Tumor-infiltrating Myeloid-derived Suppressor Cells Research Articles

Circulating and tumor-infiltrating myeloid-derived suppressor cells in cervical carcinoma patients.

Myeloid-derived suppressor cells (MDSCs) serve an immunosuppressive role in human tumors. Human Lin-/low human leukocyte antigen-antigen D related (HLA-DR-) cluster of differentiation (CD)-11b+CD33+ MDSCs are closely linked with tumor staging, progression, clinical therapeutic efficacy and prognosis for various types of tumors. The present study employed multiparametric flow cytometry to measure the proportion of Lin-/lowHLA-DR-CD11b+CD33+ MDSCs in the peripheral blood of 105 cervical cancer patients and 50 healthy subjects. The level of MDSC was higher in tumor patients than in the normal control group and this was closely associated with clinical staging. Further analysis of tumor-infiltrating MDSCs was performed in 22 patients. The MDSC proportions in tumor tissue were significantly higher than those in the corresponding adjacent tissue. The phenotypic characteristics of Lin-/lowHLA-DR-CD11b+CD33+ MDSCs were then evaluated and the results revealed that they express high CD13 and CD39, and low CD115, CD117, CD124 and programmed cell death ligand 1; they were also devoid of CD14, CD15 and CD66b. MDSCs and T-cells from peripheral blood were sorted by flow cytometry for co-culture experiments. Lin-/lowHLA-DR-CD11b+CD33+ MDSCs from patients significantly inhibited the proliferation of CD4 and CD8 T-cells. Furthermore, functional analysis verified that MDSCs from cervical cancer patients could inhibit interleukin-2 and interferon-γ production from T-cells. In addition, the associations between peripheral circulating MDSCs and tumor infiltrating MDSCs, and tumor relapse and metastasis were analyzed. The number of peripheral MDSCs and MDSCs in tumor tissue were observed to be associated with relapse-free survival. Thus, MDSCs in the peripheral blood and tumors of cervical cancer patients have a significant immunosuppressive effect, and are associated with cervical cancer staging and metastasis. These results suggest that targeting MDSCs may increase antitumor immunity and increase the efficacy of cervical cancer therapies.

Abstract OT1-01-02: A multicenter phase II trial to evaluate the efficacy and safety of pembrolizumab and gemcitabine in patients with HER2-negative advanced breast cancer: GEICAM/2015-04 PANGEA-Breast

Abstract Background: Treatment options for advanced breast cancer (ABC) are multiple but unable to properly respond to current clinical needs. In particular, improved therapies are needed for triple negative and hormone receptor (HR)-positive but heavily pretreated patients. Pembrolizumab (P) is a human monoclonal antibody that blocks the PD-1/PD-L1 interaction hence potentiates anticancer T cell responses. Gemcitabine (G) is a cytotoxic drug with well-known immunostimulatory properties. Here, we report an ongoing phase II clinical trial to identify the Recommended Phase II Dose (RP2D) and the efficacy of the combination of these two agents in ABC patients. We hypothesize that these agents may synergize to induce responses with long term clinical benefit (ClinicalTrials.gov Identifier: NCT03025880). Trial Design: Eligible patients are HER2-negative ABC patients who received prior treatment with anthracyclines and taxanes and two or more prior lines of hormone therapy, if HR-positive disease. Patients with CNS involvement are also eligible if clinically stable. Treatment consists of 21-day cycles with 200 mg P on day 1 and G on days 1 and 8. In the safety dose testing, we use a standard 6+6 design with 2 dose levels (DL) of G: 1250 mg/m2 (DL0) and 1000 mg/m2 (DL1). Patients are treated until radiologic or symptomatic progression, or unacceptable toxicity. The primary objectives are RP2D and objective response rate (ORR) of the combination; secondary objectives include evaluation of safety and tolerability and other efficacy variables (progression-free survival [PFS], clinical benefit rate [CBR], response duration [RD] and overall survival [OS]). Efficacy is measured by RECIST 1.1. and irRECIST. Safety is measured using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) 4.0. As exploratory objectives, immunological biomarkers are analyzed in tumor biopsies and blood samples and correlated with (1) clinical efficacy and (2) disease outcomes.Sequential tumor samples are collected at baseline, cycle 3 and at progression. Blood samples are drawn at baseline, cycle 3, and cycle 6, or at post-treatment visit (whatever occurs first). Tumor samples are characterized for intratumoral and stromal tumor-infiltrating lymphocytes, tumor-associated macrophages and myeloid-derived suppressor cells, PD-L1 expression in tumor cells and stroma. Moreover, molecular and genetic profiling will be performed. Blood samples are characterized for peripheral blood mononuclear cell (PBMC) phenotype (including expression of co-activatory and co-inhibitory receptors), cytokine profile, and activity of other immunosuppressive pathways (e.g., IDO1-dependent tryptophan catabolism). These results will be compared with data from a cohort of healthy volunteers. A maximum of 65 patients will be included. The study is approved by the ethical committee and Competent Authority of Spain and already open for patient recruitment in 2 of the 10 participating sites. Keywords: Breast HER2 negative Pembrolizumab Immunotherapy Citation Format: de la Cruz L, Sánchez-Margalet V, Berraondo P, Benito S, Escudero MJ, Caballero R, Carrasco E, Galluzzi L, Rojo F. A multicenter phase II trial to evaluate the efficacy and safety of pembrolizumab and gemcitabine in patients with HER2-negative advanced breast cancer: GEICAM/2015-04 PANGEA-Breast [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 OT1-01-02.

