Alexandra Sevko

Chronic inflammation promotes myeloid-derived suppressor cell activation blocking antitumor immunity in transgenic mouse melanoma model

Tumor microenvironment is characterized by chronic inflammation represented by infiltrating leukocytes and soluble mediators, which lead to a local and systemic immunosuppression associated with cancer progression. Here, we used the ret transgenic spontaneous murine melanoma model that mimics human melanoma. Skin tumors and metastatic lymph nodes showed increased levels of inflammatory factors such as IL-1β, GM-CSF, and IFN-γ, which correlated with tumor progression. Moreover, Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs), known to inhibit tumor reactive T cells, were enriched in melanoma lesions and lymphatic organs during tumor progression. MDSC infiltration was associated with a strong TCR ζ-chain down-regulation in all T cells. Coculturing normal splenocytes with tumor-derived MDSC induced a decreased T-cell proliferation and ζ-chain expression, verifying the MDSC immunosuppressive function and suggesting that the tumor inflammatory microenvironment supports MDSC recruitment and immunosuppressive activity. Indeed, upon manipulation of the melanoma microenvironment with the phosphodiesterase-5 inhibitor sildenafil, we observed reduced levels of numerous inflammatory mediators (e.g., IL-1β, IL-6, VEGF, S100A9) in association with decreased MDSC amounts and immunosuppressive function, indicating an antiinflammatory effect of sildenafil. This led to a partial restoration of ζ-chain expression in T cells and to a significantly increased survival of tumor-bearing mice. CD8 T-cell depletion resulted in an abrogation of sildenafil beneficial outcome, suggesting the involvement of MDSC and CD8 T cells in the observed therapeutic effects. Our data imply that inhibition of chronic inflammation in the tumor microenvironment should be applied in conjunction with melanoma immunotherapies to increase their efficacy.

Antitumor Effect of Paclitaxel Is Mediated by Inhibition of Myeloid-Derived Suppressor Cells and Chronic Inflammation in the Spontaneous Melanoma Model

The antitumor effects of paclitaxel are generally attributed to the suppression of microtubule dynamics resulting in defects in cell division. New data demonstrated that in ultralow noncytotoxic concentrations, paclitaxel modulated in immune cells in vitro the activity of small Rho GTPases, the key regulators of intracellular actin dynamics. However, the immunomodulatory properties of paclitaxel in vivo have not been evaluated. In this study, using the ret transgenic murine melanoma model, which mimics human cutaneous melanoma, we tested effects of ultralow noncytotoxic dose paclitaxel on functions of myeloid-derived suppressor cells (MDSCs), chronic inflammatory mediators, and T cell activities in the tumor microenvironment in vivo. Administration of paclitaxel significantly decreased accumulation and immunosuppressive activities of tumor-infiltrating MDSCs without alterations of the bone marrow hematopoiesis. This was associated with the inhibition of p38 MAPK activity, TNF-α and production, and S100A9 expression in MDSCs. The production of mediators of chronic inflammation in the tumor milieu also was diminished. Importantly, reduced tumor burden and increased animal survival upon paclitaxel application was mediated by the restoration of CD8 T cell effector functions. We suggest that the ability of paclitaxel in a noncytotoxic dose to block the immunosuppressive potential of MDSCs in vivo represents a new therapeutic strategy to downregulate immunosuppression and chronic inflammation in the tumor microenvironment for enhancing the efficacy of concomitant anticancer therapies.

Extracellular vesicle-mediated transfer of functional RNA in the tumor microenvironment.

Extracellular vesicles (EVs) have been shown to transfer various molecules, including functional RNA between cells and this process has been suggested to be particularly relevant in tumor-host interactions. However, data on EV-mediated RNA transfer has been obtained primarily by in vitro experiments or involving ex vivo manipulations likely affecting its biology, leaving their physiological relevance unclear. We engineered glioma and carcinoma tumor cells to express Cre recombinase showing their release of EVs containing Cre mRNA in various EV subfractions including exosomes. Transplantation of these genetically modified tumor cells into mice with a Cre reporter background leads to frequent recombination events at the tumor site. In both tumor models the majority of recombined cells are CD45+ leukocytes, predominantly Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs). In addition, multiple lineages of recombined cells can be observed in the glioma model. In the lung carcinoma model, recombined MDSCs display an enhanced immunosuppressive phenotype and an altered miRNA profile compared to their non-recombined counterparts. Cre-lox based tracing of tumor EV RNA transfer in vivo can therefore be used to identify individual target cells in the tumor microenvironment for further mechanistical or functional analysis.

