Myeloid-derived Suppressor Cells Populations Research Articles

Activation of VIP signaling enhances immunosuppressive effect of MDSCs on CMV-induced adaptive immunity.

Vasoactive intestinal peptide (VIP) is recognized as a potent anti-inflammatory factor which affects both the innate and adaptive arms of the immune system. These effects include, but are not limited to, inhibition of T cell proliferation and disruption of immune homeostasis. Myeloid-derived suppressor cells (MDSC) are an immune regulatory cell type that has been described in settings of cancer and infectious disease._Here we demonstrate a reduced circulating monocytic MDSCs in the VIP -/- vs. wild type MCMV. VIP-/- MDSCs secretes less NO upon stimulation with LPS and interferon that relatively lose the ability to suppress T cells activation in vitro compared to wild type MDSCs._Considering the importance of VIP in immunomodulation, the possible effect of VIP in the suppressive function of MDSC populations following CMV infection remains unknown. We describe the possible role of VIP in the regulation of anti-CMV activity of T cells through the activation of MDSCs.

Targeting phosphorylation of STAT3 delays tumor growth in HPV-negative anal squamous cell carcinoma mouse model

Although conventional chemoradiotherapy is effective for most anal squamous cell carcinoma (ASCC) patients, HPV-negative ASCC patients respond poorly to this treatment and new therapeutic approach is required. Our group has previously established an HPV-negative ASCC mouse model and demonstrated that signal transducer and activation of transcription 3 (STAT3) is hyper-activated in the model. Here, we show that in vivo inhibition of STAT3 by S3I-201 effectively delays tumor growth in ASCC mouse model indicated by significantly smaller tumor size and burden in the treatment group compared with control group at the same point. Further analysis shows that survivin and Ki67, important biomarkers for tumor cell survival and proliferation, are significantly reduced after S3I-201 treatment. Additionally, flow cytometry and immunohistofluorescent assays reveal decreased Myeloid-derived suppressor cell (MDSC) and tumor-associated macrophage (TAM) populations in the S3I-201 treatment group, which indicates a reversion of the immunosuppressive environment, unraveling the potential role for S3I-201 in immunosuppression in ASCC. Together these results for the first time demonstrated the anti-tumor effects of STAT3 inhibitor S3I-201 in HPV-negative ASCC mouse model and its multiple effects on cancer cells and immune system. Thus we conclude that S3I-201 may be a novel therapeutic approach for HPV-negative ASCC patients.

Abstract 638: Epigenetic reprograming of immune cells through selective inhibition of HDAC6 reduces suppressive phenotypes and augments anti-tumor properties of T-cells

Abstract Alteration of the epigenetic landscape of immune cells can modify the pattern of gene expression, thus resulting in phenotypic and functional changes. Small molecule inhibitors targeting epigenetic modifiers, such as histone deacetylases (HDACs), have been shown to reduce tumor growth. Besides promoting direct anti-tumor effects, HDAC inhibitors also target immune cells and alter their gene regulation. Here, we demonstrate that the HDAC6 selective inhibitors ACY-241 and ACY-1215 (ricolinostat) decrease the function of myeloid derived suppressor cells (MDSC) and T regulatory (Treg) cells, maintain an effector phenotype by CD8+ T cells, and do not reduce viability of immune cells. First, peripheral blood mononuclear cells derived from melanoma patients were treated with ACY-241, and the phenotype of MDSCs was assessed. Expression of the suppressive molecules ARG1 (p<0.01) and NOS2 (p<0.05) was decreased in CD14+HLA DR lo CD11b+ and CD14+HLA DR lo CD33+ MDSC populations, suggesting less potent MDSCs. To gain insight into other suppressive populations, we evaluated the phenotype and function of Treg cells derived from melanoma patients. Cultures of CD3+ T-cells treated with ACY-1215 or ACY-241 resulted in decreased expression of FOXP3 and reduced frequency of Tregs (CD4+CD25+CD127-; p<0.001). ACY-1215 pre-treated Tregs exhibited less suppressive activity against responding conventional T-cells in standard assays, compared to Tregs pre-treated with DMSO control (p<0.01). CD3+ T-cells exposed to ACY-241 or ACY-1215 were activated via CD3/CD28 co-stimulation to assess effects on cytokine production. Selective HDAC6 inhibition shifted T-cell differentiation towards Th1-type (Tbet+) over Th2-type (GATA3+), compared to DMSO (p<0.05). Additionally, Th2 cytokines (e.g. IL-4, IL-5, IL-6, IL-10, IL-13) were significantly decreased (p<0.05). In accordance with decreased Th2 differentiation, mTORC signal transduction, including phosphor-SGK1, was similarly reduced (p<0.05). Targeted inhibition of mTORC signaling (i.e. SGK1 inhibitor) recapitulated decreased Th2 cytokine production (p<0.05). In order to address effector immune function, tumor infiltrating lymphocytes (TILs) were harvested from melanoma biopsies and expanded in vitro in the presence of ACY-1215 or ACY-241, and IL-2. Measuring CD45RA, CD45RO, CD62L and/or CCR7, an accumulation of central memory CD4+ and CD8+ T-cells was observed (p<0.05). Activated TILs treated with ACY-241 or ACY-1215 displayed higher expression of IFN-gamma and CD107a. Collectively, these data indicate that epigenetic reprograming of immune cells by HDAC6 selective inhibitors may decrease the function of suppressive subsets (e.g. Tregs, MDSCs), while enhancing the accumulation of anti-tumor memory and effector T-cell subsets. Citation Format: Andressa L. Sodre, David M. Woods, Amod Sarnaik, Brian C. Betts, Steven Quayle, Simon Jones, Jeffrey Weber. Epigenetic reprograming of immune cells through selective inhibition of HDAC6 reduces suppressive phenotypes and augments anti-tumor properties of T-cells [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 638. doi:10.1158/1538-7445.AM2017-638

