Development Of Myeloid-derived Suppressor Cells Research Articles

Abstract A11: Myeloid-derived suppressor cells suppress immune surveillance and stimulate cancer cell proliferation/metastasis through activation of mTOR pathway in lal-/- mice

Abstract Inflammation critically contributes to cancer metastasis, in which myeloid-derived suppressor cells (MDSCs) are an important participant. Although MDSCs are known to suppress immune surveillance, their roles in directly stimulating cancer cell proliferation and metastasis currently remain unclear. MDSCs development and homeostasis is controlled by lysosomal acid lipase (LAL), a critical enzyme in the metabolic signaling pathway that hydrolyzes cholesteryl esters (CE) and triglycerides (TG) in lysosomes. Lysosomal acid lipase (LAL) deficiency causes systemic expansion and infiltration of MDSCs in multiple organs. LAL-deficient (lal-/-) MDSCs arise from dysregulated production of progenitor cells in the bone marrow. In humans, increased CD14+CD16+ and CD14+CD33+ MDSC subsets have been reported with heterozygote carriers of LAL mutations. Patients with mutations in the LAL gene has been reported to be associated with carcinogenesis. In the LAL-deficient (lal-/-) mouse model, melanoma metastasized massively in allogeneic lal-/- mice, which was suppressed in allogeneic lal+/+ mice due to immune rejection. Here we report for the first time that MDSCs isolated from lal-/- mice directly stimulated B16 melanoma cell proliferation in vitro, and growth and metastasis in vivo. Cytokines i.e., IL-1β and TNFα from MDSCs are required for B16 melanoma cell proliferative function in vitro. Myeloid-specific expression of human LAL (hLAL) in lal-/- mice rescues these malignant phenotypes in vitro and in vivo. The tumor-promoting function of lal-/- MDSCs is mediated, at least in part, through over-activation of the mammalian target of rapamycin (mTOR) pathway. mTOR is a serine/threonine protein kinase that regulates cell growth, proliferation, migration, survival, protein synthesis, and transcription in response to growth factors and mitogens. mTOR is the catalytic subunit of two distinctive complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Unique accessory proteins, regulatory-associated protein of mTOR (RAPTOR), and rapamycin-insensitive companion of mTOR (RICTOR) define the TORC1 and TORC2 complexes, respectively. Affymetrix GeneChip microarray analysis and Ingenuity Pathway Analysis of gene transcripts revealed up-regulation of multiple genes in the mTOR signaling pathway in lal-/- MDSCs. We have shown that inhibition of mTOR in lal-/- MDSCs increased apoptosis, decreased ATP synthesis, decreased ROS production, and recovered impairment of mitochondrial membrane potential. As a result, lal-/- MDSCs reversed the increased cell proliferation, corrected enhanced lal-/- MDSCs development from lineage negative progenitor cells, and reduced systemic MDSC expansion. More importantly, knockdown of mTOR, Raptor or Rictor in lal-/- MDSCs reversed the immune suppression on T cell proliferation and function, as well as suppressed their stimulation on proliferation of cancer cells (including B16 melanoma, LLC and Tramp-C2 cancer cells). Our results indicate that LAL plays a critical role in regulating MDSCs through modulation of the mTOR pathway. MDSCs possess dual functions to overcome immune rejection and facilitate cancer metastasis: 1) suppress immune surveillance, and 2) stimulate cancer cell proliferation, growth, and metastasis. Grant support: This work was supported by National Institutes of Health Grants CA138759, CA152099 (to C. Y.) and HL087001 (to H. D.). Citation Format: Hong Du, Ting Zhao, Xinchun Ding, Katlin Walls, Cong Yan. Myeloid-derived suppressor cells suppress immune surveillance and stimulate cancer cell proliferation/metastasis through activation of mTOR pathway in lal-/- mice. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A11.

Tetraspanin CD81 promotes tumor growth and metastasis by modulating the functions of T regulatory and myeloid-derived suppressor cells.

Tumor cells counteract innate and adaptive antitumor immune responses by recruiting regulatory T cells (Treg) and innate myeloid-derived suppressor cells (MDSC), which facilitate immune escape and metastatic dissemination. Here we report a role in these recruitment processes for CD81, a member of the tetraspanin family of proteins that have been implicated previously in cancer progression. We found that genetic deficiency in CD81 reduced tumor growth and metastasis in two genetic mouse backgrounds and multiple tumor models. Mechanistic investigations revealed that CD81 was not required for normal development of Treg and MDSC but was essential for immunosuppressive functions. Notably, adoptive transfer of wild-type Treg into CD81-deficient mice was sufficient to promote tumor growth and metastasis. Our findings suggested that CD81 modulates adaptive and innate immune responses, warranting further investigation of CD81 in immunomodulation in cancer and its progression.

