Evgeniy Eruslanov

Reversal of Myeloid Cell–Mediated Immunosuppression in Patients with Metastatic Renal Cell Carcinoma

Purpose: Tumor-induced immunosuppression remains a significant obstacle that limits the efficacy of biological therapy for renal cell carcinoma. Here we evaluate the role of CD33 myeloid-derived suppressor cells (MDSC) in the regulation of T-cell responses in renal cell carcinoma patients. We also examine effect of all-trans-retinoic acid (ATRA) on MDSC-mediated immune suppression. Experimental Design: CD33-positive myeloid cells were isolated from the peripheral blood of renal cell carcinoma patients with magnetic beads and tested in vitro for their ability to inhibit T-cell responses. T-cell function was evaluated using ELISPOT and CTL assays. Results: MDSC isolated from renal cell carcinoma patients, but not from healthy donors, were capable of suppressing antigen-specific T-cell responses in vitro through the secretion of reactive oxygen species and nitric oxide upon interaction with CTL. MDSC-mediated immune suppression and IFN-γ down-regulation was reversible in vitro by exposing cells to the reactive oxygen species inhibitors. Moreover, ATRA was capable of abrogating MDSC-mediated immunosuppression and improving T-cell function by direct differentiation into antigen-presenting cell precursors. Conclusions: These results may have significant implications regarding the future design of active immunotherapy protocols that may include differentiation agents as part of a multimodal approach to renal cell carcinoma immunotherapy.

Tumor-associated neutrophils stimulate T cell responses in early-stage human lung cancer

Infiltrating inflammatory cells are highly prevalent within the tumor microenvironment and mediate many processes associated with tumor progression; however, the contribution of specific populations remains unclear. For example, the nature and function of tumor-associated neutrophils (TANs) in the cancer microenvironment is largely unknown. The goal of this study was to provide a phenotypic and functional characterization of TANs in surgically resected lung cancer patients. We found that TANs constituted 5%-25% of cells isolated from the digested human lung tumors. Compared with blood neutrophils, TANs displayed an activated phenotype (CD62L(lo)CD54(hi)) with a distinct repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. TANs produced substantial quantities of the proinflammatory factors MCP-1, IL-8, MIP-1α, and IL-6, as well as the antiinflammatory IL-1R antagonist. Functionally, both TANs and neutrophils isolated from distant nonmalignant lung tissue were able to stimulate T cell proliferation and IFN-γ release. Cross-talk between TANs and activated T cells led to substantial upregulation of CD54, CD86, OX40L, and 4-1BBL costimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive-feedback loop. Together our results demonstrate that in the earliest stages of lung cancer, TANs are not immunosuppressive, but rather stimulate T cell responses.

Oxidative Stress Regulates Expression of VEGFR1 in Myeloid Cells: Link to Tumor-Induced Immune Suppression in Renal Cell Carcinoma

Metastatic renal cell carcinoma (RCC) associates with overproduction of vascular endothelial growth factor (VEGF) due to the mutation/inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. Herein we demonstrate that implantation of human RCC tumor cells into athymic nude mice promotes the appearance of VEGF receptor 1 (VEGFR1)/CD11b double-positive myeloid cells in peripheral blood. Avastin-mediated VEGF neutralization was capable of significantly reducing the numbers of circulating VEGFR1+ myeloid cells. Conversely, up-regulation of VEGFR1 by myeloid cells could also be achieved in vitro by coculturing bone marrow cells with RCC-conditioned medium or by short-term exposure of naive myeloid cells to oxidative stress. Treatment of myeloid cells with H2O2, lipid peroxidation product 4-hydroxy-2(E)-nonenal, or an inhibitor of thioredoxin reductase all resulted in increased expression of VEGFR1. Furthermore, after exposure to oxidative stress, myeloid cells acquire immunosuppressive features and become capable of inhibiting T cell proliferation. Data suggest that tumor-induced oxidative stress may promote both VEGFR1 up-regulation and immunosuppressive function in bone marrow-derived myeloid cells. Analysis of tumor tissue and peripheral blood from patients with metastatic RCC revealed that VEGFR1+ cells can be also found in cancer patients. Restoration of immunocompetence in metastatic RCC patients by pharmacological elimination of VEGFR1+ cells may have a significant impact on the therapeutic efficacy of cancer vaccines or other immune-based therapies.

