Myeloid-derived Suppressor Cells Migration Research Articles

Abstract PO-046: Dissecting the role of tumor-intrinsic Cxcl1 in mediating immune exclusion in Ras-p53 cooperative pancreatic cancer

Abstract INTRODUCTION: Ras-p53 cooperative mutations, which define >50% of human pancreatic ductal adenocarcinoma (PDAC), promote an immune-excluded tumor microenvironment. In Ras-p53 murine models that phenocopy human PDAC, overexpression of the myeloid chemo-attractant Cxcl1 is associated with T-cell exclusion. We sought to dissect the role of tumor-intrinsic Cxcl1 in mediating immune exclusion in Ras-p53 PDAC. METHODS: Single-cell immune deconvolution and Cxcl1 expression were queried in TCGA PDAC samples (n=150). Single-cell RNA sequencing (scRNAseq) data in KrasG12D/+;Trp53fl/+;Pdx1Cre (KPC WT) genetically engineered mice (GEM) were interrogated. CRISPR/Cas9-based genome editing was used to silence Cxcl1 in KPC tumor cells (KPC-Cxcl1KO). Multiparameter flow cytometry-based immunophenotyping of tumor-infiltrating and circulating myeloid/T-cell populations was performed in in-vivo orthotopic models utilizing KPCWT / KPC-Cxcl1KO cells. RESULTS: In human TCGA samples, Cxcl1 overexpression was strongly associated with expression of its cognate receptor CXCR2, reduced CD4+/CD8+ T-cells, and increased CD33+ myeloid-derived suppressor cells (MDSCs). In KPC GEMs, scRNAseq revealed that CXCR2 expression was exclusively present on the polymorphonuclear MDSC subset (PMN-MDSC). Exogenous Cxcl1 conditioning in murine bone marrow-derived cells induced PMN-MDSC polarization and upregulated CXCR2. Genetic ablation of Cxcl1 in KPC tumor cells disrupted transwell migration of splenocyte-enriched MDSC in-vitro; this effect was dependent on CXCR2 expression on MDSCs. There was a dramatic reduction in tumor weights and metastatic outgrowth (p<0.0001), as well as circulating PMN-MDSC and CXCR2+-MDSC populations in KPC-Cxcl1KO compared with KPCWT mice (p=0.0006). Significantly fewer PMN-MDSC and M2-like macrophages infiltrated KPC-Cxcl1KO tumors, which was accompanied by dramatically higher CD4+/CD8+ T-cell infiltration (p<0.0001). While cytolytic effector CD8+ T-cell populations (CD44+62L-CD107a+) remained unchanged, non-cytolytic “exhausted” PD-1+CD8+ T-cells (PD-1+CD107a-) increased in KPC-Cxcl1KO vs. KPCWT tumors, providing rationale for addition of checkpoint blockade to reverse T-cell exhaustion. CONCLUSION: Tumor-intrinsic Cxcl1 appears a major contributor to immune exclusion in Ras-p53 cooperative PDAC. Targeting Cxcl1 represents a promising strategy to overcome MDSC-mediated immunosuppression and improve susceptibility to checkpoint blockade in PDAC. Citation Format: Anna Bianchi, Siddharth Mehra, Daisy Dai, Iago de Castro Silva, Prateek Sharma, Antonio Colaprico, Austin R. Dosch, Samara Singh, Nagaraj S. Nagathihalli, Nipun B. Merchant, Jashodeep Datta. Dissecting the role of tumor-intrinsic Cxcl1 in mediating immune exclusion in Ras-p53 cooperative pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-046.

Thymosin alpha-1 blocks the accumulation of myeloid suppressor cells in NSCLC by inhibiting VEGF production

BackgroundThymosin alpha-1 (TA) has been reported to inhibit tumor growth as an immunomodulator. However, its mechanism of action in immunosuppressive cells is unclear. The purpose of this study was to investigate whether TA can reshape the immune microenvironment by inhibiting the function of myeloid-derived suppressor cells (MDSCs) in non-small cell lung carcinoma (NSCLC). MethodsThe effects of TA on peripheral blood monocytic MDSCs (M-MDSCs) in patients with NSCLC and on the apoptosis and migration of M-MDSCs were studied. A mouse subcutaneous xenograft tumor model was constructed, and the effect of TA on M-MDSC migration was evaluated. Quantitative real-time PCR, Western blotting, flow cytometry and immunohistochemistry were used to examine the mechanism by which TA affects M-MDSCs. ResultsTA not only promoted the apoptosis of M-MDSCs by reducing the Bcl-2/BAX ratio but also and more importantly inhibited the migration of MDSCs to the tumor microenvironment by suppressing the production of vascular endothelial growth factor (VEGF) through the downregulation of hypoxia-inducible factor (HIF)-1α in tumor cells. ConclusionsTA may have a novel antitumor effect mediated by decreasing M-MDSC accumulation in the tumor microenvironment through reduced VEGF production.