Abstract 3666: Chemotherapeutic sensitivity of myeloid-derived suppressor cells during cancer therapy is dictated by selective expression of clusterin

Abstract Diverse chemotherapeutic agents, including Docetaxel, have been reported by us and others to specifically target myeloid-derived suppressor cells (MDSCs) which mediate tumor-associated immune repression. However, the mechanism is unknown. To find a less toxic anticancer drug, we focused on Curcumin which exhibits natural anti-oxidant and antitumor activities with low toxicity to normal cells. Here, we demonstrate that curcumin, like Docetaxel, promotes cytotoxic function against tumor cells and depletes MDSC populations in vivo and in vitro. In 4T1 mammary tumor-bearing BALB/c mice treated with curcumin, we observed dose-dependent tumor reductions in mice treated with curcumin compared to DMSO controls, indicating that curcumin can be an effective antitumor agent. To elucidate the mechanism, we embarked on examining different populations of myeloid-derived suppressor cells (MDSCs). Flow cytometric analysis of splenic cells from tumor bearers indicated that the antitumor effect by curcumin is due to depletion of immunosuppressive Ly6G+ granulocytic (G)-MDSC lineage with selective promotion of Ly6C+ monocytic (M)-MDSC towards the CCR7+F4/80+ M1 subset. Furthermore, the increase in M1 MDSCs correlated with greater T-cell adaptive immune response and cytotoxicity against 4T1 tumor cells. Since clearance of G-MDSCs was mediated by apoptosis as evidenced by our Annexin V staining, we hypothesized that clusterin (sCLU), a potent anti-apoptotic protein, unrelated to the Bcl-2 family, may be the underlying mechanism by which M-MDSCs and M1 cells are selected out following treatment of curcumin. Western blot analysis revealed that sCLU was, indeed, differentially expressed in M1-MDSCs but not in G-MDSCs. Antisense-CLU transfection into M-MDSCs reduced chemoresistance not only to curcumin but also to Docetaxel, confirming the critical involvement of CLU in selective survival of M-MDSCs. Using both curcumin and Docetaxel we confirmed that sCLU works by binding Bax, preventing its translation to the mitochondria to initiate apoptosis. For clinical relevance, we examined human breast cancer samples and noted that the tumor microenvironment is replete with immature CD33+ MDSCs which lack sCLU expression, unlike infiltrating mature CD68+ macrophages which possess high sCLU expression. Our results taken together indicate that selective survival of M-MDSCs under chemotherapy dictate their survival advantage, allowing for maturation into M1 cells and promotion of adaptive T cell immunity against cancer. Citation Format: Nhan Tu, Thu Le Trinh, Jun-Min Zhou, Danielle L. Gilvary, Domenico Coppola, Sheng Wei, Julie Y. Djeu. Chemotherapeutic sensitivity of myeloid-derived suppressor cells during cancer therapy is dictated by selective expression of clusterin [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 3666. doi:10.1158/1538-7445.AM2017-3666

Abstract 3968: Canonical NFkB signaling in myeloid cells is required for the glioblastoma growth

Abstract Glioblastoma (GBM) development and therapeutic resistance has been accompanying with the tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). TAMs are heterogeneous cell populations of immune regulatory myeloid-derived suppressor cells (MDSCs) and polarization of anti-tumor macrophages (M1) into pro-tumor macrophages (M2). We investigated the role of myeloid cell NF-κB signaling in orthotopic GBM model using immune deficient and immune competent hosts. Interestingly, conditional deletion of canonical NF-κB signaling (p65) with Lysm-Cre (p65 KO) in myeloid cells, significantly inhibited syngeneic GL261 tumor growth in immune-competent mice compared to control mice. We studied the TAMs recruitment to the tumor and their polarization under the influence of TME. P65 KO mice displayed decreasing trend of immune cell infiltration (CD45), which phenotyped as decreased F4/80+, CD68+, CD206+ (M2) and Gr1+CD11b+ (MDSCs) macrophages, compared to control mice. This was associated with the increased CD80+ (M1) macrophages, increasing trend of CD4+ and CD8+ cytotoxic T cells, and decreased CD44+ mesenchymal cancer stem cells (CSCs) populations in the TME. Cytokine array data indicated that loss of canonical NF-κB signaling within the TAMs was implicated in increased production of IFNγ, IGF1, MCP1, MIP1α, and TNFα cytokines. Co-culture of T cells with p65 KO or control MDSCs identified increased proliferation of T cells with p65 KO MDSCs compared to control MDSCs. Conversely, GBM patient-derived xenografts and U251 GBM cell line-derived tumors showed increasing trend of growth in immune-deficient mice, following the transplantation of p65 KO bone marrow (BM) compared to control BM. Pro-tumor macrophages and CSCs were increased and T cell populations were decreased in human tumors grown in immune deficient mice transplanted with p65 KO BM, compared with control BM. In addition, analysis of human data set revealed higher expression of p65 subunit of NF-κB complex in brain tumor stroma compared to the tumor cells. The study suggests that canonical NF-κB signaling in TAMs is required for the tumor-promoting macrophage polarization and GBM growth in immunocompetent host compared to immune deficient host. Therefore, targeting myeloid-specific NFκB signaling in GBM could inhibit the immune suppressive TAMs and improve the anti-tumor immunity. Citation Format: Bhagelu R. Achyut, Jennifer Bradford, Kartik Angara, Mohammad Rashid, Meenu Jain, Thaiz Borin, ASM Iskander, Roxan Ara, Ali Arbab. Canonical NFkB signaling in myeloid cells is required for the glioblastoma growth [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 3968. doi:10.1158/1538-7445.AM2017-3968