Abstract B001: A phase 1 trial of RGX-104, a first-in-class immunotherapy targeting the liver-X nuclear hormone receptor (LXR), in patients with refractory malignancies

Abstract Background: RGX-104 is a small-molecule LXR agonist that activates the expression of the ApoE gene. We have identified the LXR/ApoE pathway as a novel innate-immune checkpoint that stimulates antitumor immunity. LXR agonist-mediated ApoE induction depletes circulating and tumor-infiltrating myeloid derived suppressor cells (MDSCs), resulting in activation of cytotoxic lymphocytes (CTLs) in murine tumor models. Treatment with RGX-104 as monotherapy elicited significant antitumor activity in a variety of preclinical models, including B16F10, LLC, and CT26. Additionally, combining RGX-104 with a PD-1 antagonist yielded synergistic antitumor activity in PD-1 resistant models. These data motivated studies of RGX-104 in patients with refractory cancer. Methods: We are conducting a first-in-human, dose escalation and expansion phase 1 study of RGX-104 in patients with refractory solid tumors or lymphomas. Safety, PK, PD, efficacy, and immunologic activity are being assessed. Results: RGX-104 was dosed orally for 21 days with a 1-week rest (28 day cycle), at 120 mg QD (n=3) and 240 mg QD (n=4). Tumor types in cohorts 1 and 2 included colon, uterine, renal, melanoma, cholangiocarcinoma, and Ewing’s sarcoma. PK analysis demonstrated dose-proportional drug increases in AUC and a t1/2 of ~7 hours with oral administration. Induction of ApoE gene expression in peripheral blood cells (median 3.57-fold; mean 11.61-fold at steady-state) correlated with RGX-104 drug exposure, indicating robust LXR target engagement. Monitoring of relevant immune-cell populations in peripheral blood revealed substantial MDSC depletion in 6/6 evaluable patients, with up to 95% depletion of granulocytic (CD33+CD15+HLA-DR-/low; median 86% decrease; mean 85%) and up to 89% depletion of monocytic (CD14+Lin-HLA-DR-/low; median 33% decrease; mean 45%) MDSCs. Treatment resulted in a significant increase in activated CTLs (PD-1+GITR+ CD8+ T-cells as a proportion of all CD8+ T-cells) in 5/6 evaluable patients (median 322% increase; mean 352%). Peak effects on ApoE, MDSCs, and CTLs began ~2 weeks after RGX-104 initiation. In order to maximize the achievement of biologically relevant RGX-104 exposure, and therefore desirable immune effects throughout the treatment period, continuous dosing at 120 mg BID is being evaluated in cohort 3. One of two evaluable patients in cohort 3 experienced reversible grade 4 neutropenia and grade 2 lipid elevations, which are “on-target” adverse events. No other grade 3-4 adverse events attributable to RGX-104 have occurred. One renal cancer and one melanoma patient-both refractory to anti-PD-1 therapy–had stable disease. Conclusions: Proof of principle has been demonstrated for the immune-stimulatory action of the first-in-class LXR-agonist RGX-104 in advanced cancer patients. RGX-104 activated ApoE in peripheral blood cells in a dose-dependent fashion and was well tolerated when administered orally at 120 mg or 240 mg QD. RGX-104 selectively depleted MDSCs, leading to activation of the relevant PD-1+ CTL population in a diverse group of cancer patients harboring PD-1 inhibitor refractory disease and immune-therapy resistant cancers. Dosing of RGX-104 at 120 mg BID continues. Future plans include studying RGX-104 both as monotherapy and in combination with a PD-1 inhibitor in select malignancies. Citation Format: Monica Mita, Alain Mita, Bartosz Chmielowski, Michael Postow, Erika Hamilton, Shubham Pant, Roger Waltzman, Eric Rowinsky, Michael Szarek, Foster Gonsalves, Isabel Kurth, Celia Andreu, Ilana Pollack, Daniel Mucida, Raissa Tanqueco, Sohail Tavazoie, Masoud Tavazoie. A phase 1 trial of RGX-104, a first-in-class immunotherapy targeting the liver-X nuclear hormone receptor (LXR), in patients with refractory malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B001.

