Targeting Myeloid-derived Suppressor Cells Research Articles

Targeting Myeloid-derived Suppressor Cells and Programmed Death Ligand 1 Confers Therapeutic Advantage of Ablative Hypofractionated Radiation Therapy Compared With Conventional Fractionated Radiation Therapy

Ablative hypofractionated radiation therapy (AHFRT) presents a therapeutic advantage compared with conventional fractionated radiation therapy (CFRT) for primary and oligometastatic cancers. However, the underlying mechanisms remain largely unknown. In the present study, we compared the immune alterations in response to AHFRT versus CFRT and examined the significance of immune regulations contributing to the efficacy of AHFRT. We established subcutaneous tumors using syngeneic lung cancer and melanoma cells in both immunocompetent and immunocompromised mice and treated them with AHFRT and CFRT under the same biologically equivalent dose. Compared with CFRT, AHFRT significantly inhibited tumor growth in immunocompetent, but not immunocompromised, mice. On the cellular level, AHFRT reduced the recruitment of myeloid-derived suppressor cells (MDSCs) into tumors and decreased the expression of programmed death-ligand 1 (PD-L1) on those cells, which unlashed the cytotoxicity of CD8+ T cells. Through the downregulation of vascular endothelial growth factor (VEGF), AHFRT inhibited VEGF/VEGF receptor signaling, which was essential for MDSC recruitment. When combined with anti-PD-L1 antibody, AHFRT presented with greater efficacy in controlling tumor growth and improving mouse survival. By altering immune regulation, AHFRT, but not CFRT, significantly delayed the growth of secondary tumors implanted outside the irradiation field. Targeting MDSC recruitment and enhancing antitumor immunity are crucial for the therapeutic efficacy of AHFRT. When combined with anti-PD-L1 immunotherapy, AHFRT was more potent for cancer treatment.

Targeting Myeloid-Derived Suppressor Cells to Bypass Tumor-Induced Immunosuppression.

The immune system has many sophisticated mechanisms to balance an extensive immune response. Distinct immunosuppressive cells could protect from excessive tissue damage and autoimmune disorders. Tumor cells take an advantage of those immunosuppressive mechanisms and establish a strongly immunosuppressive tumor microenvironment (TME), which inhibits antitumor immune responses, supporting the disease progression. Myeloid-derived suppressor cells (MDSC) play a crucial role in this immunosuppressive TME. Those cells represent a heterogeneous population of immature myeloid cells with a strong immunosuppressive potential. They inhibit an antitumor reactivity of T cells and NK cells. Furthermore, they promote angiogenesis, establish pre-metastatic niches, and recruit other immunosuppressive cells such as regulatory T cells. Accumulating evidences demonstrated that the enrichment and activation of MDSC correlated with tumor progression, recurrence, and negative clinical outcome. In the last few years, various preclinical studies and clinical trials targeting MDSC showed promising results. In this review, we discuss different therapeutic approaches on MDSC targeting to overcome immunosuppressive TME and enhance the efficiency of current tumor immunotherapies.

Abstract BS1-2: Myeloid-derived suppressor cells: many facets of regulation of tumor progression

Abstract Tumor associated macrophages (TAM), myeloid derived suppressor cells (MDSC), represented by PMN-MDSC and M-MDSC, and dendritic cells (DC) are major components of tumor microenvironment playing critical role in tumor progression. There are now compelling evidence linking MDSC with negative clinical outcomes in many types of cancer. Therefore, these cells are attractive therapeutic targets. Recently, several therapeutic strategies have been developed that target MDSC in mouse animal models and in first clinical trials. Recent findings of selective targeting of MDSC in cancer patients using agonistic TRAIL receptor DR5 antibody will be discussed. In 16 patients with advanced solid tumors this antibody demonstrated selective depletion of MDSC without affecting populations of neutrophils, monocytes, or lymphocytes. Another therapeutic strategy involves histone deacetilase inhibitor – entinostat. Recent study in tumor-bearing mice revealed that entinostat slightly increased expansion and accumulation of granulocytic cells in tumors. However, in contrast to untreated mice, these cells lacked suppressive activity. This effect was associated with potent antitumor activity of combination of entinostat with check-point inhibitors. By studying the effect of CSF1R inhibitor in tumor-bearing mice we identified the complex network regulating different groups of myeloid cells in tumor microenvironment. This network involves tumor cells and cancer associated fibroblast and protect tumors from loss of individual component of myeloid cell compartment. We determined that this network is operational not only in mice but also in cancer patients. Targeting of this network dramatically enhance the effect of cancer therapy. Citation Format: Gabrilovich DI. Myeloid-derived suppressor cells: many facets of regulation of tumor progression [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr BS1-2.