Abstract 3980: Ly6G+ neutrophils and polymorphonuclear myeloid derived suppressor cells promote the survival of tumor cells

Abstract A heterogeneous group of immature myeloid cells which suppress the immune system are termed myeloid derived suppressor cells (MDSC). Collectively a MDSC population is comprised of monocyte-like MDSC and polymorphonuclear MDSC (PMN-MDSC). MDSC are increased in the presence of a number of different tumors. We previously demonstrated that PMN-MDSC represent the majority of MDSC in tumor-bearing mice. In a mouse model of multiple myeloma (MM) depletion of myeloid cells in vivo results in increased survival. Direct co-culture of neutrophils (CD11b+Gr1+ cells from tumor-free mice) or PMN-MDSC (CD11b+Gr1+ cells from tumor-bearing mice) and MM cells increases the survival of the MM cells from chemotherapeutic-induced death. Cell-cell contact is not necessary for the protective effect. Neutrophil and PMN-MDSC supernatants protect MM cells from cell death, suggesting the factor protecting tumor cells is soluble. Results in the mouse were confirmed using human MM cells and replicated on breast cancer cells with neutrophils or PMN-MDSC. Several cytokines and growth factors, such as IL-6 and fibroblast growth factor, are implicated in chemoresistance. The role of these neutrophil and PMN-MDSC soluble factors and the identification of novel contributors to chemoresistance are under investigation. Citation Format: Sarah Herlihy, Cindy Lin, Maria Ozeriva, Alfred Garfall, Dan Vogl, Dmitry Gabrilovich, Yulia Nefedova. Ly6G+ neutrophils and polymorphonuclear myeloid derived suppressor cells promote the survival of tumor cells [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 3980. doi:10.1158/1538-7445.AM2017-3980

Abstract 2259: Evaluation of polymorphisms in myeloid-associated genes and glioma survival

Abstract BACKGROUND: Gliomas are highly infiltrated by immune cells including microglia, macrophages, and myeloid-derived suppressor cells (collectively, glioma-associated myeloid cells). These cells have been shown to be induced by the tumor to be immune-suppressive and tumor-supportive, and are a negative prognosticator for survival in mouse models. Here, we examine whether inherited variants in genes important to the function of glioma-associated myeloid cells are associated with survival following low-grade glioma diagnosis. METHODS: Subjects for this study were 484 patients with WHO grade II or grade III glioma treated at The University of Texas MD Anderson Cancer Center in Houston, Texas between 1992 and 2008 and followed up for survival through August, 2016. We selected 100 genes for analysis including transcription factors, cytokines and chemokines, receptors, enzymes, and other genes central to the function of glioma-associated myeloid cells. Genotyping was originally performed using the Illumina Human 610-Quad Bead Chip platform and 2,040 tagging SNPs as determined by Haploview Tagger software were selected for analysis with minor allele frequency (AF) ≥ 1%. Associations between selected SNPs and survival were evaluated by Cox regression analysis under an additive allelic model adjusting for age, sex, extent of surgery (biopsy only/partial resection/gross total resection), radiotherapy (yes/no), and chemotherapy (yes/no). Models were examined to ensure that proportional hazards assumptions were not violated. RESULTS: Median survival among low-grade glioma patients was 6.7 years. Age at diagnosis, extent of surgery, and having received radiotherapy or chemotherapy were each significantly associated with survival. Five SNPs were associated with survival at a significance level of p<0.001, and two remained significantly associated with low-grade glioma survival after adjustment for multiple comparisons (FDR-adjusted p-value (q)<0.10). These results indicated inferior survival for carriers of the C allele (AF=1.4%) at rs147960238 in CD163 (HR=5.47, 95% CI: 2.49-11.99, p=2.23x10-5, q=0.046) and for carriers of the G allele (AF=3.8%) at rs17138945 in MET (HR=2.27, 95% CI: 1.52-3.38, p=5.61x10-5, q=0.057). These SNPs are located in the 10th and 2nd introns of CD163 and MET, respectively. CONCLUSIONS: Here we provide preliminary evidence of an association between polymorphisms in two genes related to glioma-associated myeloid cell function and low-grade glioma survival. CD163 is a receptor that is highly expressed on macrophages and may play a role in macrophage-mediated anti-inflammatory responses, while MET is a receptor tyrosine kinase and well-studied proto-oncogene that is also involved in the expansion of myeloid-derived suppressor cell populations. Further investigation of these associations is warranted, and validation of these findings is planned in an independent population. Citation Format: Daniel I. Jacobs, Yanhong Liu, Konrad Gabrusiewicz, Spiridon Tsavachidis, E. Susan Amirian, Georgina N. Armstrong, Renke Zhou, Jun Wei, Cristina Ivan, George Calin, Michael Scheurer, Anna Dahlin, Terri Rice, Paige M. Bracci, Helen M. Hansen, John K. Wiencke, Margaret R. Wrensch, Beatrice Melin, Amy B. Heimberger, Melissa L. Bondy. Evaluation of polymorphisms in myeloid-associated genes and glioma survival [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 2259. doi:10.1158/1538-7445.AM2017-2259