Myeloid-Derived Suppressor Cells in theTumor Microenvironment: Current Knowledge and Future Perspectives.

The current knowledge on tumor-infiltrating myeloid-derived suppressor cells (MDSCs) is based mainly on the extensive work performed in murine models. Data obtained for human counterparts are generated on the basis of tumor analysis from patient samples. Both sources of information led to determination of the main suppressive mechanisms used by these cell subsets in tumor-bearing hosts. As a result of the identification of protein targets responsible for MDSCs suppressive activity, different therapeutics agents have been used to eliminate/reduce their adverse effect. In the present work, we review the current knowledge on suppressive mechanisms of MDSCs and therapeutic treatments that interfere with their differentiation, expansion or activity. Based on the accumulation of new evidences supporting their importance for tumor progression and metastasis, the interest in these cell types is increasing. We revise the methods of MDSC generation/differentiation ex vivo that may help in overcoming problems associated with limited numbers of cells available from animals and patients for their study.

Splenectomy-Induced Leukocytosis Promotes Intratumoral Accumulation of Myeloid-Derived Suppressor Cells, Angiogenesis and Metastasis

ABSTRACTBackground: Enlargement of the spleen is commonly observed in animal models of cancer. Here, in a breast cancer model, it was aimed to determine the effect of splenectomy on circulating and tumor-infiltrating myeloid-derived suppressor cells (MDSCs), tumor angiogenesis, and metastasis.Methods: Mice were inoculated with 4T1 breast cancer cells and underwent splenectomy or sham laparotomy. Tumor growth and survival of animals were followed. Macroscopic and histopathological analyses were performed to determine splenomegaly and metastasis. Immunophenotyping of myeloid cells was performed with flow cytometric analysis of CD11b, Gr-1, F4/80, CD206, CD11c, and F4/80 markers. Suppressive function of MDSCs on T cell proliferation was studied in cocultures. Tumor angiogenesis and granulocytic myeloid cell infiltration in the metastatic foci were studied by CD31 and Ly6G immunohistochemistry, respectively.Results: The mice bearing breast tumors underwent total splenectomy at an early time point of tumorigenesis when only low levels of MDSCs had accumulated in the spleen. Circulating and tumor-infiltrating MDSCs, and tumor-associated macrophages (TAMs) were increased following splenectomy. Nevertheless, splenectomy could only lead to a temporary deceleration in tumor growth but favored lung metastasis and angiogenesis in the long run.Conclusion: Our data demonstrated a link among splenectomy-induced leukocytosis, accumulation of circulating and tumor-infiltrating MDSC, and enhanced angiogenesis and metastasis. Therefore, as a part of oncological surgery, favorable and unfavorable facets of the splenectomy must be considered to improve therapeutic efficacy.

Abstract 2941: Inhibition of hypoxia-induced ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2) restrains myeloid-derived suppressor cell (MDSC) accumulation and sensitizes tumors to immune checkpoint inhibition