Cardioprotective Role of Myeloid-Derived Suppressor Cells in Heart Failure.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that expand in cancer, inflammation, and infection and negatively regulate inflammation and the immune response. Heart failure (HF) is a complex clinical syndrome wherein inflammation induction and incomplete resolution can potentially contribute to HF development and progression. However, the role of MDSCs in HF remains unclear. The percentage of MDSCs in patients with HF and in mice with pressure overload-induced HF using isoproterenol infusion or transverse aortic constriction (TAC) was detected by flow cytometry. The effects of MDSCs on isoproterenol- or TAC-induced HF were observed on depleting MDSCs with 5-fluorouracil (50 mg/kg) or gemcitabine (120 mg/kg), transferring purified MDSCs, or enhancing endogenous MDSCs with rapamycin (2 mg·kg-1·d-1). Hypertrophic markers and inflammatory factors were detected by ELISA, real-time polymerase chain reaction, or Western blot. Cardiac functions were determined by echocardiography and hemodynamic analysis. The percentage of human leukocyte antigen-D-related (HLA-DR)-CD33+CD11b+ MDSCs in the blood of patients with HF was significantly increased and positively correlated with disease severity and increased plasma levels of cytokines, including interleukin-6, interleukin-10, and transforming growth factor-β. Furthermore, MDSCs derived from patients with HF inhibited T-cell proliferation and interferon-γ secretion. Similar results were observed in TAC- and isoproterenol-induced HF in mice. Pharmaceutical depletion of MDSCs significantly exacerbated isoproterenol- and TAC-induced pathological cardiac remodeling and inflammation, whereas adoptive transfer of MDSCs prominently rescued isoproterenol- and TAC-induced HF. Consistently, administration of rapamycin significantly increased endogenous MDSCs by suppressing their differentiation and improved isoproterenol- and TAC-induced HF, but MDSC depletion mostly blocked beneficial rapamycin-mediated effects. Mechanistically, MDSC-secreted molecules suppressed isoproterenol-induced hypertrophy and proinflammatory gene expression in cardiomyocytes in a coculture system. Neutralization of interleukin-10 blunted both monocytic MDSC- and granulocytic MDSC-mediated anti-inflammatory and antihypertrophic effects, but treatment with a nitric oxide inhibitor only partially blocked the antihypertrophic effect of monocytic MDSCs. Our findings revealed a cardioprotective role of MDSCs in HF by their antihypertrophic effects on cardiomyocytes and anti-inflammatory effects through interleukin-10 and nitric oxide. Pharmacological targeting of MDSCs by rapamycin constitutes a promising therapeutic strategy for HF.

Immunosuppressive Role of Myeloid-Derived Suppressor Cells and Therapeutic Targeting in Lung Cancer.

Lung cancer is the leading cause of cancer death worldwide due to its late diagnosis and poor outcome. Immunotherapy is becoming more and more encouraging and promising in lung cancer therapy. Myeloid-derived suppressor cells (MDSCs) are the main tumor suppressor factors, and the treatment strategy of targeting MDSCs is gradually emerging. In this review, we summarize what is currently known about the role of MDSCs in lung cancer. In view of the emerging importance of MDSCs in lung cancer, the treatment of targeting MDSCs will be useful to the control of the development and progression of lung cancer. However, the occurrence, metastasis, and survival of cancer is the result of multiple factors and multiple mechanisms, so combined treatments using different strategies will become the major therapy method for lung cancer in the future.