Expansion and activation of granulocytic, myeloid-derived suppressor cells in childhood precursor B cell acute lymphoblastic leukemia.

Precursor B cell acute lymphoblastic leukemia (B-ALL) is a B cell-derived, malignant disorder with the highest incidence among children. In addition to the genetic abnormality, a dysregulated immune system also has an important role in the pathogenesis of B-ALL. Myeloid-derived suppressor cells (MDSCs) represent one of the key drivers in immune tolerance against tumor cells, including various solid tumors and hematologic malignancies. The role of MDSCs in B-ALL remains poorly understood. Here, we showed that the granulocytic (G)-MDSC population was significantly elevated in both the peripheral blood and BM of patients with B-ALL, when compared with age-matched healthy controls. G-MDSCs levels correlated positively with clinical therapeutic responses and B-ALL disease prognostic markers, including minimal residual disease, and the frequencies of CD20+ and blast cells. The immunosuppressive function of B-ALL-derived G-MDSCs was mediated through the production of reactive oxygen species and required direct cell-cell contact, with the potential participation of STAT3 signaling. Overall, the results of our study support accumulation and activation of G-MDSCs as a novel mechanism of immune evasion of tumor cells in patients with B-ALL and may be a new therapeutic target.

Myeloid derived suppressor cells (MDSC) and inflammatory biomarkers in metastatic urothelial carcinoma (mUC).

4548 Background: MDSC are potent immunosuppressive cells with prognostic implications in many solid tumors. We previously reported significant correlations between MDSC and clinicopathologic features in localized UC. We hypothesized that different MDSC populations may correlate with inflammatory biomarkers and clinicopathologic features in mUC. Methods: Peripheral blood samples were collected from 46 mUC pts. MDSCs were measured in fresh unfractionated whole blood (WB) and in peripheral blood mononuclear cells (PBMC). MDSCs were identified by flow cytometry in WB and defined as LinloCD33+/HLADR- [(T)otal MDSC]. MDSC subsets were defined as (G)ranulocytic (CD15+CD14-), (M)onocytic (CD15-CD14+), (I)mmature (CD15-CD14-), or CD11b+. MDSC populations were presented as % of live nucleated blood cells and as absolute numbers from WB. Spearman correlations (r) and Wilcoxon rank sum test were used to assess correlations between MDSC populations & clinicopathologic factors. Results: Of 46 pts:78% men, median age at diagnosis 69 (31-83), 33% never smokers, 76% pure UC, 76% bladder primary, 28% prior intravesical therapy, 35% prior neoadjuvant chemotherapy, 56% prior cystectomy, 83% overweight/obese. G-MDSC was the predominant subset in WB (43%) and PBMC (39%), although M-MDSC were almost equally predominant in PBMC (35%). There was a correlation between the WB and PBMC values of T-, I-, and M- MDSC (p≤0.05). Higher % WB I-MDSC correlated with lower blood neutrophil/lymphocyte ratio (NLR) (p = 0.009), while higher WB G-MDSC and %PBMC G-MDSC were associated with higher NLR (p = 0.03 and p = 0.02, respectively). Higher I-MDSC / G-MDSC ratio was associated with lower NLR (r = -0.35, p = .02) and with various clinicopathologic parameters. Conclusions: HigherI-MDSC / G-MDSC ratio correlates inversely with NLR, which is considered an inflammatory biomarker and had prognostic value in other studies. The mechanism of MDSC interaction with inflammatory response in mUC pts merits evaluation and is being investigated in a larger cohort of UC pts on chemotherapy or immunotherapy (with longer follow up).

