. doi:10.1158/1538-7445.AM2011-3651

Introduction: Recent evidence suggests that tumor growth and metastases are sustained by myeloid-derived suppressor cells (MDSC), which over-express arginase-1 (ARG) and cause T-cell suppression. We hypothesize that MDSC impair tumor cell radiosensitivity through ARG-mediated depletion of L-arginine, an essential substrate for the biosynthesis of the endogenous radiosensitizer nitric oxide (NO). Aim: We explored whether MDSC and ARG can be used as biomarkers for tumor growth in experimental mouse models and in the clinical setting of colorectal cancer (CRC). We further examined whether low L-arginine levels are associated with an impaired generation of NO by inducible NO synthase (iNOS) and a reduced radiosensitivity of hypoxic tumor cells. Materials & methods: MDSC (CD11b+Gr-1+) and ARG levels in mouse colon CT26 and mammary 4T1 models were analyzed by flow cytometry (FCM) and RT-PCR. Mice were inoculated I.M. with either CT26 or 4T1 carcinoma cells. After 17 days, MDSC were quantified in the spleen by FCM. In addition, ARG levels were analyzed by RT-PCR and FCM. Similar analyses were performed on human blood MDSC (CD14+HLA-DRlow) derived from CRC patients. To model the hypoxic tumor microenvironment, mouse splenocytes were cultured in 1% oxygen. Activation of iNOS was induced by LPS/IFN-γ and analysed by RT-PCR, Western blot and Griess assay. Radiosensitivity was analysed in a model of metabolic hypoxia by colony formation assay Results: The growth of both CT26 and 4T1 tumors in BALB/c mice was associated with an accumulation of MDSC in the spleen from 2-4% (in naive mice) to 8 and 35% respectively. Freshly isolated splenocytes and MDSC showed low ARG levels. Contrasting, hypoxia-conditioned splenocytes from tumor-bearing mice revealed a drastic transcriptional activation of ARG (up to 525.000-fold), and ARG+ MDSC. On the other hand splenocytes from tumor-free mice did not display ARG activation in hypoxic conditions. The iNOS/NO-mediated radiosensitization of mammary EMT6 tumor cells was strictly dependent on L-arginine levels, with a marginal effect of 1.2-fold at 10-30 μM while approaching a 2.4-fold effect at a physiological concentration of 125 μM. MDSC levels in CRC patients were increased, as compared with healthy donors (8-12% versus 4-5%). Patients with locally advanced CRC had 16-fold higher ARG expression in blood monocytes, as compared to healthy donors. 4 weeks after pre-operative radiotherapy the ARG levels decreased by 2-fold. Conclusions: Although tumor progression is clearly associated with the expansion of MDSC, their hypoxic conditioning appears to be crucial to uncover ARG as a tumor biomarker in vivo. iNOS/NO mediated radiosensitisation of hypoxic tumor cells was drastically impaired at low L-arginine levels which could be depleted by ARG+ MDSC. In a clinical setting, we consider ARG a potential biomarker in locally advanced rectal cancer. Citation Format: Wim Leonard, Inès Dufait, Heng Jiang, Femke Steenbeke, Marieke Vermeersch, Kalun Law, Guy Storme, Joeri Aerts, Valeri Verovski, Mark De Ridder. Myeloid-derived suppressor cells as a biomarker of tumor growth and radiosensitivity: Role of hypoxia-inducible arginase-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4986. doi:10.1158/1538-7445.AM2013-4986