Abstract CT095: The selective targeting of myeloid-derived suppressor cells in cancer patients using an agonistic TRAIL-R2 antibody

Abstract The goal of this current study was to clinically test the hypothesis that by targeting TRAIL-R2, myeloid-derived suppressor cells (MDSCs) can be selectively eliminated. MDSC) are one of the major contributors to immune suppression in cancer. We previously demonstrated in a preclinical study that MDSCs are sensitive to a TRAIL receptor 2 (TRAIL-R2) agonist. The TRAIL-R2 agonistic antibody (DS-8273a; provided by Daiichi Sankyo, Inc.) was tested in 16 patients with advanced solid cancers or lymphomas enrolled in a phase 1 trial. The antibody (24 mg/kg) was administered IV once every 3 weeks till disease progression, unacceptable toxicities, or withdrawal of consent. The safety and the presence of various populations of myeloid and lymphoid cells in peripheral blood and tumor tissues were evaluated using flow cytometry and immunohistochemistry. Overall, the treatment was well tolerated with only mild to moderate adverse events attributable to the study drug. Furthermore, treatment with DS-8273a resulted in reduction of the elevated numbers of MDSC in the peripheral blood of most patients to the levels observed in healthy volunteers. However, in several patients, MDSC rebounded back to the pre-treatment level by day 42. In contrast, DS-8273a did not affect the number of neutrophils, monocytes, and other populations of myeloid and lymphoid cells with decreases in MDSC levels inversely correlating with the length of progression-free survival. In tumors, DS-8273a treatment resulted in a decrease of MDSC in 50% of the patients who were able to provide pre- and on-treatment biopsies. In conclusion, targeting TRAIL-R2 using DS-8273a resulted in a temporary elimination of MDSCs without affecting mature myeloid or lymphoid cells, and these data support further use of this antibody in combination with current immmunotherapies of cancer. Citation Format: George A. Dominguez, Thomas Condamine, Sridevi Mony, Ayumi Hashimoto, Fang Wang, Qin Liu, Andres Forero, Johanna C. Bendell, Robert Witt, Neil Hockstein, Prasanna Kumar, Dmitry I. Gabrilovich. The selective targeting of myeloid-derived suppressor cells in cancer patients using an agonistic TRAIL-R2 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT095. doi:10.1158/1538-7445.AM2017-CT095

Tumor Microenvironment and Checkpoint Molecules in Primary Cutaneous Diffuse Large B-Cell Lymphoma-New Therapeutic Targets.

Programmed death ligand 1 (PD-L1) is expressed by 20% to 57% of systemic diffuse large B cell lymphomas (DLBCLs). PD-L1 expression in primary cutaneous DLBCL (pcDLBCL) has not been studied so far. Sixteen paraffin-embedded tissue samples of pcDLBCL (13 leg type [LT], 3 others [OT]) were investigated for PD-1, PD-L1, and CD33 expression and the cellular composition of the tumor microenvironment, focusing on myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages. Membrane-bound PD-L1 expression by the tumor cells was observed in all samples, albeit to a variable extent (19.9%). As expected, most DLBCL-LT (10 cases) were classified as activated B cell like type, with a higher PD-L1 score (21.9%) compared with that of the germinal center B cell like type (7.7%). The surrounding infiltrate consisted predominately of CD163(+) M2 rather than CD68(+) macrophages (CD68:CD163=1:4 to 6). Moreover, a considerable proportion of CD33(+) MDSCs with PD-L1 coexpression was admixed. Tumor cells expressed CD33 to variable degrees (2% to 60%). The number of MDSCs or M2 macrophages did not correlate with pcDLBCL subtypes LT or OT. T cells were only a minor component of the tumor microenvironment. We propose that PD-L1(+) tumor cells and PD-L1(+) MDSCs shield the tumor against PD-1(+) tumor-infiltrating lymphocytes, consequently leading to inhibition and diminution of tumor-infiltrating lymphocytes. Moreover, we found a polarization to M2 macrophages, which may contribute to the poor prognosis of DLBCL patients. Thus, targeting of tumor cells and MDSCs using anti-PD-1/anti-PD-L1 or anti-CD33 antibodies might be a worthwhile new approach to treat this aggressive form of cutaneous B-cell lymphoma.