A pilot study of Bruton’s tyrosine kinase inhibitor ibrutinib alone and in combination with PD-1 inhibitor nivolumab in patients with metastatic solid tumors.

3111 Background: Myeloid-derived suppressor cells (MDSC) are expanded in cancer and promote immune suppression. We have shown that ibrutinib inhibits migration and immunosuppressive function of MDSC. Moreover, the combination of ibrutinib and a PD-L1 inhibitor has been found to have synergistic anti-tumor effects in a multiple solid tumor mouse models. Therefore, we conducted a pilot study testing the combination of ibrutinib and nivolumab in patients with metastatic solid tumors. Methods: Sixteen patients with advanced solid tumors were recruited to this trial. Ibrutinib was dosed as an oral single agent, starting 7 days prior to cycle 1 of nivolumab and given until cycle 1, day 8 of nivolumab. Nivolumab was administered intravenously on days 1 and 15 on 28-day cycles. Patients had blood samples collected prior to initiation of ibrutinib, day 1 of cycle 1, day 8 of cycle 1, day 1 of cycle 2, and at the time of disease progression. From these specimens, we measured circulating MDSC levels, other circulating immune subsets, T cell proliferation, and cytokines/chemokines levels. Circulating MDSC levels were measured by mass spectrometry. T cell function was evaluated by CFSE to monitor proliferating cells by dye dilution and cytokine/chemokine levels were measured with a U-PLEX assay. Data were analyzed using two-tailed, paired Student's t-tests to assess statistical significance. Results: An increase in circulating MDSC (22% to 28%; SD 9.158) levels was observed following 7 days of single-agent ibrutinib compared to baseline. However, in combination therapy, MDSC levels decreased (19%; SD 13.17) prior to cycle 2. Despite increasing levels of circulating MDSC, T cell function improved throughout the study. Furthermore, plasma levels of chemokines associated with MDSC recruitment and migration significantly decreased with ibrutinib treatment (IL-12, CCL2, CCL3, and CCL4). Of the 16 patients, four achieved a partial response and four achieved stable disease. Median progression free survival was 3.5 months and median overall survival was 11.5 months. Conclusions: The combination of ibrutinib and nivolumab was well tolerated, demonstrated early signs of immune modulation, and showed preliminary signs of promising clinical activity in patients with metastatic solid tumors. Clinical trial information: NCT03525925 .

Myeloid-derived suppressor cells modulate aortic aneurysm formation via CXCR2 and IL-17 signaling

Abstract Aortic aneurysm formation is associated with vascular inflammation and remodeling mediated via CD4+ T cell-secreted IL-17A production. Our hypothesis is that innate myeloid-derived suppressor cells (MDSCs), which are a heterogeneous population of myeloid precursor and progenitor cells, exhibit an immunoregulatory role in the progression of aortic inflammation during abdominal aortic aneurysm (AAA) formation. The trafficking and migration of MDSCs to the aortic tissue and role of CXCR2 and programmed death-1 (PD-1) receptor signaling was deciphered using an in vivo elastase-treatment model of AAA as well as in vitro co-culture experiments. We used an established murine AAA model of topical elastase-treatment in the present study. Preliminary data showed that elastase-treatment of C57BL/6 (wild-type; WT) mice resulted in significant increase in infrarenal aortic diameter causing AAA formation on day 14 that was significantly inhibited by a combined treatment with anti-CXCR2 and PD-1 antibodies. A multi-fold increase in MDSCs (CD11b+Gr-1+) in aortic tissue of elastase-treated WT mice on days 7 and 14 occurred compared to controls. Co-culture of MDSCs with aortic endothelial cells in the presence of anti-CXCR2 antibody inhibited the transmigration of MDSCs through the endothelial barrier. Moreover, transient elastase treatment of co-cultures of anti-CD3/CD28 treated mononuclear cells with MDSCs significantly enhanced IL-17A secretion compared to cultures without MDSCs. Our results indicate that MDSCs may play a contributory role in aortic inflammation by trafficking in a CXCR2-dependent manner and promotion of IL-17A secretion, signifying the importance of MDSC-mediated immunoregulation of aortic aneurysms.