Pivotal Advance: Tumor-mediated induction of myeloid-derived suppressor cells and M2-polarized macrophages by altering intracellular PGE2 catabolism in myeloid cells

Recent studies suggest that tumor‐infiltrated myeloid cells frequently up‐regulate COX‐2 expression and have enhanced PGE2 metabolism. This may affect the maturation and immune function of tumor‐infiltrated antigen‐presenting cells. In vitro studies demonstrate that tumor‐derived factors can skew GM‐CSF‐driven differentiation of Th1‐oriented myeloid APCs into M2‐oriented Ly6C+F4/80+ MDSCs or Ly6C–F4/80+ arginase‐expressing macrophages. These changes enable myeloid cells to produce substantial amounts of IL‐10, VEGF, and MIP‐2. The tumor‐mediated inhibition of APC differentiation was associated with the up‐regulated expression of PGE2‐forming enzymes COX‐2, mPGES1 in myeloid cells, and the simultaneous repression of PGE2‐catabolizing enzyme 15‐PGDH. The presence of tumor‐derived factors also led to a reduced expression of PGT but promoted the up‐regulation of MRP4, which works as a PGE2 efflux receptor. Addition of COX‐2 inhibitor to the BM cell cultures could prevent the tumor‐induced skewing of myeloid cell differentiation, partially restoring cell phenotype and down‐regulating the arginase expression in the myeloid APCs. Our study suggests that tumors impair the intracellular PGE2 catabolism in myeloid cells through simultaneous stimulation of PGE2‐forming enzymes and inhibition of PGE2‐degrading systems. This tumor‐induced dichotomy drives the development of M2‐oriented, arginase‐expressing macrophages or the MDSC, which can be seen frequently among tumor‐infiltrated myeloid cells.

Tumor-Associated Macrophages Mediate Immunosuppression in the Renal Cancer Microenvironment by Activating the 15-Lipoxygenase-2 Pathway

Renal cell carcinoma (RCC), the most common human kidney cancer, is frequently infiltrated with tumor-associated macrophages (TAM) that can promote malignant progression. Here, we show that TAMs isolated from human RCC produce substantial amounts of the proinflammatory chemokine CCL2 and immunosuppressive cytokine IL-10, in addition to enhanced eicosanoid production via an activated 15-lipoxygenase-2 (15-LOX2) pathway. TAMs isolated from RCC tumors had a high 15-LOX2 expression and secreted substantial amounts of 15(S)-hydroxyeicosatetraenoic acid, its major bioactive lipid product. Inhibition of lipoxygenase activity significantly reduced production of CCL2 and IL-10 by RCC TAMs. In addition, TAMs isolated from RCC were capable of inducing in T lymphocytes, the pivotal T regulatory cell transcription factor forkhead box P3 (FOXP3), and the inhibitory cytotoxic T-lymphocyte antigen 4 (CTLA-4) coreceptor. However, this TAM-mediated induction of FOXP3 and CTLA-4 in T cells was independent of lipoxygenase and could not be reversed by inhibiting lipoxygenase activity. Collectively, our results show that TAMs, often present in RCCs, display enhanced 15-LOX2 activity that contributes to RCC-related inflammation, immunosuppression, and malignant progression. Furthermore, we show that TAMs mediate the development of immune tolerance through both 15-LOX2-dependent and 15-LOX2-independent pathways. We propose that manipulating LOX-dependent arachidonic acid metabolism in the tumor microenvironment could offer new strategies to block cancer-related inflammation and immune escape in patients with RCC.

Circulating and tumor-infiltrating myeloid cell subsets in patients with bladder cancer

Both cancer‐related inflammation and tumor‐induced immune suppression are associated with expansion of myeloid cell subsets including myeloid‐derived suppressor cells. However, little known regarding characteristics of myeloid cells in patients with bladder cancer. In this study, we analyzed myeloid cells from peripheral blood (PBMC) and tumor tissue that were collected from patients with superficial noninvasive and invasive urothelial carcinomas. Our results demonstrate that PBMC from bladder cancer patients contain two major CD11b myeloid cell subsets: granulocyte‐type CD15high CD33low cells and monocyte‐type CD15low CD33high cells. The number of circulating granulocytic but not monocytic myeloid cells in cancer patients was markedly increased when compared to healthy individuals. Both myeloid cell subsets from cancer patients were highly activated and produced substantial amounts of proinflammatory chemokines/cytokines including CCL2, CCL3, CCL4, G‐CSF, IL‐8 and IL‐6. Granulocytic myeloid cells were able to inhibit in vitro T cell proliferation through induction of CD4+Foxp3+ T regulatory cells. Analysis of bladder cancer tissues revealed that tumors were infiltrated with monocyte–macrophage CD11b+HLA‐DR+ and granulocytic CD11b+CD15+HLA‐DR‐ myeloid cells. Collectively, this study identifies myeloid cell subsets in patients with bladder cancer. We demonstrate that these highly activated inflammatory myeloid cells represent a source of multiple chemokines/cytokines and may contribute to inflammation and immune dysfunction in bladder cancer.

Effects of CXCR4 antagonist CTCE-9908 on prostate tumor growth†

Recent reports have linked the survival‐promoting effect of CXCR4 to the up regulation of Bcl‐2 protein expression.