Targeting myeloid derived suppressor cells with all-trans retinoic acid is highly time-dependent in therapeutic tumor vaccination

ABSTRACTTumor immune escape is a critical problem which frequently accounts for the failure of therapeutic tumor vaccines. Among the most potent suppressors of tumor immunity are myeloid derived suppressor cells (MDSCs). MDSCs can be targeted by all-trans-retinoic-acid (atRA), which reduced their numbers and increased response rates in several vaccination studies. However, not much is known about the optimal administration interval between atRA and the vaccine as well as about its mode of action.Here we demonstrate in 2 different murine tumor models that mice unresponsive to a therapeutic vaccine harbored higher MDSC numbers than did responders. Application of atRA overcame MDSC-mediated immunosuppression and restored tumor control. Importantly, atRA was protective only when administered 3 d after vaccination (delayed treatment), whereas simultaneous administration even decreased the anti-tumor immune response and reduced survival.When analyzing the underlying mechanisms, we found that delayed, but not simultaneous atRA treatment with vaccination abrogated the suppressive capacity in monocytic MDSCs and instead caused them to upregulate MHC-class-II. Consistently, MDSCs from patients with colorectal carcinoma also failed to upregulate HLA-DR after ex vivo treatment with TLR-ligation.Overall, we demonstrate that atRA can convert non-responders to responders to vaccination by suppressing MDSCs function and not only by reducing their number. Moreover, we identify a novel, strictly time-dependent mode of action of atRA to be considered during immunotherapeutic protocols in the future.

Targeting myeloid derived suppressor cells: Phase 0/1 trial of low dose capecitabine + bevacizumab in patients with recurrent glioblastoma.

e13507 Background: Glioblastoma (GBM) creates an immunosuppressive environment that allows tumor growth. Myeloid derived suppressor cells (MDSCs), a heterogeneous class of immunosuppressive cells, mediate immune suppression in GBMs. MDSCs are up-regulated in the blood of patients with GBM and multiple other tumor types. We have developed a novel strategy to target GBM immunosuppression using low dose 5-fluorouracil (5-FU) that, targets immune cells and does not depend on blood brain or blood tumor barrier penetration. Marked MDSC depletion occurs at 5-FU doses in mice equivalent to < 10% of the normal human dosing. Goal: proof of concept that MDSC suppression in feasible in GBM pts with low-dose capecitabine [cap], an oral 5-FU analogue). Methods: Eligibility: Recurrent GBM in need of surgical resection; no prior cap or bevacizumab (bev). Cohorts of 3-6 patients receive low-dose cap 150 mg/m2/d (dose level [DL] 1) for 7 days before surgery. After surgery, patients resume cap for one cycle after which bev is added. Blood MDSC, immune cell levels, and relevant secreted factors are measured at baseline; pre- and post-op; and after the addition of bev. Tumors are assayed for MDSCs and glioma stem-like cells (GSCs). Primary endpoint: MDSC and T-Regulatory cell reduction after cap. Secondary endpoints: Tumor concentrations of MDSCs, GSCs, and T-reg cells; safety; and PFS6. Results: Three of the 4 patients enrolled have data available. All patients received 5 days of pre-op cap at DL 1 with MDSC reductions of 20, 26, and 79% from baseline. The first patient reached a reduction of 93% (measured 2 days after pre-op course) whereas the other 2 experienced return to baseline. The regulatory T cells (T-reg) also fell approximately 15, 20, and 50%, respectively. CD8 concentrations appeared to rise at the time of MDSC and T-reg reduction. No patient experienced grade 3 or higher toxicity. Conclusions: Low dose capecitabine appears to reduce MDSC concentrations with minimal toxicity. During this course T-regs also fell and CD8 concentrations rose. Dose escalation continues. (NCT02669173) (Supported by Musella Foundation, Blast GBM, Sontag Foundation, Velosano, Mylan Pharmaceuticals) Clinical trial information: NCT02669173.

Glycolysis regulates the expansion of myeloid-derived suppressor cells in tumor-bearing hosts through prevention of ROS-mediated apoptosis

Immunotherapy aiming to rescue or boost antitumor immunity is an emerging strategy for treatment of cancers. The efficacy of immunotherapy is strongly controlled by the immunological milieu of cancer patients. Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature myeloid cell populations with immunosuppressive functions accumulating in individuals during tumor progression. The signaling mechanisms of MDSC activation have been well studied. However, there is little known about the metabolic status of MDSCs and the physiological role of their metabolic reprogramming. In this study, we discovered that myeloid cells upregulated their glycolytic genes when encountered with tumor-derived factors. MDSCs exhibited higher glycolytic rate than their normal cell compartment did, which contributed to the accumulation of the MDSCs in tumor-bearing hosts. Upregulation of glycolysis prevented excess reactive oxygen species (ROS) production by MDSCs, which protected MDSCs from apoptosis. Most importantly, we identified the glycolytic metabolite, phosphoenolpyruvate (PEP), as a vital antioxidant agent able to prevent excess ROS production and therefore contributed to the survival of MDSCs. These findings suggest that glycolytic metabolites have important roles in the modulation of fitness of MDSCs and could be potential targets for anti-MDSC strategy. Targeting MDSCs with analogs of specific glycolytic metabolites, for example, 2-phosphoglycerate or PEP may diminish the accumulation of MDSCs and reverse the immunosuppressive milieu in tumor-bearing individuals.