Serial measurements of myeloid derived suppressor cells (MDSC) in metastatic urothelial carcinoma (mUC) patients (pts) treated with immune checkpoint inhibitors (CI).

e16005 Background: MDSC are a heterogeneous population of immunosuppressive cells with potentially predictive implications in UC pts receiving CI. We hypothesized that MDSC populations may change after CI exposure. Methods: Serial peripheral blood samples were collected from mUC pts treated with CI. MDSC were measured in fresh unfractionated whole blood (WB) and in peripheral blood mononuclear cells (PBMC). MDSC were identified by flow cytometry in WB and defined as LinloCD33+/HLADR- [(T)otal MDSC]. MDSC subsets were defined as (G)ranulocytic (CD15+CD14-), (M)onocytic (CD15-CD14+), (I)mmature (CD15-CD14-), or CD11b+. MDSC populations were presented as % of live nucleated blood cells and as absolute numbers from WB. The Wilcoxon signed rank and rank sum tests were used to assess changes in MDSC populations while on CI. Results: 17 pts treated with CI (9 atezolizumab [A], 8 pembrolizumab [P]) had ≥ 2 MDSC samples for analysis. Median age at diagnosis was 71 (46-81), 12 men, 29% never smokers; 53% / 29% / 18% ECOG PS 0/1/2 and 59% visceral metastasis at the time of 1st sample collection. 10 pts received CI as 1st line therapy (Tx) in metastatic setting; 7 pts received chemotherapy as 1st-line Tx for mUC (6 platinum-based, 1 docetaxel) and CI as 2nd-line Tx. In 16 pts with samples before 1stdose, there was a relative decrease (median 36.3%, range -59.7 to +21.2) in PBMC % I-MDSCs between 1st and 2nd samples (p=0.06). Interestingly, PBMC %M-MDSC and %I-MDSC tended to increase compared to baseline in pts treated with P, while they tended to decrease in pts treated with A (Table). Conclusions: In this cohort of pts with mUC treated with CIs,MDSC changes differed based on CI (anti-PDL1 or anti-PD1). Further study in larger cohort with various prior Tx lines and longer follow up as well as correlations with Tx response, toxicity and outcomes are ongoing. [Table: see text]

Real-world experience with atezolizumab (atezo) in advanced urothelial cancer (UC).

e16031 Background: Atezo was recently FDA-approved for patients (pts) with UC (IMvigor210 trial). We assessed the safety and efficacy of Atezo in the real world setting and explored the role of putative biomarkers. Methods: Data from 44 consecutive non-trial pts with UC treated with ≥1 dose of Atezo 1200mg IV q3w at Cleveland Clinic (May - Dec 2016) were available. Demographic, clinicopathologic, biomarker data, including blood neutrophil to lymphocyte ratio (NLR) and myeloid derived suppressor cell (MDSC) populations [LinloCD33+/HLADR-(Total), and Granulocytic(CD15+CD14-), Monocytic(CD15-CD14+), Immature(CD15-CD14-), CD11b+ subsets], were collected. Response, progression-free survival (PFS) and overall survival (OS) were measured (RECIST v1.1). Adverse events (AE) were reported (CTCAE v4.03). Outcome data were analyzed with Fisher’s exact test, chi-square tests, log rank test and proportional hazards models. Results: Median age was 68y (34-88), 68% men, 68% pure urothelial histology, 27% never smokers; 61% PS > 0, 61% visceral metastases, 57% CrCl < 60 mL/min, 84% bladder primary, 61% prior cystectomy. In metastatic setting, 25% had ≥2 prior systemic therapies, 84% prior platinum-based regimen, and 9% no prior therapy. With median follow-up of 3.3 months ( < 1 - 6.6) and median number of 3 doses, in 38 pts evaluable for response, 21% had PR; 24% SD; 61% progressed, 27% died; estimated median (m)PFS was 3 months (95%CI 2.2 - 4.3) and mOS was 5.2 months (95%CI 4.6 - NR). Only 3/27 eligible pts got subsequent treatment. Number of metastatic sites, 2 Bellmunt prognostic factors, and NLR ≥ 5.0 negatively impacted response, PFS and OS. MDSC populations (data available in 14 pts) demonstrated non-significant correlation with worse outcome and elevated NLR. Most frequent all-cause AE ( > 20%) were fatigue (60%, G3:9%), anorexia (33%, G3:7%), elevated creatinine (26%, G3:5%); 1 pt had G4 hyperbilirubinemia. There were no treatment-related deaths. Conclusions: To our knowledge, this is the largest real-world experience with Atezo in UC. Response and toxicity were comparable to IMvigor210 trial; estimated mOS was shorter but with limited follow up. Higher NLR had negative prognostic value, while the role of MDSCs merits further exploration.

Metastasized murine oral squamous cell carcinoma cells induce intratumoral polymorphonuclear myeloid derived suppressor cells.

Myeloid derived suppressor cells (MDSCs) localize to hematopoietic organs and peripheral blood during inflammation or tumor tissues and lymph nodes in the presence of a tumor. However, whether there are differences in MDSCs found in the primary tumor and metastases is unknown. In the present study, we established a cell line of metastasized tumor cells to a lymph node, L5-11, which were derived from the Sq-1979 mouse buccal mucosa squamous cell carcinoma cell line. We then analyzed tumor immunogenicity, especially with regard to MDSCs, to clarify the differences between the primary tumor and metastases, using an isogenic heterotopic tumor transplantation model. Our data showed that the population of intratumoral MDSCs, especially polymorphonuclear MDSCs in the lymph node metastasis model were significantly increased compared with syngeneic grafts from the primary cell line Sq-1979 after 21 days. Furthermore, we identified that the lymph node metastasis cell line had increased expression of genes that promote the expansion of MDSCs, tumor growth and metastasis. Hence, these data suggest that tumor immunosuppression can occur via activation of MDSCs. However, further examination is required to clarify whether all or a subset of these factors from the lymph node metastasis tumor cells are required to induce intratumoral MDSCs.