Abstract Background and Objective: Rapidly expanding knowledge on cancer immunology has introduced promising anti-cancer therapeutic approaches which involve the activation of T cells to combat cancer cells. Accumulating studies have indicated that the efficacy of immunotherapies is critically determined by the stromal cell components in tumors. Myeloid-derived suppressor cells (MDSCs), are regarded as one of the major immune cell types that possess immunosuppressive activities against T cells which allow cancers to escape immune surveillance and become non-responsive to immune checkpoints blockade. To increase the efficacy of immunotherapy, novel strategies to target MDSC in tumors are warranted. Hypoxia, oxygen (O2) shortage, frequently occurs in tumors due to abnormal vasculature. Using hepatocellular carcinoma (HCC) as a model, we have previously observed MDSC preferentially accumulates in hypoxic regions of human HCC tissues. Here, we aim to identify hypoxia-induced therapeutic targets that are critical for MDSC accumulation in tumors. Experimental Procedures: Transcriptome sequencing in multiple HCC cell lines exposed to hypoxia and normoxia and HCC clinical specimens was performed to identify potential hypoxia-induced genes relevant to HCC development. MDSCs were isolated from HCC-bearing mice by magnetic bead sorting for different functional assays. LC-MS was performed to evaluate the level of extracellular metabolites. Flow cytometry was used to detect the frequencies of tumor-infiltrating MDSCs in orthotopic and subcutaneous HCC mouse models. Results: We showed that hypoxia, through stabilization of hypoxia-inducible factor-1 (HIF-1), induced ectoenzyme, ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2/ CD39L1), in cancer cells, causing its over-expression in HCC clinical specimens. Over-expression of ENTPD2 was found as a poor prognostic indicator for HCC patients. Mechanistically, we demonstrated that ENTPD2 converted extracellular ATP to 5’-AMP which prevents the differentiation of monocytic MDSCs to dendritic cells, therefore promoting the maintenance of MDSCs in vitro and in vivo. Therapeutically, we found that ENTPD2 inhibitor POM-1 restrained MDSC accumulation and tumor growth, substantially enhancing the efficiency and efficacy of immune checkpoints inhibitors. Conclusion: Our study reveals a novel mechanism whereby hypoxia/HIF-1 in cancer cells governs tumor-infiltrating MDSCs. Our data suggest that ENTPD2 may be a good prognostic marker and therapeutic target for cancer patients especially those receiving immune therapy. Citation Format: David Kung-Chun Chiu, Aki Pui-Wah Tse, Iris Ming-Jing Xu, Robin Kit-Ho Lai, Hui-yu Koh, Felice Ho-Ching Tsang, Larry Lai Wei, Chun-Ming Wong, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. Inhibition of hypoxia-induced ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2) restrains myeloid-derived suppressor cell (MDSC) accumulation and sensitizes tumors to immune checkpoint inhibition [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 2941. doi:10.1158/1538-7445.AM2017-2941

Efficacy of vaccination with tumor-exosome loaded dendritic cells combined with cytotoxic drug treatment in pancreatic cancer

ABSTRACT Pancreatic cancer (PaCa) has a dismal prognosis and adjuvant immunotherapy frequently is of low efficacy due to immunosuppressive features of PaCa and PaCa-stroma. We here explored, whether the efficacy of vaccination with tumor-exosome (TEX)-loaded dendritic cells (DC) can be improved by combining with drugs affecting myeloid-derived suppressor cells (MDSC). Experiments were performed with the UNKC6141 PaCa line. UNKC6141 TEX-loaded DC were weekly intravenously injected, mice additionally receiving Gemcitabine (GEM) and/or ATRA and/or Sunitinib (Sun). UNKC6141 grow aggressively after subcutaneous and orthotopic application and are consistently recovered in peripheral blood, bone marrow, lung and frequently liver. Vaccination with DC-TEX significantly prolonged the survival time, the efficacy of DC-TEX exceeding that of the cytotoxic drugs. However, ATRA, Sun and most efficiently GEM, sufficed for a pronounced reduction of MDSC including tumor-infiltrating MDSC, which was accompanied by a decrease in migrating and metastasizing tumor cells. When combined with DC-TEX vaccination, a higher number of activated T cells was recovered in the tumor and the survival time was prolonged compared with only DC-TEX vaccinated mice. As ATRA, GEM and Sun affect MDSC at distinct maturation and activation stages, a stronger support for DC-TEX vaccination was expected by the drug combination. Intrapancreatic tumor growth was prevented beyond the death of control mice. However, tumors developed after a partial breakdown of the immune system by the persisting drug application. Nonetheless, in combination with optimized drug tuning to prevent MDSC maturation and activation, vaccination with TEX-loaded DC appears a most promising option in PaCa therapy.