Abstract B52: KRAS-IRF2 axis drives immune suppression and immune therapy resistance in colorectal cancer

Abstract The precise biologic functions and mechanisms of oncogenic KrasG12D (Kras*) in therapeutic resistance, including immune therapy, are not understood. Here, utilizing a colorectal cancer (CRC) mouse model engineered with an inducible Kras* transgene and conditional null APC and Trp53 alleles (iKAP) that we recently established (Boutin et al., Gene & Development 2017), we observed that KRAS*-expressing invasive tumors possess a prominent immune-suppressive profile characterized by a preponderance of myeloid-derived suppressor cells (MDSCs). Integrated transcriptomic and genomic analyses identified the Interferon Regulatory Factor 2 (IRF2) gene as a key downstream target of KRAS* mediating tumor progression and immune suppression. Mechanistically, integrated transcriptomics, cytokine profiling and ChIP-seq analysis identified CXCL3 as a key direct target of IRF2, and pharmacologic inhibition of CXCL3 or its receptor CXCR2 resulted in suppression of MDSC migration and infiltration in KRAS*-expressing CRC tumors. Translational studies established that de novo anti-PD1 resistance of KRAS*-expressing tumors can be overcome by enforced IRF2 expression or by treatment with small-molecule CXCR2 antagonist SX-682. Correspondingly, CRC patient tumors with higher IRF2 expression show increased response to anti-PD1 therapy. This KRAS*-mediated immune suppressive mechanism provides a framework for patient selection and combination therapies to enhance the effectiveness of immune checkpoint blockade therapy in advanced CRC. Citation Format: Wenting Liao, Michael Overman, Adam Boutin, Scott Kopetz, Ron DePinho, Alan Wang. KRAS-IRF2 axis drives immune suppression and immune therapy resistance in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B52.

Abstract IA27: Context-dependent role of KRAS in GI malignancies

Abstract Oncogenic Kras (Kras*) plays a key role in tumor maintenance and therapeutic resistance in several cancer types, although the precise biologic functions and mechanisms are not completely understood. We had previously shown in pancreatic ductal adenocarcinoma that some tumors underwent spontaneous relapse and were devoid of Kras* expression and downstream canonical MAPK signaling, instead acquiring amplification and overexpression of the transcriptional coactivator Yap1. Functional studies established the role of Yap1 and the transcriptional factor Tead2 in driving Kras*-independent tumor maintenance. In a second previous study, we developed a faithful colorectal cancer (CRC) mouse model and showed that Kras* drove invasion and maintenance in CRC. Here, utilizing this CRC mouse model, we demonstrate that KRAS* promotes a pronounced immune-suppressive profile distinguished by a predominance of myeloid-derived suppressor cells (MDSCs). We performed integrated transcriptomic and genomic analyses that identified IRF2 (Interferon Regulatory Factor 2) as a key KRAS* effector mediating immune suppression. IRF2 suppresses MDSC migration and infiltration in KRAS*-expressing CRC tumors by targeting the CXCL3/CXCR2 axis. We also observed that de novo resistance of KRAS*-expressing tumors to anti-PD1 therapy could be overcome by enforced IRF2 expression or by treatment with SX-682, a small-molecule CXCR2 antagonist. In addition, CRC patient tumors with higher IRF2 expression showed increased response to anti-PD1 therapy. This KRAS*-mediated immune-suppressive mechanism provides a framework for CRC patient selection and combination therapies to enhance the effectiveness of immune checkpoint blockade therapy in advanced disease. Citation Format: Wen-Ting Liao, Michael J. Overman, Adam Boutin, Prasenjit Dey, Di Zhao, Guocan Wang, Jiexi Li, Zhengdao Lan, Jun Li, Xiaoying Shang, Ming Tang, Shan Jiang, Xingdi Ma, Peiwen Chen, Riham Katkhuda, Krittiya Korphaisarn, Deepavali Chakravarti, Qing Chang, Jianhua Zhang, Dipen M. Maru, Dean Y. Maeda, John A. Zebala, Scott Kopetz, Y. Alan Wang, Ronald A. DePinho. Context-dependent role of KRAS in GI malignancies [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA27.

Activated hepatic stellate cells regulate MDSC migration through the SDF-1/CXCR4 axis in an orthotopic mouse model of hepatocellular carcinoma.