Highlights of This Issue

Because of poor lysosomal degradation, HER2-directed antibody-drug conjugate (ADC), T-DM1 is efficient only in patients with high HER2 overexpression. Thus, there is a need for HER2-directed ADCs effective in patients expressing low/moderate levels of HER2. Prolactin receptor (PRLR) is rapidly and constitutively degraded in lysosomes. Cross-linking HER2 to PRLR, using a HER2 x PRLR bispecific ADC, dramatically enhances lysosomal degradation of HER2. Accordingly, anti-HER2 x PRLR bispecific ADC significantly improves upon T-DM1 efficacy in cells expressing intermediate levels of HER2. These results demonstrate that coupling a tumor-specific ADC target to a rapidly internalizing protein may be a useful approach to enhance efficacy of ADCs.Delta-24-RGD is an oncolytic virus whose replication depends upon a defective p16/RB/E2F pathway, an alternation found in ∼85% of pancreatic adenocarcinomas (PDAC). Dai and colleagues report that exposure of human PDAC cells to Delta-24-RGD induced dramatic cytotoxicity accompanied by robust exposure of phosphatidylserine (PS) on PDAC cell membranes. Sequential combination of Delta-24-RGD followed by a PS-targeting antibody, 1N11, enhanced anticancer activity and favorably altered the innate immune tumor microenvironment. These results suggest that combining these agents sequentially could achieve positive clinical results when used in a molecularly-defined subset of pancreatic cancer.The presence of mature mast cells within tumor tissue, expression of stem cell factor by many tumor cells and previous reports of interactions between mast cells and other innate immune cells suggest that KIT signaling within mast cells may influence tumor growth. Using a KIT-specific inhibitory antibody in syngeneic mouse tumor models, Garton and colleagues demonstrated that anti-KIT treatment enhanced the anti-tumor activity of immune checkpoint inhibitors (anti-CTLA-4 or anti-PD-1), and promoted immune responses by selectively reducing the immunosuppressive monocytic myeloid-derived suppressor cell (M-MDSC) population. These data provide a rationale for clinical investigation of a KIT-directed antibody in combination with checkpoint inhibitors.Using high-throughput profiling of cancer cell lines with candidate anti-cancer agents, Lee and colleagues identified determinants of the response to inhibitors of the Chk1 kinase, a mediator of the DNA damage response. While sensitivity to Chk1 inhibition was previously linked to p53 mutation status, these new findings revealed an unexpected requirement for Chk1 in cells experiencing genotoxic stress after Ras-MEK pathway activation. Chk1 inhibition combined with DNA-targeted chemotherapeutics led to enhanced cell killing in some osteosarcoma, ovarian, and breast cancer cells. These findings provide insight on how to improve the efficacy of Chk1 inhibition in a subpopulation of patients.

Anti-KIT Monoclonal Antibody Treatment Enhances the Antitumor Activity of Immune Checkpoint Inhibitors by Reversing Tumor-Induced Immunosuppression

The receptor tyrosine kinase KIT is an established oncogenic driver of tumor growth in certain tumor types, including gastrointestinal stromal tumors, in which constitutively active mutant forms of KIT represent an actionable target for small-molecule tyrosine kinase inhibitors. There is also considerable potential for KIT to influence tumor growth indirectly based on its expression and function in cell types of the innate immune system, most notably mast cells. We have evaluated syngeneic mouse tumor models for antitumor effects of an inhibitory KIT mAb, dosed either alone or in combination with immune checkpoint inhibitors. Anti-KIT mAb treatment enhanced the antitumor activity of anti-CTLA-4 and anti-PD-1 mAbs, and promoted immune responses by selectively reducing the immunosuppressive monocytic myeloid-derived suppressor cell population and by restoring CD8+ and CD4+ T-cell populations to levels observed in naïve mice. These data provide a rationale for clinical investigation of the human KIT-specific mAb KTN0158 in novel immuno-oncology combinations with immune checkpoint inhibitors and other immunotherapeutic agents across a range of tumor types. Mol Cancer Ther; 16(4); 671-80. ©2017 AACR.

Assessment of blood and tissue myeloid derived suppressor cells (MDSC), clinicopathologic factors, and treatment response in urothelial carcinoma (UC) patients (pts) undergoing surgery.