Cyclophosphamide Promotes Chronic Inflammation-Dependent Immunosuppression and Prevents Antitumor Response in Melanoma

Low-dose cyclophosphamide (CP) therapy induces immunogenic tumor cell death and decreases regulatory T cell (Treg) numbers in mice with transplantable tumors. Using the ret transgenic murine melanoma model that resembles human melanoma, we detected no beneficial antitumor effects with such treatment, despite a decrease in Tregs. On the contrary, low-dose CP enhanced the production of chronic inflammatory mediators in melanoma lesions associated with increased accumulation of Gr1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs), which exhibit elevated suppressive activity and nitric oxide (NO) production as well as inhibition of T-cell proliferation. Moreover, the frequencies of CD8(+) T cells in the tumors and their ability to produce perforin were decreased. To study whether the observed CP-induced MDSC expansion and activation also occurs under chronic inflammatory tumor-free conditions, mice exhibiting chronic inflammation were treated with CP. Similar to tumor-bearing mice, CP-treated inflamed mice displayed elevated levels of MDSCs with enhanced production of NO, reactive oxygen species, and a suppressed in vivo natural killer (NK) cell cytotoxic activity indicating CP effects on the host immune system independent of the tumor. We suggest that melanoma therapy with low-dose CP could be efficient only when combined with the neutralization of MDSC immunosuppressive function and chronic inflammatory microenvironment.

Tumor Microenvironment and Myeloid-Derived Suppressor Cells

Tumor progression has been demonstrated to be supported by chronic inflammatory conditions developed in the tumor microenvironment and characterized by the long-term secretion of various inflammatory soluble factors (including cytokines, chemokines, growth factors, reactive oxygen and nitrogen species, prostaglandins etc.) and strong leukocyte infiltration. Among leukocytes infiltrating tumors, myeloid-derived suppressor cells (MDSCs) represent one of the most important players mediating immunosuppression. These cells may not only strongly inhibit an anti-tumor immune reactions mediated by T cells but also directly stimulate tumorigenesis, tumor growth and metastasis by enhancing neoangiogenesis and creating a suitable environment for the metastatic formation. This review provides an overview of interactions between MDSCs and tumor cells leading to MDSC generation, activation and migration to the tumor site, where they can strongly enhance tumor progression. Better understanding of the MDSC-tumor interplay is critical for the development of new strategies of tumor immunotherapy.

Skin melanoma development in ret transgenic mice despite the depletion of CD25+Foxp3+ regulatory T cells in lymphoid organs.

CD4+CD25+Foxp3+ regulatory T cells (Treg) known to mediate self-tolerance were also shown to contribute to tumor progression. In mouse melanoma transplantation models, Treg depletion resulted in the stimulation of antitumor immune responses and tumor eradication. To study Treg in conditions close to the clinical situation, we used a ret transgenic mouse spontaneous melanoma model, which, in contrast to transplantation models, resembles human melanoma regarding clinical development. Significantly higher numbers of Treg were found in skin tumors and metastatic lymph nodes at early stages of melanoma progression compared with more advanced stages accompanied by the elevated CCR4 expression on Treg and higher production of its ligand CCL2 in tumor lesions. Numbers of tumor infiltrating Treg inversely correlated with Treg amounts in the bone marrow, suggesting their possible recruitment to melanoma lesions from this organ. The immunosuppressive function of Treg from transgenic tumor-bearing mice was similar to that from transgenic tumor-free mice or nontransgenic littermates. Although anti-CD25 mAb injections resulted in the efficient Treg depletion from lymphoid organs of transgenic mice, melanoma development was not significantly delayed. Furthermore, the treatment of mice with macroscopical tumors also failed to inhibit tumor progression, which correlated with the inability to deplete intratumoral Treg. We suggest that in the autochthonous melanoma genesis, other immunosuppressive cells could play an important role and replace immunosuppressive, tumor-promoting functions of Treg. Therefore, effective melanoma immunotherapy should include the inhibition of Treg migration into the tumor combined with neutralization of other immunosuppressive cells and factors in the tumor microenvironment.