Hepatic stellate cells (HSCs) are important stromal cells and pivotal mediators involved in the pathogenesis and immunosuppression of hepatocellular carcinoma (HCC). The liver has been demonstrated to be a site for accumulation of tumor-induced myeloid-derived suppressor cells (MDSCs). We previously reported that HSCs induced an increase in the number of MDSCs in HCC. However, how MDSCs are recruited in HCC remains largely unclear. In the present study, we found that HSC-conditioned medium (HSC-CM) induced bone marrow-derived cell and splenocyte migration, especially MDSC migration. Using chemokine-neutralizing antibodies and chemokine receptor inhibitors, we found that HSCs promoted MDSC migration through the SDF-1/CXCR4 axis. Subsequently, we used an orthotopic mouse liver tumor model to determine how HSCs mediated MDSC migration to HCC in vivo. The in vivo results indicated that pretreatment of MDSCs with a CXCR4 inhibitor or injection with SDF-1-knocked down HSCs inhibited MDSC migration to the spleen and liver of the tumor-bearing mice. Together, our findings indicate a central role for HSCs in MDSC migration mediated by the SDF-1/CXCR4 axis, thus revealing a potentially effective approach for modulating the tumor microenvironment by targeting HSCs in HCC.

LDK378 inhibits the recruitment of myeloid-derived suppressor cells to spleen via the p38-GRK2-CCR2 pathway in mice with sepsis.

Myeloid-derived suppressor cells (MDSCs) are functionally immunosuppressive cells that are persistently increased in abundance and associated with adverse clinical outcomes in sepsis. Here, we investigated the therapeutic potential of an anaplastic lymphoma kinase inhibitor, LDK378, in cecal ligation and puncture (CLP)-induced polymicrobial sepsis and examined its effects on the recruitment of MDSCs. LDK378 significantly improved the survival of CLP-induced polymicrobial septic mice, which was paralleled by reduced organ injury, decreased release of inflammatory cytokines and decreased recruitment of MDSCs to the spleen. Importantly, LDK378 inhibited the migration of MDSCs to the spleen by blocking the CLP-mediated upregulation of CC chemokine receptor 2 (CCR2), a chemokine receptor critical for the recruitment of MDSCs. Mechanistically, LDK378 treatment blocked the CLP-induced CCR2 upregulation of MDSCs via partially inhibiting the phosphorylation of p38 and G-protein-coupled receptor kinase-2 (GRK2) in bone marrow MDSCs of septic mice. Furthermore, invitro experiments also showed that lipopolysaccharide (LPS)-induced migration of MDSCs was similarly owing to the activation of GRK2 and upregulation of CCR2 by LPS, whereas the treatment with LDK378 partially blocked the LPS-induced phosphorylation of p38 and GRK2 and decreased the expression of CCR2 on the cell surface, therefore leading to the suppression of MDSC migration. Together, these findings unravel a novel function of LDK378 in the host response to infection and suggest that LDK378 could be a potential therapeutic agent for sepsis.

Targeting myeloid-derived suppressor cells in the treatment of hepatocellular carcinoma: current state and future perspectives.

Systemic therapy for advanced hepatocellular carcinoma (HCC) has been focusing on overcoming tumor angiogenesis and immunosuppression. Myeloid-derived suppressor cells (MDSCs) promote both angiogenesis and immunosuppression in the tumor microenvironment (TME). Multiple clinical studies have demonstrated the prognostic implications of and suggested the translational significance of MDSCs in patients with HCC. In preclinical HCC models, targeting MDSCs has been shown to enhance antitumor efficacy of sorafenib or immune checkpoint inhibitors. Reversing the protumor effects of MDSCs could be achieved by depleting MDSCs, blocking MDSC trafficking and migration into TME, and inhibiting the immunosuppressive functions of MDSCs. To date, these strategies have not yet been validated to be clinically useful in patients with malignancy including HCC. Future studies should focus on identifying specific markers for human MDSCs and developing combination approaches incorporating MDSC-targeting therapy in the treatment of HCC.

Development of myeloid-derived suppressor cells (MDSC) targeted therapy for the treatment of cisplatin-resistant bladder cancer.