Transcriptional regulation of myeloid-derived suppressor cells.

Myeloid-derived suppressor cells are a heterogeneous group of pathologically activated immature cells that play a major role in the negative regulation of the immune response in cancer, autoimmunity, many chronic infections, and inflammatory conditions, as well as in the regulation of tumor angiogenesis, tumor cell invasion, and metastases. Accumulation of myeloid-derived suppressor cells is governed by a network of transcriptional regulators that could be combined into 2 partially overlapping groups: factors promoting myelopoiesis and preventing differentiation of mature myeloid cells and factors promoting pathologic activation of myeloid-derived suppressor cells. In this review, we discuss the specific nature of these factors and their impact on myeloid-derived suppressor cell development.

Lysosomal acid lipase in cancer

Lysosomal Acid Lipase Regulates Myeloid-derived Suppressor Cells to Control Cancer Cell Proliferation and Metastasis. Inflammation critically contributes to cancer growth and metastasis, in which myeloid-derived suppressor cells (MDSCs) are an important participant. MDSCs are known to suppress immune surveillance to promote tumorigenesis [1]. Lysosomal acid lipase (LAL), a critical enzyme in controlling neutral lipid metabolic signaling, hydrolyzes cholesteryl esters and triglycerides in the lysosome of cells to generate free fatty acids and cholesterol. Deficiency of this metabolic enzyme causes abnormal hematopoietic development, skewing progenitor cell differentiation towards an overabundance of myeloid cells in the bone marrow. As a result, immature MDSCs expand dramatically and accumulate in the bone marrow, peripheral blood, immune organs (e.g. thymus, spleen) and distal organs (e.g. lung, liver) [2]. Systemic infiltration of lal−/− MDSCs is facilitated by changed ECs functions through various mechanisms. lal−/− MDSCs promote ECs angiogenesis, tube formation, and proliferation [3]. Upon exiting the bone marrow and entering circulation and distal organ compartments, lal−/− MDSCs promote tumorigenesis by two mechanisms. First, infiltration of lal−/− MDSCs leads to abnormal organization of the thymus and spleen, impair progression of T cell development in the thymus, and retard T cell maturation in the spleen. In the blood and distal organs, lal−/− MDSCs strongly suppress peripheral T cell proliferation and lymphokine release [4]. This is largely due to increased apoptosis and decreased proliferation of lal−/− T cells in thymus and peripheral compartments. As a consequence, the anti-tumor immunity is significantly weakened. Second, lal−/− MDSCs directly stimulate tumor cell proliferation, growth and metastasis even in an allogenic mouse model, showing that lal−/− MDSCs are able to overcome host immune rejection [5]. Therefore, MDSCs facilitate tumorigenesis at least by two mechanisms: 1) suppressing immune surveillance; and 2) stimulating cancer cell proliferation, growth and metastasis directly. Based on Affymetrix GeneChip microarray and Ingenuity Pathway analyses, the mammalian target of rapamycin (mTOR) signaling and peroxisome proliferator-activated receptor gamma (PPARγ) signaling are two major pathways that mediate pathogenic phenotypes of lal−/− MDSCs in suppressing immune surveillance and stimulating tumorigenesis [6]. These activities are associated with alteration of other genes involved in cell growth, cell cycle entry, cell survival, cell migration, histone epigenetics, bioenergetic pathways, and ROS production [6]. Inhibition of mTOR by pharmacological inhibitors and siRNA interference not only reduces immunosuppressive function of lal−/− MDSCs, but also suppresses their stimulation on tumor proliferation, progression and metastasis [5, 7]. mTOR inhibitor treatment of lal−/− mice reverses the increased proliferation, decreased apoptosis, increased ATP synthesis, and increased cell cycling in lal−/− MDSCs. Pharmacological and siRNA suppression of mTOR, Raptor, Rictor, and Akt1 also correct lal−/− MDSCs development from Lin-progenitor cells, decrease ROS production, and recover from impairment of mitochondrial membrane potential. On the other hand, derivatives of free fatty acid metabolites serve as hormonal ligands for nuclear receptors (e.g. PPARγ) that possess the anti-inflammatory function. Myeloid-specific expression of human LAL (hLAL) to restore the PPARγ function in lal−/− mice reverses both immunosuppression and tumor stimulation [2, 5]. Reintroducing PPARγ ligands into lal−/− mice also reverses immunosuppression and tumor stimulation (unpublished result). To confirm the functional role of PPARγ in myeloid cells, dominant negative PPARγ (dnPPARγ) was overexpressed in a myeloid-specific bitransgenic mouse model [8]. Overexpression of dnPPARγ in myeloid lineage cells abnormally elevates frequencies and total numbers of LK, LSK, CMP and GMP progenitor populations in the bone marrow. As a result, MDSCs are systemically increased in association with activation of Stat3, NF-kB, Erk1/2 and p38 molecules. MDSCs from this system inhibit the proliferation and lymphokine production of T cells. Both CD4+ and CD8+ T cell populations are decreased in conditional bitransgenic mice. Multiple forms of carcinoma and sarcoma in the lung, liver, spleen and lymph nodes are observed. Bone marrow transplantation reveals that a myeloid autonomous defect is responsible for MDSCs expansion, immunosuppression and tumorigenesis in this conditional bitransgenic model [8]. Therefore, the metabolic signaling controlled by the LAL/PPARγ/mTOR axis is essential for MDSCs development, homeostasis and functions during tumorigenesis, which can facilitates pharmacological drug and immunotherapy designs in clinical application.