The expression profiles and regulation of PD-L1 in tumor-induced myeloid-derived suppressor cells

Abstract Programmed death-ligand 1 (PD-L1) is an inhibitory ligand that binds to PD-1 to suppress T cell activation. PD-L1 is constitutively expressed and inducible in tumor cells, but the expression profiles and regulatory mechanism of PD-L1 in myeloid-derived suppressor cells (MDSCs) are largely unknown. We report that PD-L1 is abundantly expressed in tumor-infiltrating immune cells in human colon carcinoma, and 25–67% CD11b+CD33+ myeloid-derived suppressor cells (MDSCs) from peripheral blood of human colon cancer patients are PD-L1+. Using a spontaneous colon cancer mouse model, we observed that PD-L1+MDSCs increased significantly in tumor-bearing mice as compared to MDSCs from tumor-free mice. Interestingly, the highest PD-L1+ MDSCs were observed in the tumor microenvironment in which 56–71% tumor-infiltrating MDSCs are PD-L1+ in vivo. In contrast, PD-L1+ MDSCs are significantly less in secondary lymphoid organs and peripheral blood as compared to the tumor tissues, whereas, bone marrow MDSCs are essentially PD-L1− in tumor-bearing mice. IFNg is highly expressed in cells of the tumor tissues and IFNg neutralization significantly decreased PD-L1+ MDSCs in the tumor microenvironment in vivo. However, IFNg-activated pSTAT1 does not bind to the cd274 promoter in CD11b+Gr1+PD-L1+ myeloid cells. Instead, pSTAT1 activates expression of IRF1, IRF5, IRF7 and IRF8 in MDSCs, and only pSTAT1-activated IRF1 binds to an unique IRF-binding sequence element in vitro and chromatin in vivo in the cd274 promoter to activate PD-L1 transcription. Our data determine that PD-L1 is highly expressed in tumor-infiltrating MDSCs and in a less degree in lymphoid organs, and the pSTAT1-IRF1 axis regulates PD-L1 expression in MDSCs.

Myeloid-derived suppressor cell and macrophage exert distinct angiogenic and immunosuppressive effects in breast cancer.

The immunosuppressive tumor microenvironment is a key obstacle to hinder a cancer immunotherapy. Myeloid-derived suppressor cells (MDSCs) have been considered as a major player in immunosuppression. In this study, we find that tumor-infiltrating MDSCs (tiMDSCs) are less immunosuppressive than tumor-associated macrophages (TAMs) in multiple murine orthotopic breast tumor models. Compared to TAMs, tiMDSCs produce higher levels of pro-inflammatory factors and lower levels of anti-inflammatory factors. Furthermore, tiMDSCs are preferentially located in hypoxic areas and are more pro-angiogenic than TAMs. Consistent with these functional disparities, a shift from tiMDSCs to TAMs is observed during the progression of breast cancer. Moreover, infiltration of tiMDSCs is also noted in distal colonization of breast cancer cells in the lung. Taken together, our findings indicate that tiMDSCs are more pro-angiogenic and promote tumor initiation, while TAMs are more immunosuppressive and facilitate tumor immune evasion. This study suggests that selectively targeting on TAMs could alleviate the immunosuppressive tumor microenvironment and potentiate cancer immunotherapy.

Estrogen-induced SDF-1α production promotes the progression of ER-negative breast cancer via the accumulation of MDSCs in the tumor microenvironment.

Estrogen plays a role in the processes of tumorigenesis, metastasis, and drug resistance in estrogen receptor (ER)-positive breast cancer (BC). Whether estrogen contributes to ER-negative BC is unclear. Here, we aimed to investigate whether estrogen could stimulate the secretion of stromal-derived factor-1 (SDF-1α) by cancer-associated fibroblasts (CAFs) to promote the progression of ER-negative BC. We transplanted ER-negative BC cells into ovariectomized mice, which was followed by continuous injection of estrogen, and found that estrogen promoted the tumorigenesis of BC. Furthermore, High levels of SDF-1α and tumor-infiltrating myeloid-derived suppressor cells (MDSCs) were detected in the estrogen treatment group. Estrogen stimulates secretion of SDF-1α by CAFs extracted from BC patients. Recombinant SDF-1α could recruit MDSCs isolated from bone marrow cells of mice. In addition, the co-culture of CAFs and MDSCs demonstrated that the recruitment of MDSCs was increased when CAFs were exposed to estrogen. Using AMD3100 to block the SDF-1α/CXCR4 axis or gemcitabine to delete MDSCs, we observed that both of these agents could neutralize the effect of estrogen on tumorigenesis. Together, these results suggest that estrogen may promote the progression of ER-negative BC by stimulating CAFs to secrete SDF-1α, which can recruit MDSCs to the tumor microenvironment to exert tumor-promoting effects.