362 Background: MDSC are heterogeneous immunosuppressive cells with potential predictive/prognostic role in cancer. The association between MDSC, clinicopathologic factors and pathologic response in pts with UC merits evaluation. Methods: Peripheral blood and/or tissue was collected from 120 pts. MDSC were measured in fresh unfractionated whole blood (WB), in peripheral blood mononuclear cells (PBMC) and fresh tumor tissue. MDSCs were identified by flow cytometry in WB and defined as LinloCD33+/HLADR- ((T)otal MDSC). MDSC subsets were defined as LinloCD33+/HLADR- and (G)ranulocytic (CD15+CD14-), (M)onocytic (CD15-CD14+), (I)mmature (CD15-CD14-, CD11b+ ). MDSC populations were presented as % of live nucleated blood cells and as absolute numbers from WB. Spearman correlations (r) and Wilcoxon rank sum test were used to assess correlations between MDSC populations, clinicopathologic factors and pT0N0%. Results: Of 120 pts, 82 were non-metastatic: 58 had only blood, 23 had blood & tissue, 1 had only tissue available for analysis. Of these 82 non-metastatic pts, 70 were men, median age 68; 81 pts had primary UC histology, 1 small cell cancer, 24 had mixed UC histology; 24 had prior intravesical therapy, 34 had neoadjuvant therapy (79% cisplatin-based, 21% unknown), 4 pts had post-op recurrence. At cystectomy: 15/82 pT0, 22/82 pT3/4; 37/82 CIS; 8/78 pN+. Significant associations were seen between MDSC blood levels and mixed histology, CIS, pN+, and lower pT0N0% (Table). Tumor M-MDSCs were associated with pN+ (p=0.05). There was significant correlation between tumor and WB % M-MDSC (r=0.55, p=0.007), and tumor and WB % G-MDSC (r=0.46, p=0.03). Conclusions: Blood MDSC levels correlate with several clinicopathologic factors and may predict pathological complete response (pT0N0). Assessment of association between MDSC levels, outcome and immunotherapy response is ongoing including in metastatic pts. [Table: see text]

Abstract P2-04-12: Targeting of phosphatidylserine by monoclonal antibodies enhances the activity of immune checkpoint lag3 targeting antibodies in murine breast tumors

Abstract Previous preclinical modeling has demonstrated that antibodies targeting the programmed cell death ligand 1 (PD-1) have activity in murine breast cancers (BC), and this activity is significantly enhanced when combined with phosphatidylserine (PS) targeting antibodies. This suggested that the addition of PS targeting antibodies could be capable of augmenting anti-tumorigenic properties of other checkpoint inhibitors and alternative immune activating therapies in BC. The ability of PS targeting antibodies to enhance the activity of anti-PD-1 therapy occurs in part through increasing tumor infiltrating lymphocytes (TILs), boosting the Th1 immuno-profile, and the suppression of tumor promoting cytokines induced by anti-PD-1 therapy. PS normally resides in the inner leaflet of the plasma membrane in many types of cells. Conditions that incite cellular stress in the tumor microenvironment, such as ROS, hypoxia, and irradiation, promote PS externalization and exposure on tumor associated endothelial cells and tumor cells, where it is recognized by specific receptors, including members of TIM and TAM family. This PS recognition promotes an innate system driven immunosuppressive condition in part by promoting the recruitment of myeloid derived suppressor cells (MDSCs), M2-like macrophages, and suppressing dendritic cells maturation, while inducing anti-inflammatory cytokines. To identify additional immuno-therapeutic targets that may have activity when used in combination with PS targeting antibodies, we employed bioinformatics analysis on tumors from murine triple negative breast cancer (TNBC) treated with PS and PD-1 targeting antibodies, alone or in combination. Interestingly, expression of the lymphocyte activation gene 3 (LAG3) increased in each treatment arm, suggesting it may be a potential target for combinational therapy in breast cancers. To test this hypothesis, immune competent mice harboring the murine TNBC line E0771 were treated with a PS targeting antibody or a LAG3 targeting antibody as single agent or in combination. Our data demonstrate that while PS-blocking and anti-LAG3 therapies each have efficacy in E0771 as single agents, combinational treatment significantly improved growth inhibition and was capable of increasing TILs, including CD8 + and CD3 + T-cells, while reducing the population of MDSCs. Overall, our data suggest that LAG3 targeting may also represent a viable option for the treatment of breast cancer and that the addition of PS targeting antibodies to LAG3 therapy can effectively increase the anti-tumor and immune-activating effects mediated by additional T-cell checkpoint therapies. Citation Format: Gray MJ, Gong J, Hutchins JT, Freimark BD. Targeting of phosphatidylserine by monoclonal antibodies enhances the activity of immune checkpoint lag3 targeting antibodies in murine breast tumors [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-12.