In Brief: Myeloid-derived suppressor cells in cancer.

The role of myeloid‐derived suppressor cells (MDSCs) in cancer development has become clear over recent years, and MDSC targeting is an emerging opportunity for enhancing the effectiveness of current anticancer therapies. As MDSCs are not only able to limit anti‐tumour T‐cell responses, but also to promote tumour angiogenesis and invasion, their monitoring has prognostic and predictive value. Herein, we review the key features of MDSCs in cancer promotion. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Effects of in vitro ATRA treatment on human MDSC expansion and function.

125 Background: Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of immature immunosuppressive myeloid cells that are expanded in tumor bearing hosts. Melanoma patients with high frequencies of MDSCs have decreased overall survival and an increased risk of death and disease progression, as well as impaired responses to ipilimumab. Targeting MDSCs to decease their frequency or suppressive activity may provide an effective means to improve the efficacy of anti-cancer therapies. All-trans retinoic acid (ATRA) is a vitamin A derivative currently used to treat APL that may target MDSCs. ATRA differentiates immature myeloid cells into macrophages, dendritic cells, or granulocytes. ATRA has been tested as an MDSC targeting agent in two completed caner clinical trials and is being investigated in several other open and completed trials. Methods: We purified myeloid cells from LRS chambers collected from normal donors. The purified cells were treated with GM-CSF+IL-6, GM-CSF+IL-4, or melanoma conditioned media with or without ATRA. After 5 days of incubation, PCR was used to determine the expression of immunoregulatory genes. Additionally, we used a MLR to determine the effect of ATRA on MDSC’s T cell suppressive function. Results: Here we report that in vitro ATRA treatment decreases the expression of ARG1, NOX1, INOS, and PD-L1. Further, we report that ATRA treatment improved T cell proliferation. Conclusions: Controlling MDSC suppressive function and accumulation may provide a mechanism to improve the efficacy of many current cancer therapies. Here we show that ATRA reduces the expression of the immunosuppressive genes ARG1, NOX1, INOS, and PD-L1 in MDSCs. Additionally, we show that treatment of MDSCs with ATRA decreases MDSC’s ability to suppress T cell proliferation. These results indicate that ATRA may improve the efficacy of conventional and immnunotherapeutic treatments.

Targeting the glucose metabolism of monocytic myeloid-derived suppressor cells to stimulate cancer immunity.

126 Background: Myeloid derived suppressor cells (MDSCs) inhibit the expansion of tumor antigen-specific effector CD8+ T cells via different mechanisms including increased expression of arginase, transforming growth factor – β (TGF – β) and indoleamine 2,3-dioxygenase (IDO). Recently, MDSCs were found to over-express hypoxia inducible factor 1 alpha (HIF-1α) which is required for their differentiation. An essential transcriptional target of HIF-1α is 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) which synthesizes fructose 2,6-bisphosphate, an allosteric stimulator of glycolysis and of proliferation via stimulation of cyclin dependent kinase-1 (CDK1). We hypothesized that MDSCs might over-express PFKFB3 which in turn might be required for their function as T cell suppressors. Methods: We used monocytic MDSCs (M-MDSCs) induced by co-culture with A375 melanoma cells, M-MDSCs from metastatic melanoma patients, murine bone marrow MDSCs and splenic M-MDSCs from B16 F10 tumor bearing mice for our studies. T cell suppression assays were performed to analyze M-MDSC suppression and reversal following PFKFB3 blockade. Results: We found that M-MDSCs have increased PFKFB3 expression. We also found that PFK-158 administration in B16 (wild-type) melanoma-bearing mice results in a marked reduction in MDSCs and a simultaneous increase in CD8+ T cell infiltration in the tumors. We analyzed three advanced cancer patients for circulating MDSCs before and after PFK-158 administration as part of a multi-center phase 1 clinical trial. And, we found that the MDSCs were markedly reduced in each patient. In addition, we have generated data for MDSC suppressive activity following in vitro treatment with PFK-158 showing reversal of suppressive activity. Conclusions: Taken together, these data indicate that selective inhibition of PFKFB3 may be a novel approach to target MDSCs and combinations of PFKFB3 inhibitors with immunotherapies may be a rational strategy to promote durable immune-mediated remissions in cancer patients.