The expression profiles and regulation of PD-L1 in tumor-induced myeloid-derived suppressor cells

ABSTRACTProgrammed death-ligand 1 (PD-L1) is an inhibitory ligand that binds to PD-1 to suppress T cell activation. PD-L1 is constitutively expressed and inducible in tumor cells, but the expression profiles and regulatory mechanism of PD-L1 in myeloid-derived suppressor cells (MDSCs) are largely unknown. We report that PD-L1 is abundantly expressed in tumor-infiltrating leukocytes in human patients with both microsatellite instable and microsatellite stable colon cancer. About 60% CD11b+CD33+HLA-DR− MDSCs from peripheral blood of human colon cancer patients are PD-L1+. PD-L1+ MDSCs are also significantly higher in tumor-bearing mice than in tumor-free mice. Interestingly, the highest PD-L1+ MDSCs were observed in the tumor microenvironment in which 56–71% tumor-infiltrating MDSCs are PD-L1+ in vivo. In contrast, PD-L1+ MDSCs are significantly less in secondary lymphoid organs and peripheral blood as compared to the tumor tissues, whereas bone marrow MDSCs are essentially PD-L1− in tumor-bearing mice. IFNγ is highly expressed in cells of the tumor tissues and IFNγ neutralization significantly decreased PD-L1+ MDSCs in the tumor microenvironment in vivo. However, IFNγ-activated pSTAT1 does not bind to the cd274 promoter in MDSCs. Instead, pSTAT1 activates expression of IRF1, IRF5, IRF7 and IRF8 in MDSCs, and only pSTAT1-activated IRF1 binds to a unique IRF-binding sequence element in vitro and chromatin in vivo in the cd274 promoter to activate PD-L1 transcription. Our data determine that PD-L1 is highly expressed in tumor-infiltrating MDSCs and in a lesser degree in lymphoid organs, and the pSTAT1-IRF1 axis regulates PD-L1 expression in MDSCs.

Indoleamine 2,3-dioxygenase regulates anti-tumor immunity in lung cancer by metabolic reprogramming of immune cells in the tumor microenvironment.

Indoleamine 2,3-dioxygenase (IDO) has been implicated in immune evasion by tumors. Upregulation of this tryptophan (Trp)-catabolizing enzyme, in tumor cells and myeloid-derived suppressor cells (MDSCs) within the tumor microenvironment (TME), leads to Trp depletion that impairs cytotoxic T cell responses and survival; however, exact mechanisms remain incompletely understood. We previously reported that a combination therapy of gemcitabine and a superoxide dismutase mimetic promotes anti-tumor immunity in a mouse model of lung cancer by inhibiting MDSCs, enhancing polyfunctional response of CD8+ memory T cells, and extending survival. Here, we show that combination therapy targets IDO signaling, specifically in MDSCs, tumor cells, and CD8+ T cells infiltrating the TME. Deficiency of IDO caused significant reduction in tumor burden, tumor-infiltrating MDSCs, GM-CSF, MDSC survival and infiltration of programmed death receptor-1 (PD-1)-expressing CD8+ T cells compared to controls. IDO−/− MDSCs downregulated nutrient-sensing AMP-activated protein kinase (AMPK) activity, but IDO−/− CD8+ T cells showed AMPK activation associated with enhanced effector function. Our studies provide proof-of-concept for the efficacy of this combination therapy in inhibiting IDO and T cell exhaustion in a syngeneic model of lung cancer and provide mechanistic insights for IDO-dependent metabolic reprogramming of MDSCs that reduces T cell exhaustion and regulates anti-tumor immunity.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 1448: MDSC depletion delays primary tumor growth in syngeneic models of oral cavity cancer

Abstract 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.

Pancreatic adenocarcinoma up-regulated factor (PAUF) enhances the accumulation and functional activity of myeloid-derived suppressor cells (MDSCs) in pancreatic cancer.