Abstract 3358: SOCS3 deficiency in myeloid cells promotes tumor development: Involvement of STAT3 activation and myeloid-derived suppressor cells

Abstract Suppressor Of Cytokine Signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway, and generally function as tumor suppressors. The absence of SOCS3 in particular leads to heightened activation of the STAT3 transcription factor, which has a striking ability to promote tumor survival while suppressing anti-tumor immunity. We report for the first time that genetic deletion of SOCS3 specifically in myeloid cells significantly enhances tumor growth, which correlates with elevated levels of myeloid-derived suppressor cells (MDSC) in the tumor microenvironment, and diminished CD8+ T-cell infiltration in tumors. Furthermore, SOCS3-deficient bone-marrow-derived cells exhibit heightened STAT3 activation and preferentially differentiate into the Gr-1+CD11b+Ly6G+ MDSC phenotype. Importantly, we identify granulocyte-colony stimulating factor (G-CSF) as a critical factor secreted by the tumor microenvironment that promotes development of MDSC via a STAT3-dependent pathway. Abrogation of tumor-derived G-CSF reduces the proliferation and accumulation of Gr-1+CD11b+ MDSC and inhibits tumor growth. These findings highlight the critical function of SOCS3 as a negative regulator of MDSC development and function, via inhibition of STAT3 activation. Note: This abstract was not presented at the meeting. Citation Format: Hao Yu, Hongwei Qin, Etty (Tika) Benveniste. SOCS3 deficiency in myeloid cells promotes tumor development: Involvement of STAT3 activation and myeloid-derived suppressor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3358. doi:10.1158/1538-7445.AM2015-3358

SOCS3 Deficiency in Myeloid Cells Promotes Tumor Development: Involvement of STAT3 Activation and Myeloid-Derived Suppressor Cells.

Suppressor of cytokine signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway and generally function as tumor suppressors. The absence of SOCS3 in particular leads to heightened activation of the STAT3 transcription factor, which has a striking ability to promote tumor survival while suppressing antitumor immunity. We report for the first time that genetic deletion of SOCS3, specifically in myeloid cells, significantly enhances tumor growth, which correlates with elevated levels of myeloid-derived suppressor cells (MDSC) in the tumor microenvironment, and diminishes CD8(+) T-cell infiltration in tumors. The importance of MDSCs in promoting tumor growth is documented by reduced tumor growth upon depletion of MDSCs. Furthermore, SOCS3-deficient bone-marrow-derived cells exhibit heightened STAT3 activation and preferentially differentiate into the Gr-1(+)CD11b(+)Ly6G(+) MDSC phenotype. Importantly, we identify G-CSF as a critical factor secreted by the tumor microenvironment that promotes development of MDSCs via a STAT3-dependent pathway. Abrogation of tumor-derived G-CSF reduces the proliferation and accumulation of Gr-1(+)CD11b(+) MDSCs and inhibits tumor growth. These findings highlight the critical function of SOCS3 as a negative regulator of MDSC development and function via inhibition of STAT3 activation.