415 Background: The emergence of immune checkpoint inhibitors (ICI) has brought hope for cure and survival for those suffering from various cancers, including bladder cancer. However, the response rate of ICI monotherapy is modest, and recent reports indicate that myeloid-derived suppressor cells (MDSC) might play a role in the resistant mechanism of ICI. In this study, we assess the effect of chemokine signal on the proliferation of bladder cancer and investigate whether MDSC could be a new target for the treatment of cisplatin-resistant bladder cancer. Methods: We established a cisplatin resistant strain (MB49R) of mice bladder cancer cell line MB49, and examined the alteration of the expression levels of inflammatory chemokines by chemokine array. Next, we isolated MDSCs from spleen and tumor in tumor-bearing mice to examine gene expression levels of chemokine receptors (CXCR2 and CCR2) and immunosuppression genes (Arg-1 and iNOS). Furthermore, we assessed the efficacy of CDDP, α-PD-L1 and chemokine antagonists against the proliferation of tumors in MB49 and MB49R xenograft models. Results: Expression levels of CCL2 and CXCL1/2, which are involved in the migration of MDSC, were significantly increased in the culture supernatant of MB49R compared to those in MB49 cell lines. This result was confirmed by real-time RT-qPCR of tumor extract, and this increase was also observed in human bladder cancer cell lines (T24 and T24R). CXCR2 and CCR2 were highly expressed in PMN-MDSC and M-MDSC, respectively, which were isolated from spleen or tumors in tumor-bearing mice, and gene expression levels of Arg-1 and iNOS were dramatically increased in M-MDSCs from the tumor tissues compared to those from spleen. Also, analysis by flow cytometry revealed that PMN-MDSC dramatically decreased in MB49R compared to parental MB49 tumors, while the proportion of M-MDSC was not changed in MB49R, which indicates that M-MDSC could be a target for the treatment of CDDP resistant bladder cancer. Conclusions: The results in the present study might indicate that the combination treatment with ICI and MDSC-targeting therapy could be an option for the treatment of cisplatin-resistant bladder cancer.

Visualization and quantification of in vivo homing kinetics of myeloid-derived suppressor cells in primary and metastatic cancer.

Myeloid-derived suppressor cells (MDSCs) are immunosuppressive cells of the myeloid compartment and major players in the tumor microenvironment (TME). With increasing numbers of studies describing MDSC involvement in cancer immune escape, cancer metastasis and the dampening of immunotherapy responses, MDSCs are of high interest in current cancer therapy research. Although heavily investigated in the last decades, the in vivo migration dynamics of MDSC subpopulations in tumor- or metastases-bearing mice have not yet been studied extensively. Therefore, we have modified our previously reported intracellular cell labeling method and applied it to in vitro generated MDSCs for the quantitative in vivo monitoring of MDSC migration in primary and metastatic cancer. MDSC migration to primary cancers was further correlated to the frequency of endogenous MDSCs. Methods: Utilizing a 64Cu-labeled 1,4,7-triazacyclononane-triacetic acid (NOTA)-modified CD11b-specific monoclonal antibody (mAb) (clone M1/70), we were able to label in vitro generated polymorphonuclear (PMN-) and monocytic (M-) MDSCs for positron emission tomography (PET) imaging. Radiolabeled PMN- and M-MDSCs ([64Cu]PMN-MDSCs and [64Cu]M-MDSCs, respectively) were then adoptively transferred into primary and metastatic MMTV-PyMT-derived (PyMT-) breast cancer- and B16F10 melanoma-bearing experimental animals, and static PET and anatomical magnetic resonance (MR) images were acquired 3, 24 and 48 h post cell injection. Results: The internalization of the [64Cu]NOTA-mAb-CD11b-complex was completed within 3 h, providing moderately stable radiolabeling with little detrimental effect on cell viability and function as determined by Annexin-V staining and T cell suppression in flow cytometric assays. Further, we could non-invasively and quantitatively monitor the migration and tumor homing of both [64Cu]NOTA-αCD11b-mAb-labeled PMN- and M-MDSCs in mouse models of primary and metastatic breast cancer and melanoma by PET. We were able to visualize and quantify an increased migration of adoptively transferred [64Cu]M-MDSCs than [64Cu]PMN-MDSCs to primary breast cancer lesions. The frequency of endogenous MDSCs in the PyMT breast cancer and B16F10 melanoma model correlated to the uptake values of adoptively transferred MDSCs with higher frequencies of PMN- and M-MDSCs in the more aggressive B16F10 melanoma tumors. Moreover, aggressively growing melanomas and melanoma-metastatic lesions recruited higher percentages of both [64Cu]PMN- and [64Cu]M-MDSCs than primary and metastatic breast cancer lesions as early as 24 h post adoptive MDSC transfer, indicating an overall stronger recruitment of cancer-promoting immunosuppressive MDSCs. Conclusion: Targeting of the cell surface integrin CD11b with a radioactive mAb is feasible for labeling of murine MDSCs for PET imaging. Fast internalization of the [64Cu]NOTA-αCD11b-mAb provides presumably enhanced stability while cell viability and functionality was not significantly affected. Moreover, utilization of the CD11b-specific mAb allows for straightforward adaptation of the labeling approach for in vivo molecular imaging of other myeloid cells of interest in cancer therapy, including monocytes, macrophages or neutrophils.