Pancreatic cancer is characterized by an immunosuppressive tumor microenvironment (TME) with a profound immune infiltrate populated by a significant number of myeloid-derived suppressor cells (MDSCs). MDSCs have been increasingly recognized for their role in immune evasion and cancer progression as well as their potential as a target for immunotherapy. However, not much is known about the mechanisms regulating their behavior and function in the pancreatic TME. Here we report that pancreatic adenocarcinoma up-regulated factor (PAUF), a soluble protein involved in pancreatic tumorigenesis and metastasis, plays a role as an enhancer of tumor-infiltrating MDSC and its functional activity. We show that PAUF enhanced the accumulation of MDSCs in the spleen and tumor tissues of PAUF-overexpressing tumor cell-injected mice. In addition, PAUF was found to enhance the immunosuppressive function of MDSCs via the TLR4-mediated signaling pathway, which was demonstrated by PAUF-induced increased levels of arginase, nitric oxide (NO), and reactive oxygen species (ROS). The role of PAUF in modulating the functional properties of MDSCs was further demonstrated by the use of a PAUF-neutralizing antibody that caused a decreased number of tumor-infiltrating MDSCs and reduced MDSC immunosuppressive activity. The observations made in mice were confirmed in human pancreatic cancer patient-derived MDSCs, supporting the clinical relevance of our findings. Collectively, we conclude that the PAUF is a powerful and multifunctional promoter of tumor growth through increase and functional activation of MDSCs, suggesting therapeutic potential for targeting PAUF in pancreatic cancers.

Stromal cell-derived factor-1 (SDF-1) as a target in liver diseases.

The chemokine stromal cell-derived factor-1 (SDF-1) or CXCL12 is constitutively expressed in healthy liver. However, its expression increases following acute or chronic liver injury. Liver sinusoidal endothelial cells (LSEC), hepatic stellate cells (HSC), and malignant hepatocytes are important sources of SDF-1/CXCL12 in liver diseases. CXCL12 is able to activate two chemokine receptors with different downstream signaling pathways, CXCR4 and CXCR7. CXCR7 expression is relevant on LSEC, while HSC, mesenchymal stem cells, and tumor cells mainly respond via CXCR4. Here, we summarize recent developments in the field of liver diseases involving this chemokine and its receptors. SDF-1-dependent signaling contributes to modulating acute liver injury and subsequent tissue regeneration. By activating HSC and recruiting mesenchymal cells from bone marrow, CXCL12 can promote liver fibrosis progression, while CXCL12-CXCR7 interactions endorse proregenerative responses in chronic injury. Moreover, the SDF-1 pathway is linked to development of hepatocellular carcinoma (HCC) by promoting tumor growth, angiogenesis, and HCC metastasis. High hepatic CXCR4 expression has been suggested as a biomarker indicating poor prognosis of HCC patients. Tumor-infiltrating myeloid-derived suppressor cells (MDSC) also express CXCR4 and migrate toward CXCL12. Thus CXCL12 inhibition might not only directly block HCC growth but also modulate the tumor microenvironment (angiogenesis, MDSC), thereby sensitizing HCC patients to conventional or emerging novel cancer therapies (e.g., sorafenib, regorafenib, nivolumab, pembrolizumab). We herein summarize the current knowledge on the complex interplay between CXCL12 and CXCR4/CXCR7 in liver diseases and discuss approaches on the therapeutic targeting of these axes in hepatitis, fibrosis, and liver cancer.

Combination of IL-12 and radiotherapy induced regression of large orthotopic liver cancers through modulation of immune cells in tumor microenvironment

Abstract Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide with few therapeutic options. Radiation is wildly used for cancer treatment, but local relapse often occurs. Interleukin 12 (IL-12) has potent antitumor activities by activating natural killer cells (NK) and CD8+ T cells, reprogramming myeloid cells to an immune active phenotype, and inhibiting new blood vessel formation. We hypothesized that combined radiation and IL-12 treatment may change tumor suppressive microenvironment and induce antitumor immune responses. Our data showed that only combination of radiation and intratumoral injection of adenovirus encoding IL-12 (Ad/IL-12) led to tumor regression in mice with large subcutaneous or orthotopic HCC, and significantly prolonged their survival. Mechanistic studies revealed that combination therapy enhanced antigen (Ag) presentation ability of DCs by increasing MHC class II, CD40 and CD86 expression. Combination therapy also significantly reduced numbers of tumor-infiltrating myeloid-derived suppressor cells (MDSCs) and impaired their suppressive functions by reducing reactive oxygen species (ROS) production. Accordingly, combination therapy significantly increased the cell numbers of tumor-infiltrating CD8+ T cells and their expressions of CD107a and TNF-α. The cell numbers of tumor-infiltrating NK cells was not affected, but the expression of activation markers was significantly increased after treatment. Together, our results demonstrate that combination therapy effectively against advanced HCC through activating tumor-infiltrating NK cells and tumor-specific CD8+ T cells, enhancing Ag presentation ability of DCs while reducing the suppressive functions of MDSCs.

Myeloid-Derived Suppressor Cell Survival and Function Are Regulated by the Transcription Factor Nrf2.