The expansion and activity of myeloid-derived suppressor cells in nasopharyngeal carcinoma mediated by up-regulating COX-2 (TUM6P.964)

Abstract The expansion of myeloid-derived suppressor cells (MDSCs) is linked to tumor metastasis, but the molecular mechanism related to this link remains unclear. Here, we investigated the interactions between MDSCs and tumor cells in patients with nasopharyngeal carcinoma (NPC) and demonstrated that NPC cells induced the expansion of tumor-induced MDSCs (T-MDSCs); in turn T-MDSCs enhanced NPC cell migration and metastasis. Up-regulation of tumor cyclooxygenase-2 (COX-2) expression positively correlated with the expansion of MDSCs in NPC patients and both of COX-2 and MDSCs were poor predictors for disease-free survival. In vitro, knockdown of COX-2 expression in TW03 cells hampered T-MDSC induction by blocking IL-6. In addition, T-MDSCs promoted NPC cell proliferation, invasion and migration by triggering the epithelial-mesenchymal transition (EMT) on cell-to-cell contact; furthermore, T-MDSCs enhanced tumor lung metastasis in nude mice. Interestingly, T-MDSCs-mediated EMT was through enhancing COX-2 expression and activating the b-catenin/TCF4 pathway in NPC cells; Blockage of TGFb and iNOS significantly abolished the T-MDSC-mediated up-regulation of COX-2 and EMT in NPC cells, whereas the administration of TGFb and L-arginine supplements up-regulated COX2 expression and the b-catenin/TCF4 pathway in NPC cells. Taken together, these findings unveil a pivotal role for COX-2 in the development and cancerous contributions of MDSCs in NPC.

SOCS3 deficiency in myeloid cells promotes tumor development: involvement of STAT3 activation and myeloid-derived suppressor cells (TUM6P.1000)

Abstract Suppressor Of Cytokine Signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway, and generally function as tumor suppressors. The absence of SOCS3 in particular leads to heightened activation of the STAT3 transcription factor, which has a striking ability to promote tumor survival while suppressing anti-tumor immunity. We report for the first time that genetic deletion of SOCS3 specifically in myeloid cells significantly enhances tumor growth, which correlates with elevated levels of myeloid-derived suppressor cells (MDSC) in the tumor microenvironment, and diminished CD8+ T-cell infiltration in tumors. Furthermore, SOCS3-deficient bone-marrow-derived cells exhibit heightened STAT3 activation and preferentially differentiate into the Gr-1+CD11b+Ly6G+ MDSC phenotype. Importantly, we identify granulocyte-colony stimulating factor (G-CSF) as a critical factor secreted by the tumor microenvironment that promotes development of MDSC via a STAT3-dependent pathway. Abrogation of tumor-derived G-CSF reduces the proliferation and accumulation of Gr-1+CD11b+ MDSC and inhibits tumor growth. These findings highlight the critical function of SOCS3 as a negative regulator of MDSC development and function, via inhibition of STAT3 activation.

Abstract B51: SOCS3 deficiency in myeloid cells promotes prostate tumor development

Abstract Suppressor Of Cytokine Signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway, and have been implicated as functioning as tumor suppressors, particularly SOCS3. We have previously demonstrated that specific deletion of SOCS3 in myeloid cell leads to heightened activation of STAT3 and downstream STAT3 target gene expression. STAT3 activation has been strongly implicated in contributing to prostate cancer progression and metastasis. STAT3 signaling in myeloid cells has been demonstrated to attenuate anti-tumor immunity. Thus, we evaluated the impact of SOCS3 deletion in myeloid cells in a murine prostate tumor model. We demonstrate that prostate tumor growth is significantly enhanced in myeloid-specific SOCS3-deficient mice, which correlates with elevated levels of Myeloid Derived Suppressor Cells (MDSC) in both the spleen and tumor microenvironment, and less CD8+ T-cell infiltration in tumors. Furthermore, SOCS3-deficient bone-marrow-derived mononuclear cells exhibit heightened STAT3 activation and are preferentially differentiate into MDSC phenotype. Importantly, we identify granulocyte-colony stimulating factor (G-CSF) as a critical factor secreted by the prostate tumor microenvironment that promotes development of Gr-1+CD11b+ cells via a STAT3-depentent pathway. Abrogation of tumor-derived G-CSF reduced the proliferation and accumulation of Gr-1+CD11b+ cells and inhibited prostate tumor growth. Therefore, the tumor-derived cytokine, G-CSF, is a prospective candidate upstream of the STAT3/SOCS3 pathway in MDSC differentiation from the bone marrow. These findings highlight the critical function of SOCS3 as a negative regulator of MDSC development and function, which may contribute to tumor suppressor attributes of SOCS3. Citation Format: Hao Yu, Yudong Liu, Jessy Deshane, Etty (Tika) Benveniste, Hongwei Qin. SOCS3 deficiency in myeloid cells promotes prostate tumor development. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B51. doi:10.1158/1538-7445.CHTME14-B51