Changes in the local tumor microenvironment in recurrent cancers may explain the failure of vaccines after surgery

Each year, more than 700,000 people undergo cancer surgery in the United States. However, more than 40% of those patients develop recurrences and have a poor outcome. Traditionally, the medical community has assumed that recurrent tumors arise from selected tumor clones that are refractory to therapy. However, we found that tumor cells have few phenotypical differences after surgery. Thus, we propose an alternative explanation for the resistance of recurrent tumors. Surgery promotes inhibitory factors that allow lingering immunosuppressive cells to repopulate small pockets of residual disease quickly. Recurrent tumors and draining lymph nodes are infiltrated with M2 (CD11b+F4/80hiCD206hi and CD11b+F4/80hiCD124hi) macrophages and CD4+Foxp3+ regulatory T cells. This complex network of immunosuppression in the surrounding tumor microenvironment explains the resistance of tumor recurrences to conventional cancer vaccines despite small tumor size, an intact antitumor immune response, and unaltered cancer cells. Therapeutic strategies coupling antitumor agents with inhibition of immunosuppressive cells potentially could impact the outcomes of more than 250,000 people each year.

Altered Expression of 15-Hydroxyprostaglandin Dehydrogenase in Tumor-Infiltrated CD11b Myeloid Cells: A Mechanism for Immune Evasion in Cancer

Many cancers are known to produce high amounts of PGE2, which is involved in both tumor progression and tumor-induced immune dysfunction. The key enzyme responsible for the biological inactivation of PGE2 in tissue is NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). It is well established that cancer cells frequently show down-regulated expression of 15-PGDH, which plays a major role in catabolism of the PGE2. Here we demonstrate that tumor-infiltrated CD11b cells are also deficient for the 15-PGDH gene. Targeted adenovirus-mediated delivery of 15-PGDH gene resulted in substantial inhibition of tumor growth in mice with implanted CT-26 colon carcinomas. PGDH-mediated antitumor effect was associated with attenuated tumor-induced immune suppression and substantially reduced secretion of immunosuppressive mediators and cytokines such as PGE2, IL-10, IL-13, and IL-6 by intratumoral CD11b cells. We show also that introduction of 15-PGDH gene in tumor tissue is sufficient to redirect the differentiation of intratumoral CD11b cells from immunosuppressive M2-oriented F4/80+ tumor-associated macrophages (TAM) into M1-oriented CD11c+ MHC class II-positive myeloid APCs. Notably, the administration of the 15-PGDH gene alone demonstrated a significant therapeutic effect promoting tumor eradication and long-term survival in 70% of mice with preestablished tumors. Surviving mice acquired antitumor T cell-mediated immune response. This study for the first time demonstrates an important role of the 15-PGDH in regulation of local antitumor immune response and highlights the potential to be implemented to enhance the efficacy of cancer therapy and immunotherapy.

Lectin-type oxidized LDL receptor-1 distinguishes population of human polymorphonuclear myeloid-derived suppressor cells in cancer patients

PMN-MDSC in cancer patients can be distinguished from neutrophils by a genomic signature and by expression of the LOX-1 receptor. Stressing myeloid-derived suppressor cells in cancer Immunotherapies for cancer have shown promising results in part because they overcome the suppressive effects of the tumor microenvironment on immune cells. Condamine et al. now report that polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) can be distinguished from neutrophils in the same cancer patient by the expression of the lipid metabolism–related molecule lectin-type oxidized LDL receptor-1 (LOX-1). LOX-1–expressing neutrophils were nearly undetectable in healthy individuals but were found prominently in tumor tissues. Moreover, exposing neutrophils from healthy individuals to endoplasmic reticulum stress resulted in up-regulation of LOX-1 and increased suppressive function. These data support the specific targeting of LOX-1–expressing PMN-MDSC for cancer immunotherapy. Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are important regulators of immune responses in cancer and have been directly implicated in the promotion of tumor progression. However, the heterogeneity of these cells and the lack of distinct markers hamper the progress in understanding the biology and clinical importance of these cells. Using partial enrichment of PMN-MDSC with gradient centrifugation, we determined that low-density PMN-MDSC and high-density neutrophils from the same cancer patients had a distinct gene profile. The most prominent changes were observed in the expression of genes associated with endoplasmic reticulum (ER) stress. Unexpectedly, low-density lipoprotein (LDL) was one of the most increased regulators, and its receptor oxidized LDL receptor 1 (OLR1) was one of the most overexpressed genes in PMN-MDSC. Lectin-type oxidized LDL receptor-1 (LOX-1) encoded by OLR1 was practically undetectable in neutrophils in peripheral blood of healthy donors, whereas 5 to 15% of total neutrophils in cancer patients and 15 to 50% of neutrophils in tumor tissues were LOX-1+. In contrast to their LOX-1− counterparts, LOX-1+ neutrophils had gene signature, potent immunosuppressive activity, up-regulation of ER stress, and other biochemical characteristics of PMN-MDSCs. Moreover, induction of ER stress in neutrophils from healthy donors up-regulated LOX-1 expression and converted these cells to suppressive PMN-MDSCs. Thus, we identified a specific marker of human PMN-MDSC associated with ER stress and lipid metabolism, which provides new insights into the biology and potential therapeutic targeting of these cells.