Expansion of HLA-G-Expressing Myeloid-Derived Suppressor Cells in Patients with Chronic Hepatitis B Virus Infection

Both human leukocyte antigen-G (HLA-G) and myeloid-derived suppressor cells (MDSCs) was associated with the pathogenesis of infectious diseases. Whether peripheral MDSCs express HLA-G during virus infection remains unknown. We investigated the frequency of HLA-G+ MDSCs and its subsets in patients infected with chronic hepatitis B (CHB). In this study, frequencies of peripheral MDSCs (Lin1-HLA-DR-CD33+CD11b+) and HLA-G expressing subsets from 50 CHB patients and 27 normal controls were analyzed using flow cytometry. Data revealed the median percentage of MDSCs was not significantly different between the CHB patients and controls (0.30% vs. 0.29%; p=0.884). Among MDSCs, similar frequency was observed for CD14+ monocytic MDSC (mMDSCs; 31.25% vs. 23.35%; p=0.063) and CD15+ granulocytic MDSC (gMDSCs; 22.60% vs. 21.55%; p=0.558) between the two groups. However, HLA-G+ MDSCs was significantly increased in CHB patients compared with that of controls (3.30% vs. 0.50%; p<0.001). Furthermore, both HLA-G+ mMDSCs (0.99% vs. 0.00%; p<0.001) and HLAG+ gMDSC (0.78% vs. 0.00%; p<0.001) were also dramatically increased in CHB patients. Particularly, HLA-G+ gMDSC was inversely correlated to the viral DNA loads and significantly increased in HBeAb positive patients. Summary, this work reports for the first time HLA-G+ MDSCs, a new population of peripheral MDSCs, were expanded in CHB patients; however, its clinical relevance yet to be further explored.

Myeloid-derived suppressor cells and myeloid regulatory cells in cancer and autoimmune disorders.

Myeloid-derived suppressor cells (MDSCs) were originally described as a heterogeneous population of immature cells derived from myeloid progenitors with immune-suppressive functions in tumor-bearing hosts. In recent years, increasing number of studies have described various populations of myeloid cells with MDSC-like properties in murine models of cancer and autoimmune diseases. These studies have observed that the populations of MDSCs are increased during inflammation and autoimmune conditions. In addition, MDSCs can effectively suppress T cell responses and modulate the activity of natural killer cells and other myeloid cells. MDSCs have also been implicated in the induction of regulatory T cell production. Furthermore, these cells have the potential to suppress the autoimmune response, thereby limiting tissue injury. Myeloid regulatory cells (Mregs) are recently attracting increasing attention, since they function in proinflammatory and immune suppression in autoimmune diseases, as well as in various types of cancer. Currently, research focus is directed from MDSCs to Mregs in cancer and autoimmune diseases. The present study reviewed the suppressive roles of MDSCs in various autoimmune murine models, the immune modulation of MDSCs to T helper 17 lymphocytes, as well as the proinflammatory and immunosuppressive roles of Mregs in various types of cancer and autoimmune diseases.

Immature myeloid-derived suppressor cells: A bridge between inflammation and cancer (Review).

Chronic inflammation is considered to be one of the hallmarks of tumor initiation and progression. Changes occurring in the microenvironment of progressing tumors resemble the process of chronic inflammation, which begins with ischemia followed by interstitial and cellular edema, appearance of immune cells, growth of blood vessels and tissue repair, and development of inflammatory infiltrates. Moreover, long‑term production and accumulation of inflammatory factors lead to local and systemic immunosuppression associated with cancer progression. Of the several mechanisms described to explain this anergy, the accumulation of myeloid cells in the tumor, spleen, and peripheral blood of cancer patients has gained considerable interest. A population of suppressive CD11b+Gr-1+cells has in fact been designated as myeloid-derived suppressor cells (MDSCs). MDSCs are a unique category of the myeloid lineage, and they induce the prevention of the development of cytotoxic T lymphocytes(CTLs) invitro, and the induction of antigen-specific CD8+T-cell tolerance invivo. Therapeutic approaches directed toward the manipulation of the MDSC population and their function may improve chemoimmune-enhancing therapy for advanced malignancies.

Histone deacetylase inhibitors deplete myeloid-derived suppressor cells induced by 4T1 mammary tumors in vivo and in vitro.

Myeloid-derived suppressor cells (MDSC) have been identified as a population of immature myeloid cells that suppress anti-tumor immunity. MDSC are increased in tumor-bearing hosts; thus, depletion of MDSC may enhance anti-tumor immunity. Histone deacetylase inhibitors (HDACi) are chemical agents that are primarily used against hematologic malignancies. The ability of these agents to modulate anticancer immunity has recently been extensively studied. However, the effect of HDACi on MDSC has remained largely unexplored. In the present study, we provide the first demonstration that HDACi treatment decreases MDSC accumulation in the spleen, blood and tumor bed but increases the proportion of T cells (particularly the frequency of IFN-γ- or perforin-producing CD8+ T cells) in BALB/C mice with 4T1 mammary tumors. In addition, HDACi exposure of bone marrow (BM) cells significantly eliminated the MDSC population induced by GM-CSF or the tumor burden in vitro, which was further demonstrated as functionally important to relieve the inhibitory effect of MDSC-enriched BM cells on T cell proliferation. Mechanistically, HDACi increased the apoptosis of Gr-1+ cells (almost MDSC) compared with that of Gr-1- cells, which was abrogated by the ROS scavenger N-acetylcysteine, suggesting that the HDACi-induced increase in MDSC apoptosis due to increased intracellular ROS might partially account for the observed depletion of MDSC. These findings suggest that the elimination of MDSC using an HDACi may contribute to the overall anti-tumor properties of these agents, highlighting a novel property of HDACi as potent MDSC-targeting agents, which may be used to enhance the efficacy of immunotherapeutic regimens.