Abstract A25: SOCS3 deficiency in myeloid cells promotes prostate tumor development

Abstract Suppressor Of Cytokine Signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway, and have been implicated as functioning as tumor suppressors, particularly SOCS3. We have previously demonstrated that specific deletion of SOCS3 in myeloid cells leads to heightened activation of STAT3 and downstream STAT3 target gene expression. STAT3 activation has been strongly implicated in contributing to prostate cancer progression and metastasis. STAT3 signaling in myeloid cells has been demonstrated to attenuate anti-tumor immunity. Thus, we evaluated the impact of SOCS3 deletion in myeloid cells in a murine prostate tumor model. We demonstrate that prostate tumor growth is significantly enhanced in myeloid-specific SOCS3-deficient mice, which correlates with elevated levels of Myeloid Derived Suppressor Cells (MDSC) in both the spleen and tumor microenvironment, and less CD8+ T-cell infiltration in tumors. Furthermore, SOCS3-deficient bone-marrow-derived mononuclear cells exhibit heightened STAT3 activation and are preferentially differentiate into MDSC phenotype. Importantly, we identify granulocyte-colony stimulating factor (G-CSF) as a critical factor secreted by the prostate tumor microenvironment that promotes development of Gr-1+CD11b+ cells via a STAT3-depentent pathway. Abrogation of tumor-derived G-CSF reduced the proliferation and accumulation of Gr-1+CD11b+ cells and inhibited prostate tumor growth. Therefore, the tumor-derived cytokine, G-CSF, is a prospective candidate upstream of the STAT3/SOCS3 pathway in MDSC differentiation from the bone marrow. These findings highlight the critical function of SOCS3 as a negative regulator of MDSC development and function, which may contribute to tumor suppressor attributer of SOCS3. Citation Format: Hao Yu, Yudong Liu, Braden C. McFarland, Etty N. Benveniste, Hongwei Qin. SOCS3 deficiency in myeloid cells promotes prostate tumor development. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A25. doi:10.1158/1538-7445.CHTME14-A25

Hepatic stellate cell–conditioned myeloid cells provide a novel therapy for prevention of factor VIII antibody formation in mice

A major complication of factor VIII (F.VIII) infusion therapies for the treatment of hemophilia A is the formation of antibodies (inhibitors) against F.VIII, a T-cell-dependent, B-cell-mediated process. To date, attempts to inhibit formation of the inhibitors have been limited in success. We have shown that hepatic stellate cells (HSCs) promote the development of myeloid-derived suppressor cells (MDSCs). The HSC-induced MDSCs are potent regulators of T-cell and B-cell responses. Here we show that MDSCs can be propagated from hemophilia A mouse bone marrow cells in coculture with HSCs. These cells exhibit a suppressive phenotype and display a marked ability to inhibit T-cell proliferation induced by dendritic cells in response to F.VIII. MDSCs can also inhibit proliferation and activation of B cells stimulated by immunoglobulin M and interleukin 4. Administration of HSC-induced MDSCs induces CD4(+) T cell and B220(+) B-cell hyporesponsiveness to F.VIII and reduces inhibitor formation in hemophilia A mice. These results suggest that MDSCs could serve as a form of immunotherapy for preventing inhibitor formation via induction of immune tolerance.

HMGB1 enhances immune suppression by facilitating the differentiation and suppressive activity of myeloid-derived suppressor cells.

Chronic inflammation often precedes malignant transformation and later drives tumor progression. Likewise, subversion of the immune system plays a role in tumor progression, with tumoral immune escape now well recognized as a crucial hallmark of cancer. Myeloid-derived suppressor cells (MDSC) are elevated in most individuals with cancer, where their accumulation and suppressive activity are driven by inflammation. Thus, MDSCs may define an element of the pathogenic inflammatory processes that drives immune escape. The secreted alarmin HMGB1 is a proinflammatory partner, inducer, and chaperone for many proinflammatory molecules that MDSCs develop. Therefore, in this study, we examined HMGB1 as a potential regulator of MDSCs. In murine tumor systems, HMGB1 was ubiquitous in the tumor microenvironment, activating the NF-κB signal transduction pathway in MDSCs and regulating their quantity and quality. We found that HMGB1 promotes the development of MDSCs from bone marrow progenitor cells, contributing to their ability to suppress antigen-driven activation of CD4(+) and CD8(+) T cells. Furthermore, HMGB1 increased MDSC-mediated production of IL-10, enhanced crosstalk between MDSCs and macrophages, and facilitated the ability of MDSCs to downregulate expression of the T-cell homing receptor L-selectin. Overall, our results revealed a pivotal role for HMGB1 in the development and cancerous contributions of MDSCs.