MDSCs drive the process of endometriosis by enhancing angiogenesis and are a new potential therapeutic target.

Endometriosis affects women of reproductive age via unclear immunological mechanism(s). Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of myeloid cells with potent immunosuppressive and angiogenic properties. Here, we found MDSCs significantly increased in the peripheral blood of patients with endometriosis and in the peritoneal cavity of a mouse model of surgically induced endometriosis. Majority of MDSCs were granulocytic, produced ROS, and arginase, and suppressed T-cell proliferation. Depletion of MDSCs by antiGr-1 antibody dramatically suppressed development of endometrial lesions in mice. The chemokines CXCL1, 2, and 5 were expressed at sites of lesion while MDSCs expressed CXCR-2. These CXC-chemokines promoted MDSC migration toward endometriotic implants both in vitro and in vivo. Also, CXCR2-deficient mice show significantly decreased MDSC induction, endometrial lesions, and angiogenesis. Importantly, adoptive transfer of MDSCs into CXCR2-KO mice restored endometriotic growth and angiogenesis. Together, this study demonstrates that MDSCs play a role in the pathogenesis of endometriosis and identifies a novel CXC-chemokine and receptor for the recruitment of MDSCs, thereby providing a potential target for endometriosis treatment.

Blockade of CCL2 enhances immunotherapeutic effect of anti-PD1 in lung cancer

Myeloid derived suppressor cells (MDSC) play a pivotal role in tumor immune evasion and MDSC levels increased in patients with cancer. Studies confirmed the associations between MDSC and various cytokines in the peripheral blood. However, little is known about the association between parenchymal MDSC subsets and cytokines, or the mechanism drawing MDSC into tumor parenchyma. This study was to analyze the correlation between MDSC subsets and CCL2 level in lung cancer model. G-MDSC and M-MDSC from the blood and parenchyma were analyzed by flow cytometry and western blot in the lung tumor model. CCL2 was detected by ELISA assay, real-time PCR, western blot and flow cytometry. Furthermore, the therapeutic effects of combination treatment combining CCL2 antagonist and anti-PD1 antibody were assessed. Results showed that MDSC subsets had a positive correlation with CCL2, suggesting CCL2 may attract MDSC into the parenchyma. Gene and protein expression of CCL2, as well as the CCL2 surface expression significantly increased in blood and tumor of tumor-bearing mice. Anti-CCL2 treatment decreased G-MDSC and M-MDSC in the periphery and tumor through inhibiting the protein expression of arginase 1 and iNOS. In addition, combination therapy enhanced CD4+ and CD8+ T cell infiltration, as well as the production of interferon gamma (IFNγ), and increased the survival time of tumor-bearing mice. Our study provided potential new target to enhance the efficacy of immunotherapy in patients with lung cancer, in addition to elucidate a possible association between MDSC subsets and the cytokine drawing MDSC migration into the tumor tissue.

CCR5+ Myeloid-Derived Suppressor Cells Are Enriched and Activated in Melanoma Lesions.

Accumulation of myeloid-derived suppressor cells (MDSC) in melanoma microenvironment is supported by chemokine receptor/chemokine signaling. Although different chemokines were suggested to be involved in this process, the role of CCR5 and its ligands is not established. Using a Ret transgenic mouse melanoma model, we found an accumulation of CCR5+ MDSCs in melanoma lesions associated with both increased concentrations of CCR5 ligands and tumor progression. Tumor-infiltrating CCR5+ MDSCs displayed higher immunosuppressive activity than their CCR5- counterparts. Upregulation of CCR5 expression on CD11b+Gr1+ myeloid cells was induced in vitro by CCR5 ligands and other inflammatory factors. In melanoma patients, CCR5+ MDSCs were enriched at the tumor site and correlated with enhanced production of CCR5 ligands. Moreover, they exhibited a stronger immunosuppressive pattern compared with CCR5- MDSCs. Blocking CCR5/CCR5 ligand interactions increased survival of tumor-bearing mice and was associated with reduced migration and immunosuppressive potential of MDSCs in tumor lesions. Our findings define a critical role for CCR5 in recruitment and activation of MDSCs, suggesting a novel strategy for melanoma treatment.Significance: These findings validate the importance of the CCR5/CCR5 ligand axis not only for MDSC recruitment but also for further activation of their immunosuppressive functions in the tumor microenvironment, with potentially broad therapeutic implications, given existing clinically available inhibitors of this axis. Cancer Res; 78(1); 157-67. ©2017 AACR.

Ubiquitin Conjugation Probed by Inflammation in Myeloid-Derived Suppressor Cell Extracellular Vesicles.