Myeloid-derived suppressor cells in malignant melanoma

Melanoma is known for its rapid progression, metastasis to distant organs and therapeutic resistance. Despite high melanoma immunogenicity, the results of immunotherapeutic clinical studies are mostly unsatisfactory. One explanation is the development of strong immunosuppression mediated by highly immunosuppressive regulatory leukocytes, in particular, myeloid‐derived suppressor cells (MDSCs). These cells were found to be enriched and activated in the melanoma microenvironment, inducing a profound impairment of anti‐tumor immune responses and leading to the tumor progression. Therefore, understanding the mechanisms of MDSC generation, migration to the tumor site and activation as well as their targeting is important for the development of novel strategies for effective melanoma immunotherapy. We suggest that such therapeutic approaches should involve the inhibition of MDSC‐mediated immunosuppressive melanoma microenvironment combined with other immunologic treatments.

Serum inflammatory factors and circulating immunosuppressive cells are predictive markers for efficacy of radiofrequency ablation in non‐small‐cell lung cancer

In recent years, percutaneous radiofrequency ablation (RFA) has been developed as a new tool in the treatment of non‐small‐cell lung cancer (NSCLC) in non‐surgical patients. There is growing evidence that RFA‐mediated necrosis can modulate host immune responses. Here we analysed serum inflammatory factors as well as immunosuppressive cells in the peripheral blood to discover possible prognostic indicators. Peripheral blood and serum samples were collected before RFA and within 3 months after the treatment in a total of 12 patients. Inflammatory cytokines and growth factors were measured in serum by the Bio‐Plex assay. Myeloid‐derived suppressor cells (MDSCs) and regulatory T cells (Tregs) were evaluated in the peripheral blood via flow cytometry. In patients developing local or lymphogenic tumour relapse (n = 4), we found an early significant increase in the concentration of tumour necrosis factor (TNF)‐α as well as chemokine (C‐C motif) ligand (CCL)‐2 and CCL‐4 compared to patients without relapse (n = 4) and healthy donors (n = 5). These changes were associated with an elevated activity of circulating MDSC indicated by an increased nitric oxide (NO) production in these cells. Elevated serum levels of TNF‐α, CCL‐2 and CCL‐4 associated with an increased NO production in circulating MDSCs might be an early indicator of the incomplete RFA and subsequently a potential tumour relapse in NSCLC.

Tadalafil has biologic activity in human melanoma. Results of a pilot trial with Tadalafil in patients with metastatic Melanoma (TaMe)

ABSTRACT Myeloid-derived suppressor cells (MDSCs) are known to play a critical role in the suppression of T cell antitumor responses. Our preclinical data showed that the phosphodiesterase (PDE)-5 inhibitor sildenafil impaired MDSC functions, enhanced intratumoral T cell activity and prolonged survival of melanoma-bearing mice. In this study, we evaluated biologic effects, safety and efficacy of palliative treatment with the PDE-5 inhibitor tadalafil in metastatic melanoma patients. We conducted an open-label, dose de-escalation trial with tadalafil in pretreated metastatic melanoma patients. Tumor and peripheral blood samples were taken before and 4 weeks after the start of treatment. Samples were investigated by immunohistochemistry and FACS analysis, for different immune subsets with numbers of CD8+ tumor-infiltrating lymphocytes (TIL) as primary end point. Stable disease was achieved in 3/12 patients (25%). Median progression-free survival was 4.6 mo (range 0.7–7.1), median overall survival (OS) 8.5 mo (range 2.7–23.7). The treatment was well tolerated. Stable patients displayed significantly higher numbers of CD8+ TIL in the center of metastases before treatment as compared with progressive patients. Upon the therapy, they showed increased expression of ζ-chain (used as a marker of T cell activation) in CD8+ and CD4+TILs and CD8+T cells in the peripheral blood as compared with baseline. Our study suggests that the PDE-5 inhibitor tadalafil can improve clinical outcome of advanced melanoma patients by enhancing antitumor immunity and highlights its potential application in combined melanoma immunotherapy.

Ret transgenic mouse model of spontaneous skin melanoma: focus on regulatory T cells

Ret transgenic mouse model of skin malignant melanoma is characterized by the overexpression of the human ret transgene in melanin‐containing cells. Transgenic mice spontaneously develop skin tumors with metastases in lymph nodes, lungs, liver, brain, and the bone marrow. Tumor lesions show typical melanoma morphology and express melanoma‐associated antigens. Although transgenic mice demonstrate an accumulation of melanoma antigen‐specific memory and effector T cells, their anti‐tumor effects could be blocked by highly immunosuppressive leukocytes enriched in the tumor microenvironment and in the periphery. Here, we discuss the role of one of the most potent immunosuppressive subset, regulatory T cells, in the melanoma progression in this model.