Targeting Myeloid-Derived Suppressor Cells in Cancer.

Myeloid derived suppressor cells (MDSC) represent only a minor fraction of circulating blood cells but play an important role in tumor formation and progression. They are a heterogeneous group of cells that influence the tumor microenvironment by depletion of amino acids, oxidative stress, decreased trafficking of antitumor effector cells, and increased regulatory T and regulatory dendritic cell responses. Investigational treatment strategies targeting MDSCs have attempted to inhibit MDSC development and expansion (stem cell factor blockade, modulate of cell signaling, and target MDSC migration and recruitment), inhibit MDSC function (nitric oxide inhibition and reactive oxygen and nitrogen species inhibition), differentiate MDSCs into more mature cells (Vitamins A and D, all-trans retinoic acid, interleukin-2, toll-like receptor 9 inhibitors, taxanes, beta-glucan particles, tumor-derived exosome inhibition, and very small size proteoliposomes), and destroy MDSCs (cytotoxic agents, ephrin A2 degradation, anti-interleukin 13, and histamine blockers). To date, there are no Food and Drug Administration approved therapies selectively targeting MDSCs, but such therapies are likely to be implemented in the future, due to the key role of MDSCs in antitumor immunity.

Abstract A135: Novel immunotherapy for intractable pancreatic cancer focusing on the myeloid derived suppressor cells in the tumor microenvironment

Abstract Pancreatic cancer is one of malignant diseases with poor prognosis. Despite the recent progress for preoperative neoadjuvant chemoradiotherapy, the long term prognosis is still poor to make forward new treatment strategy. We have recently reported novel mechanism of anticancer reagents to impact patients' prognosis improving tumor microenvironment other than its direct cytotoxic effects (Ann Surg Oncol 2013, Cancer Sci 2014). The underlying mechanism include inhibition of HLA class I down regulation and Treg infiltration, increase of CD4 and CD8 T cell infiltration (Int J Clin Oncol 2015, Cancer Res 2015). Here we report that GM-CSF production was significantly enhanced in various PDAC cell lines after treatment with chemotherapy including gemcitabine or 5-FU, which induced the differentiation of CD14 positive monocytes into myeloid-derived suppressor cells (MDSC). This differentiation was promoted through MAPK signal pathway or NFkB pathway. Furthermore, blockade of GM-CSF with monoclonal antibodies helped to restore T-cell proliferation when co-cultured with monocytes stimulated with tumor supernatants. GM-CSF expression was also observed in primary tumors and correlated with poor prognosis in 68 PDAC patients analyzed by immunohistochemistry. We expect that postoperative prognosis of pancreatic cancer would be more improved by multidisciplinary strategy targeting MDSC and GM-CSF followed by curative surgical intervention. Citation Format: Takahiro Tsuchikawa, Shintaro Takeuchi, Toru Nakamura, Mizuna Takahashi, kazuho Inoko, Toshihiro Kushibiki, Koji Hontani, Masato Ono, Shota Kuwabara, Toshiaki Shichinohe, Satoshi Hirano. Novel immunotherapy for intractable pancreatic cancer focusing on the myeloid derived suppressor cells in the tumor microenvironment [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A135.

Intratumoral CD4+ cell depletion sensitizes poorly immunogenic melanomas to immunotherapy with an OX40 agonist