Abstract 1079: COX-2 blockade immunologically suppresses brain metastasis of lung cancer

Abstract Epidemiological studies have highlighted associations between the regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) and reduced cancer risks in human. In addition, NSAIDs reduce VEGF-D production levels, which in turn inhibit lymphatic vessel dilation and eventually cancer metastasis. Most NSAIDs function as cyclooxygenase-2 (COX-2) inhibitors that prevent the production of prostaglandin E2 (PGE2). The PGE2 induces myeloid-derived suppressor cells (MDSCs) in the bone marrow of cancer-bearing hosts, which is known to be responsible for T-cell suppression in chronic inflammation. Based on these findings, we hypothesized that COX-2 blockade would suppress brain metastasis of cancers by inhibiting MDSC development and their chemokine-mediated accumulation in the tumor microenvironment (TME). In both mouse and human brain metastasis cases, the expression levels of COX-2 and CCL2, a chemokine primarily attracting MDSCs, were increased in the brain tumor tissues compared with normal brain or peripheral bloods. Concomitantly, the infiltration of MDSCs (CD11+Gr-1+ for mice and CD14-CD15+HLA-DRlow for human, respectively) was observed at high levels in the tumor tissues. In the mouse brain tumor models using LL/2 murine lung cancer cell line, treatment with NSAIDs inhibited the PGE2 production levels and the formation of brain metastases. Furthermore, the NSAID treatment reduced the CCL2 in the TME, which was associated with a reduction in Ly6ChighLy6Glow monocytic and Ly6ClowLy6Ghigh granulocytic MDSCs in both the bone marrow and the TME. Mice genetically deficient for CCR2, a receptor for CCL2, exhibited low penetration of brain metastases along with reduced infiltration of MDSCs. Collectively, our findings show that the COX-2 pathway promotes brain metastases of lung cancers by supporting systemic development of MDSC and their accumulation in the TME mediated by the CCL2-CCR2 chemokine axis. Citation Format: Mitsugu Fujita, Susumu Nakata, Takeshi Okuda, Amami Kato, Osamu Yoshie. COX-2 blockade immunologically suppresses brain metastasis of lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1079. doi:10.1158/1538-7445.AM2014-1079

Abstract 1068: Socs3 deficiency in myeloid cells promotes prostate tumor development

Abstract Suppressor Of Cytokine Signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway, and have been implicated as functioning as tumor suppressors, particularly SOCS3. We have previously demonstrated that specific deletion of SOCS3 in myeloid cells leads to heightened activation of STAT3 and downstream STAT3 target gene expression. STAT3 activation has been strongly implicated in contributing to prostate cancer progression and metastasis. STAT3 signaling in myeloid cells has been demonstrated to attenuate anti-tumor immunity. Thus, we evaluated the impact of SOCS3 deletion in myeloid cells in a murine prostate tumor model. We demonstrate that prostate tumor growth is significantly enhanced in myeloid-specific SOCS3-deficient mice, which correlates with elevated levels of Myeloid Derived Suppressor Cells (MDSC) in both the spleen and tumor microenvironment, and less CD8+ T-cell infiltration in tumors. Furthermore, SOCS3-deficient bone-marrow-derived mononuclear cells exhibit heightened STAT3 activation and are preferentially differentiate into MDSC phenotype. Importantly, we identify granulocyte-colony stimulating factor (G-CSF) as a critical factor secreted by the prostate tumor microenvironment that promotes development of Gr-1+CD11b+ cells via a STAT3-depentent pathway. Abrogation of tumor-derived G-CSF reduced the proliferation and accumulation of Gr-1+CD11b+ cells and inhibited prostate tumor growth. Therefore, the tumor-derived cytokine, G-CSF, is a prospective candidate upstream of the STAT3/SOCS3 pathway in MDSC differentiation from the bone marrow. These findings highlight the critical function of SOCS3 as a negative regulator of MDSC development and function, which may contribute to tumor suppressor attributer of SOCS3. Citation Format: Hao Yu, Hongwei Qin. Socs3 deficiency in myeloid cells promotes prostate tumor development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1068. doi:10.1158/1538-7445.AM2014-1068