Ubiquitinated proteins carried by the extracellular vesicles (EV) released by myeloid-derived suppressor cells (MDSC) have been investigated using proteomic strategies to examine the effect of tumor-associated inflammation. EV were collected from MDSC directly following isolation from tumor-bearing mice with low and high inflammation. Among the 1092 proteins (high inflammation) and 925 proteins (low inflammation) identified, more than 50% were observed as ubiquitinated proteoforms. More than three ubiquitin-attachment sites were characterized per ubiquitinated protein, on average. Multiple ubiquitination sites were identified in the pro-inflammatory proteins S100 A8 and S100 A9, characteristic of MDSC and in histones and transcription regulators among other proteins. Spectral counting and pathway analysis suggest that ubiquitination occurs independently of inflammation. Some ubiquitinated proteins were shown to cause the migration of MDSC, which has been previously connected with immune suppression and tumor progression. Finally, MDSC EV are found collectively to carry all the enzymes required to catalyze ubiquitination, and the hypothesis is presented that a portion of the ubiquitinated proteins are produced in situ.

Inhibition of IL-18-mediated myeloid derived suppressor cell accumulation enhances anti-PD1 efficacy against osteosarcoma cancer

Myeloid derived suppressor cells (MDSC) are very important in tumor immune evasion and they dramatically increased in peripheral blood of patients with osteosarcoma cancer. The association between MDSC and various cytokines has been studied in the peripheral blood. However, little is known about the mechanism drawing MDSC into tumor parenchyma. This study was to analyze the correlation between MDSC subsets and interleukin 18 (IL-18) level in osteosarcoma tumor model and its effect on the immunotherapy. MDSC were isolated from the blood and parenchyma and analyzed in the osteosarcoma tumor model. IL-18 levels were detected by enzyme-linked immunosorbent assay (ELISA) assay, real-time PCR, western blot and flow cytometry. Moreover, combination treatment with IL-18 inhibition and anti-PD1 was conducted to assess the therapeutic effects of IL-18 blockade. Results showed MDSC levels had a positive correlation with IL-18, suggesting IL-18 may attract MDSC into the parenchyma. IL-18 gene and protein expression significantly increased in blood and tumor lysates of tumor-bearing mice. Anti-IL-18 treatment significantly decreased G-MDSC and M-MDSC in the peripheral blood and tumor. Furthermore, combination therapy decreased the tumor burden and increased CD4+ and CD8+ T cell infiltration, as well as the production of interferon gamma (IFNγ) and granzyme B. Our study revealed a possible correlation between MDSC subsets and IL-18 inducing MDSC migration into the tumor tissue, in addition to provide the potential target to enhance the efficacy of immunotherapy in patients with osteosarcoma.

Calreticulin Fragment 39-272 Promotes B16 Melanoma Malignancy through Myeloid-Derived Suppressor Cells In Vivo

Calreticulin (CRT), a multifunctional Ca2+-binding glycoprotein mainly located in the endoplasmic reticulum, is a tumor-associated antigen that has been shown to play protective roles in angiogenesis suppression and anti-tumor immunity. We previously reported that soluble CRT (sCRT) was functionally similar to heat shock proteins or damage-associated molecular patterns in terms of ability to activate myeloid cells and elicit strong inflammatory cytokine production. In the present study, B16 melanoma cell lines expressing recombinant CRT fragment 39-272 (sCRT/39-272) in secreted form (B16-CRT), or recombinant enhanced green fluorescence protein (rEGFP) (B16-EGFP), were constructed for investigation on the roles of sCRT in tumor development. When s.c. inoculated into C57BL/6 mice, the B16-CRT cells were significantly more aggressive (in terms of solid tumor growth rate) than B16-EGFP controls in a TLR4- and myeloid-derived suppressor cells (MDSC)-dependent manner. The B16-CRT-bearing mice showed increased Gr1+ MDSC infiltration in tumor tissues, accelerated proliferation of CD11b+Ly6G+Ly6Clow (G-MDSC) precursors in bone marrow, and higher percentages of G-MDSCs in spleen and blood, which was mirrored by decreased percentage of dendritic cells (DC) in periphery. In in vitro studies, recombinant sCRT/39-272 was able to promote migration and survival of tumor-derived MDSCs via interaction with TLR4, inhibit MDSC differentiation into DC, and also elicit expression of inflammatory proteins S100A8 and S100A9 which are essential for functional maturation and chemotactic migration of MDSCs. Our data provide solid evidence for CRT as a double-edged sword in tumor development.

A selective sphingosine-1-phosphate receptor 1 agonist SEW-2871 aggravates gastric cancer by recruiting myeloid-derived suppressor cells.