OX40 which belongs to the TNF receptor superfamily and is expressed by activated T cells, have potent antitumor activities in preclinical models. However, previous studies have shown that the antitumor effects of OX40 agonists depend on the immunogenicity of the tumor and that poorly immunogenic tumors such as B16F10 melanomas do not respond to OX40 agonist treatment. In this study, we have shown that intratumoral administration of an anti-CD4 mAb (for CD4+ cell depletion) sensitized poorly immunogenic B16F10 melanomas to immunotherapy with an OX40 agonist. CD4+ cell depletion markedly enhanced the infiltrations of both CD8+ T cells and natural killer (NK) cells into tumor tissues. Cytokines with antitumor activities, such as IFN-γ, TNF-α, IL-2, and IL-12, were significantly upregulated by CD4+ cell depletion. However, unexpectedly, the number of CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) within tumor tissues also significantly increased as a result of CD4+ cell depletion. As a countermeasure against CD8+ T cell accumulation, CCR2-positive CD11b+Gr-1int (monocytic) MDSCs predominantly increased. Treatment with an OX40 agonist under CD4+ cell depletion neither reduced MDSCs nor increased CD8+ T cells and NK cells, but further enhanced the expression of cytotoxic molecules from tumor-infiltrating effector cells. Our results suggest that combined immunotherapy using both an OX40 agonist and CD4+ cell depletion in situ could be a promising therapeutic strategy for poorly immunogenic tumors and might be more effective if further combined with a therapeutic strategy targeting MDSCs.

Targeting myeloid-derived suppressor cells using a novel adenosine monophosphate-activated protein kinase (AMPK) activator

ABSTRACTMyeloid-derived suppressor cells (MDSC) are a heterogeneous population of early myeloid cells that accumulate in the blood and tumors of patients with cancer. MDSC play a critical role during tumor evasion and promote immune suppression through variety of mechanisms, such as the generation of reactive oxygen and nitrogen species (ROS and RNS) and cytokines. AMPactivated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that regulates energy homeostasis and metabolic stress. However, the role of AMPK in the regulation of MDSC function remains largely unexplored. This study was designed to investigate whether treatment of MDSC with OSU-53, a PPAR-inactive derivative that stimulates AMPK kinase, can modulate MDSC function. Our results demonstrate that OSU-53 treatment increases the phosphorylation of AMPK, significantly reduces nitric oxide production, inhibits MDSC migration, and reduces the levels of IL-6 in murine MDSC cell line (MSC2 cells). OSU53 treatment mitigated the immune suppressive functions of murine MDSC, promoting T-cell proliferation. Although OSU-53 had a modest effect on tumor growth in mice inoculated with EMT-6 cells, importantly, administration of OSU53 significantly (p < 0.05) reduced the levels of MDSC in the spleens and tumors. Furthermore, mouse MDSC from EMT-6 tumor-bearing mice and human MDSC isolated from melanoma patients treated with OSU-53 showed a significant reduction in the expression of immune suppressive genes iNOS and arginase. In summary, these results demonstrate a novel role of AMPK in the regulation of MDSC functions and provide a rationale of combining OSU-53 with immune checkpoint inhibitors to augment their response in cancer patients.

Abstract 4399: Hypoxia as a driver of myeloid-derived suppressor cell recruitment in hepatocellular carcinoma via CCL26/CX3CR1

Abstract Background and Objective: Myeloid-derived suppressor cells (MDSCs) accumulate in tumors and highly pro-tumorigenic. These MDSCs exhibit inhibitory functions against effector T cells and natural killer cells in tumor sites, as well as secrete pro-angiogenic factors or differentiate to endothelial cells to promote angiogenesis and metastasis. While it is appreciated that depletion of MDSCs could bring tumoricidal effects, there are significant gaps in knowledge about the underlying mechanisms responsible for MDSC recruitment to tumor sites. Hypoxia, O2 deprivation, is an important factor in the tumor microenvironment of HCC that modifies the stromal components. Using hepatocellular carcinoma (HCC) as a model, we investigated whether hypoxia is a driver of MDSC recruitment in HCC. Experimental Procedures: Gene profiling of HCC cells exposed to hypoxia and normoxia were analyzed by transcriptome sequencing to identify potential hypoxia-induced chemokines for MDSC recruitment. MDSCs were isolated from HCC-bearing mice by magnetic bead sorting for different functional assays. Boyden chambers were used to evaluate the invasive ability of MDSCs. Flow cytometry was used to detect the frequencies of tumor-associated MDSCs in orthotopic HCC mouse models. Results: We observed that MDSCs preferentially infiltrated into hypoxic regions in human HCC tissues and hypoxia-induced MDSC infiltration was dependent on hypoxia-inducible factors (HIFs). HIFs activated the transcription of chemokine (C-C motif) ligand 26 (CCL26) in HCC cells to recruit chemokine (C-X3-C motif) receptor 1 (CX3CR1)-expressing MDSCs to the primary tumor. Knockdown of CCL26 in HCC cells profoundly reduces MDSC recruitment, angiogenesis, and tumor growth. Therapeutically, blockade of CCL26 production in HCC cells by HIF inhibitor, digoxin, or blockade of CX3CR1 in MDSCs by CX3CR1 neutralizing antibody could substantially suppress MDSC recruitment and tumor growth. Conclusion: This study unprecedentedly reveals a novel molecular mechanism by which HCC cells direct MDSC homing to primary tumor and suggests that targeting MDSC recruitment represents an attractive therapeutic approach against HCC. Citation Format: David Kung-Chun Chiu, Iris Ming-Jing Xu, Robin Kit-Ho Lai, Aki Pui-Wah Tse, Larry Lai Wei, Hui-Yu Koh, Regina Cheuk-Lam Lo, Chun-Ming Wong, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. Hypoxia as a driver of myeloid-derived suppressor cell recruitment in hepatocellular carcinoma via CCL26/CX3CR1. [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 4399.