Development of a Translation‐Ready Combination Cancer Immunotherapy Regimen Based on Clinically Available Drugs

Objectives: Immunosuppressive immunocytes induced by cancer-associated inflammation, such as myeloid derived suppressor cells (MDSC) and T-regulatory cells (Treg), mediate immune-escape critical to development of solid tumors, including melanoma and oral squamous cell carcinoma (OSCC). Novel therapeutic approaches, which block tumor-mediated immunosuppression, unmasking endogenous anti-tumor immune responses, are thus a rational treatment approach. The aims of the study were: (1) Demonstrate that inhibition of inducible nitric oxide synthase (iNOS, critical for MDSC development) and Treg depletion in preclinical cancer models leads to potent anti-tumor activity; (2) Design a regimen suitable for testing in clinical trials. Methods: Mouse bone marrow cells were co-cultured with MT-RET melanoma supernatants ex vivo to generate MDSC in the presence of doxycycline, an iNOS inhibitor. C57/BL6 wild-type mice were injected with syngeneic MT-RET-1 melanoma. Tumor-bearing mice were treated with injection(s) of cyclophosphamide and/or doxycycline. Tumor growth and survival times were measured, and tumors and spleens harvested for CD8+ T-cell and other immunocyte levels. Results: In co-cultures, induction of MDSC was significantly reduced in the presence of doxycycline. Treatment of MT-RET melanoma-bearing C57/B16 mice with doxycycline resulted in tumor-infiltrating CD8+ T cells enhancement and tumor growth suppression. Treatment with cyclophosphamide suppressed intratumoral Treg accumulation. Dual treatment with cyclophosphamide and doxycycline suppressed tumor growth more efficiently than either alone. Conclusions: Treatment with the immunomodulators doxycycline and cyclophosphamide is a promising approach to reversing tumor-mediated immunosuppression and suppressing cancer growth in solid tumors, like melanoma and OSCC. Thus, we have designed a window of opportunity clinical trial of doxycycline and cyclophosphamide before surgical resection of OSCC.

Inflammatory lymphangiogenesis in postpartum breast tissue remodeling.

Like many cancers, mammary carcinomas use lymphatic vessels to disseminate, and numerous clinical and experimental studies have documented a strong correlation between peritumoral lymphangiogenesis and tumor dissemination. At the same time, many other factors can affect the incidence, invasiveness, and mortality of breast cancer, including lactation history. Although lactation reduces overall cancer risk, patients diagnosed within 5 years of pregnancy have an increased incidence of metastatic disease. In this issue of the JCI, Lyons and colleagues demonstrate that postpartum breast tissue remodeling during involution coincides with inflammatory lymphangiogenesis. In mouse models, cyclooxygenase-2 (COX-2) inhibition during involution reduced the risk of cancer metastasis and correlated with decreased lymphangiogenesis. In addition to lymphangiogenesis, COX-2 inhibition reduces many of the immune-suppressive features of the tumor microenvironment, including development of myeloid-derived suppressor cells and regulatory T cells; therefore, these results support the notion that inhibiting COX-2 during lactation weaning may lessen the incidence of breast cancer metastasis.

Suppressive Macrophages Control the Induction of Transplantation Tolerance.

Myeloid derived suppressor cells (MDSC) were initially described as key inhibitors of protective anti-tumor immunity, but emerging evidence suggests that MDSCs are also important mediators of transplant tolerance. Progress in this field has been limited by an inability to discern MDSC from within the greater population of myeloid cells and by a dearth of information regarding mechanisms of MDSC development in vivo. Herein we demonstrate that CD11b+CSF1R+Ly6CloLy6GnegCD169+ MDSC specifically accumulate in allografts of anti-CD154 mAb tolerized transplant recipients, and that these cells prevent CD8+ T cell mediated alloreactivity, promote CD4+Foxp3+ Treg expansion and are required for prolonged allograft survival. The transcriptome analysis of these graft-infiltrating MDSC differs from dendritic cells and corresponds to that of macrophages with inhibitory function. We reveal that CSF1-induced F4/80 expression and cell cycle entry are required for differentiation of these suppressive macrophages from their precursors. Mechanistically, we demonstrate that DC-SIGN (CD209a) controls the immune regulatory function of suppressive macrophages including the ability to induce transplantation tolerance.