Tumor-induced myeloid-derived suppressor cells (MDSC) contribute to immune suppression in tumor-bearing individuals and are a major obstacle to effective immunotherapy. Reactive oxygen species (ROS) are one of the mechanisms used by MDSC to suppress T cell activation. Although ROS are toxic to most cells, MDSC survive despite their elevated content and release of ROS. NF erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates a battery of genes that attenuate oxidative stress. Therefore, we hypothesized that MDSC resistance to ROS may be regulated by Nrf2. To test this hypothesis, we used Nrf2(+/+)and Nrf2(-/-)BALB/c and C57BL/6 mice bearing 4T1 mammary carcinoma and MC38 colon carcinoma, respectively. Nrf2 enhanced MDSC suppressive activity by increasing MDSC production of H2O2, and it increased the quantity of tumor-infiltrating MDSC by reducing their oxidative stress and rate of apoptosis. Nrf2 did not affect circulating levels of MDSC in tumor-bearing mice because the decreased apoptotic rate of tumor-infiltrating MDSC was balanced by a decreased rate of differentiation from bone marrow progenitor cells. These results demonstrate that Nrf2 regulates the generation, survival, and suppressive potency of MDSC, and that a feedback homeostatic mechanism maintains a steady-state level of circulating MDSC in tumor-bearing individuals.

Targeting myeloid-derived suppressor cells with colony stimulating factor-1 receptor blockade can reverse immune resistance to immunotherapy in indoleamine 2,3-dioxygenase-expressing tumors

Tumor indoleamine 2,3-dioxygenase (IDO) promotes immunosuppression by direct action on effector T cells and Tregs and through recruitment, expansion and activation of myeloid-derived suppressor cells (MDSCs). Targeting of MDSCs is clinically being explored as a therapeutic strategy, though optimal targeting strategies and biomarkers predictive of response are presently unknown. Maturation and tumor recruitment of MDSCs are dependent on signaling through the receptor tyrosine kinase CSF-1R on myeloid cells. Here, we show that MDSCs are the critical cell population in IDO-expressing B16 tumors in mediating accelerated tumor outgrowth and resistance to immunotherapy. Using a clinically relevant drug, we show that inhibition of CSF-1R signaling can functionally block tumor-infiltrating MDSCs and enhance anti-tumor T cell responses. Furthermore, inhibition of CSF-1R sensitizes IDO-expressing tumors to immunotherapy with T cell checkpoint blockade, and combination of CSF-1R blockade with IDO inhibitors potently elicits tumor regression. These findings provide evidence for a critical and functional role for MDSCs on the in vivo outcome of IDO-expressing tumors.

High-mobility group box protein 1 promotes the survival of myeloid-derived suppressor cells by inducing autophagy.

Myeloid-derived suppressor cells are immune-suppressive cells that are elevated in most individuals with cancer, where their accumulation and suppressive activity are driven by inflammation. As myeloid-derived suppressor cells inhibit anti-tumor immunity and promote tumor progression, we are determining how their viability is regulated. Previous studies have established that the damage-associated molecular pattern molecule high-mobility group box protein 1 drives myeloid-derived suppressor cell accumulation and suppressive potency and is ubiquitously present in the tumor microenvironment. As high-mobility group box protein 1 also facilitates tumor cell survival by inducing autophagy, we sought to determine if high-mobility group box protein 1 regulates myeloid-derived suppressor cell survival through induction of autophagy. Inhibition of autophagy increased the quantity of apoptotic myeloid-derived suppressor cells, demonstrating that autophagy extends the survival and increases the viability of myeloid-derived suppressor cells. Inhibition of high-mobility group box protein 1 similarly increased the level of apoptotic myeloid-derived suppressor cells and reduced myeloid-derived suppressor cell autophagy, demonstrating that in addition to inducing the accumulation of myeloid-derived suppressor cells, high-mobility group box protein 1 sustains myeloid-derived suppressor cell viability. Circulating myeloid-derived suppressor cells have a default autophagic phenotype, and tumor-infiltrating myeloid-derived suppressor cells are more autophagic, consistent with the concept that inflammatory and hypoxic conditions within the microenvironment of solid tumors contribute to tumor progression by enhancing immune-suppressive myeloid-derived suppressor cells. Overall, these results demonstrate that in addition to previously recognized protumor effects, high-mobility group box protein 1 contributes to tumor progression by increasing myeloid-derived suppressor cell viability by driving them into a proautophagic state.