Therapeutic targeting of myeloid-derived suppressor cells involves a novel mechanism mediated by clusterin.

Myeloid-derived suppressor cells (MDSCs) constitute a key checkpoint that impedes tumor immunity against cancer. Chemotherapeutic intervention of MDSCs has gained ground as a strategy for cancer therapy but its mechanism remains obscure.We report here a unique mechanism by which monocytic (M)-MDSCs are spared, allowing them to polarize towards M1 macrophages for reactivation of immunity against breast cancer. We first demonstrated that curcumin, like docetaxel (DTX), can selectively target CD11b+Ly6G+Ly6Clow granulocytic (G)-MDSCs, sparing CD11b+Ly6G−Ly6Chigh M-MDSCs, with reduced tumor burden in 4T1-Neu tumor-bearing mice. Curcumin treatment polarized surviving M-MDSCs toward CCR7+ Dectin-1−M1 cells, accompanied by IFN-γ production and cytolytic function in T cells. Selective M-MDSC chemoresistence to curcumin and DTX was mediated by secretory/cytoplasmic clusterin (sCLU). sCLU functions by trapping Bax from mitochondrial translocation, preventing the apoptotic cascade. Importantly, sCLU was only found in M-MDSCs but not in G-MDSCs. Knockdown of sCLU in M-MDSCs and RAW264.7 macrophages was found to reverse their natural chemoresistance. Clinically, breast cancer patients possess sCLU expression only in mature CD68+ macrophages but not in immature CD33+ immunosuppressive myeloid cells infiltrating the tumors. We thus made the seminal discovery that sCLU expression in M-MDSCs accounts for positive immunomodulation by chemotherapeutic agents.

Hypoxia induces myeloid-derived suppressor cell recruitment to hepatocellular carcinoma through chemokine (C-C motif) ligand 26.

A population of stromal cells, myeloid-derived suppressor cells (MDSCs), is present in tumors. Though studies have gradually revealed the protumorigenic functions of MDSCs, the molecular mechanisms guiding MDSC recruitment remain largely elusive. Hypoxia, O2 deprivation, is an important factor in the tumor microenvironment of solid cancers, whose growth often exceeds the growth of functional blood vessels. Here, using hepatocellular carcinoma as the cancer model, we show that hypoxia is an important driver of MDSC recruitment. We observed that MDSCs preferentially infiltrate into hypoxic regions in human hepatocellular carcinoma tissues and that hypoxia-induced MDSC infiltration is dependent on hypoxia-inducible factors. We further found that hypoxia-inducible factors activate the transcription of chemokine (C-C motif) ligand 26 in cancer cells to recruit chemokine (C-X3-C motif) receptor 1-expressing MDSCs to the primary tumor. Knockdown of chemokine (C-C motif) ligand 26 in cancer cells profoundly reduces MDSC recruitment, angiogenesis, and tumor growth. Therapeutically, blockade of chemokine (C-C motif) ligand 26 production in cancer cells by the hypoxia-inducible factor inhibitor digoxin or blockade of chemokine (C-X3-C motif) receptor 1 in MDSCs by chemokine (C-X3-C motif) receptor 1 neutralizing antibody could substantially suppress MDSC recruitment and tumor growth. This study unprecedentedly reveals a novel molecular mechanism by which cancer cells direct MDSC homing to primary tumor and suggests that targeting MDSC recruitment represents an attractive therapeutic approach against solid cancers. (Hepatology 2016;64:797-813).