Myeloid expression of angiotensin-converting enzyme facilitates myeloid maturation and inhibits the development of myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells which accumulate in cancer, infection and chronic inflammation. These cells suppress T-cell function and the immune response. Angiotensin-converting enzyme (ACE) is a peptidase that is now known to regulate aspects of myelopoiesis. Here, we show that ACE expression correlates with myeloid maturation in vitro. Forced ACE overexpression in monocytic cells reduces the generation of MDSCs. In vivo, mice with a genetic change resulting in myeloid cell ACE overexpression have reduced numbers of blood and splenic MDSCs in a tumor model and in a model of chronic inflammation induced by complete Freund's adjuvant. In contrast, ACE-null mice produce large numbers of MDSCs during chronic inflammation. Macrophages from mice with myeloid ACE overexpressing are more pro-inflammatory and have more tumor-killing activity than cells from wild-type mice. Thus, manipulating myeloid ACE activity can interfere with MDSC development and the maturation of myeloid cells.

Role of Rip2 in development of tumor-infiltrating MDSCs and bladder cancer metastasis.

Tumor invasion and metastases represent a complex series of molecular events that portends a poor prognosis. The contribution of inflammatory pathways mediating this process is not well understood. Nod-like receptors (NLRs) of innate immunity function as intracellular sensors of pathogen motifs and danger molecules. We propose a role of NLRs in tumor surveillance and in programming tumor-infiltrating lymphocytes (TILs). In this study, we examined the downstream serine/threonine and tyrosine kinase Rip2 in a murine model of bladder cancer. In Rip2-deficient C57Bl6 mice, larger orthotopic MB49 tumors developed with more numerous and higher incidence of metastases compared to wild-type controls. As such, increased tumor infiltration of CD11b+Gr1hi myeloid-derived suppressor cells (MDSCs) with concomitant decrease in T cells and NK cells were observed in Rip2-deficient tumor bearing animals using orthotopic and subcutaneous tumor models. Rip2-deficient tumors showed enhanced epithelial-to-mesenchymal transition, with elevated expression of zeb1, zeb2, twist, and snail in the tumor microenvironment. We found that the absence of Rip2 plays an intrinsic role in fostering the development of granulocytic MDSCs by an autocrine and paracrine effect of granulocytic colony stimulating factor (G-CSF) expression. Our findings suggest that NLR pathways may be a novel modality to program TILs and influence tumor metastases.

Pancreatic Cancer Cells Isolated from Muc1-Null Tumors Favor the Generation of a Mature Less Suppressive MDSC Population

Mucin 1 (MUC1) is a transmembrane mucin glycoprotein that is over-expressed and aberrantly glycosylated in >80% of human pancreatic ductal adenocarcinoma (PDA) and is associated with poor prognosis. To understand the role of MUC1 in PDA, we have recently developed two mouse models of spontaneous PDA, one that expresses full-length human MUC1 transgene (KCM mice) and one that is null for MUC1 (KCKO mice). We have previously reported that KCM mice express high levels of myeloid derived suppressor cells (MDSCs) in their tumors and develop highly aggressive PDA. To further understand the underlying mechanism for high MDSC levels in KCM-tumors, we generated primary cell lines from KCM and KCKO-tumors. In this study, we report that MDSCs derived using KCM cells express significantly higher levels of arginase 1 and inducible nitric oxide synthase (markers associated with immune suppression) and lower levels of CD115 (a marker associated with maturation of myeloid cells) as compared to KCKO-derived MDSCs. Functionally, KCM-derived MDSCs secrete significantly higher levels of urea and nitric oxide (NO) when co-cultured with normal splenic cells as compared to KCKO-derived MDSCs. Data indicates that KCM-derived MDSCs remain immature and are more suppressive as compared to KCKO-derived MDSCs. This was further corroborated in vivo where MDSCs isolated from KCM-tumor-bearing mice retained their immature state and were highly suppressive as compared to MDSCs derived from KCKO-tumor-bearing mice. Finally, we show that KCM cells secrete significantly higher levels of prostaglandin E2 (PGE2), a COX-2 metabolite and a known driver of suppressive MDSCs as compared to KCKO cells. Thus, inhibiting PGE2 with a specific COX-2 inhibitor reverses the immunosuppressive and immature phenotype of KCM-derived MDSCs. This is the first report that clearly suggests a functional role of pancreatic tumor-associated MUC1 in the development of functional MDSCs.