The immune status of tumor microenvironment in gastric cancer is poorly understood, which limits the development of novel strategies in this field. Sphingosine-1-phosphate (S1P) acts as an immune modulator, but the role of S1P in gastric cancer is elusive. Here, we aim to investigate S1P receptor 1 (S1P1)-mediated effect of S1P in gastric cancer. We generated a xenograft mouse model and used SEW-2871, a S1P1 specific agonist to activate S1P1 signalling. Tumor-infiltrating lymphocytes (TILs) were isolated and analysed using flow cytometry. Chemokine expression of tumor cells was evaluated using quantitative real-time polymerase chain reaction. Myeloid-derived suppressor cells (MDSCs) migration was assessed using Transwell chambers. SEW-2871 promoted tumor growth in our mouse model, and induced a higher level of MDSC and a reduced level of CD8+CD69+ T cells within tumor. Consistently, the anti-tumoral function of cytotoxic T lymphocytes was impaired in mice with SEW-2871 treatment. Additionally, SEW-2871 enhanced expression of several MDSC recruitment-associated chemokines (CXCL12, CXCL5 and CCL2) in tumor cells. These chemokines facilitated MDSC migration by interaction with CCR2, CXCR2 and CXCR4. S1P1 signalling promoted gastric cancer by enhancing chemokine expression in tumor cells and recruiting MDSC to tumor microenvironment, which impaired anti-tumoral function of TILs.

C-C Chemokine Receptor Type 2-Dependent Migration of Myeloid-Derived Suppressor Cells in Protection of Islet Transplants.

Islet transplantation is a promising therapeutic approach to restore the physical response to blood glucose in type 1 diabetes. Current chronic use of immunosuppressive reagents for preventing islet allograft rejection is associated with severe complications. In addition, many of the immunosuppressive drugs are diabetogenic. The induction of transplant tolerance to eliminate the dependency on immunosuppression is ideal, but remains challenging. Addition of hepatic stellate cells allowed generation of myeloid-derived suppressor cells (MDSC) from precursors in mouse bone marrow. Migration of MDSC was examined in an islet allograft transplant model by tracking the systemic administered MDSC from CD45.1 congenic mice. The generated MDSC were expressed C-C chemokine receptor type 2 (CCR2), which was enhanced by exposure to interferon-γ. A single systemic administration of MDSC markedly prolonged survival of islet allografts without requirement of immunosuppression. Tracking the administered MDSC showed that they promptly migrated to the islet graft sites, at which point they exerted potent immune suppressive activity by inhibiting CD8 T cells, enhancing regulatory T cell activity. MDSC generated from CCR2 mice failed to be mobilized and lost tolerogenic activity in vivo, but sustained suppressive activity in vitro. MDSC migration was dependent on expression of CCR2, whereas CCR2 does not directly participate in immune suppression. Expression of CCR2 needs to be closely monitored for quality control purpose when MDSC are generated in vitro for immune therapy.

CCR5 in recruitment and activation of myeloid-derived suppressor cells in melanoma.

Malignant melanoma is characterized by the development of chronic inflammation in the tumor microenvironment, leading to the accumulation of myeloid-derived suppressor cells (MDSCs). Using ret transgenic mouse melanoma model, we found a significant migration of MDSCs expressing C-C chemokine receptor (CCR)5 into primary tumors and metastatic lymph nodes, which was correlated with tumor progression. An increased CCR5 expression on MDSCs was associated with elevated concentrations of CCR5 ligands in melanoma microenvironment. In vitro experiments showed that the upregulation of CCR5 expression on CD11b+Gr1+ immature myeloid cells was induced by CCR5 ligands, IL-6, GM-CSF, and other inflammatory factors. Furthermore, CCR5+ MDSCs infiltrating melanoma lesions displayed a stronger immunosuppressive pattern than their CCR5- counterparts. Targeting CCR5/CCR5 ligand signaling via a fusion protein mCCR5-Ig, which selectively binds and neutralizes all three CCR5 ligands, increased the survival of tumor-bearing mice. This was associated with a reduced migration and immunosuppressive potential of tumor MDSCs. In melanoma patients, circulating CCR5+ MDSCs were increased as compared to healthy donors. Like in melanoma-bearing mice, we observed an enrichment of these cells and CCR5 ligands in tumors as compared to the peripheral blood. Our findings define a critical role for CCR5 not only in the recruitment but also in the activation of MDSCs in tumor lesions, suggesting that novel strategies of melanoma treatment could be based on blocking CCR5/CCR5 ligand interactions.