Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent antitumor immunity in melanoma.

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.

Generation of Myeloid-Derived Suppressor Cells Mediated by MicroRNA-125a-5p in Melanoma.

The ability of tumor-derived extracellular vesicles (EVs) to modulate the function of myeloid cells is widely recognized. Hence, a comprehensive understanding of the distinct components associated with EVs and the signals that they deliver to myeloid cells could provide potential approaches to impede the immunosuppression by myeloid-derived suppressor cells (MDSCs). We investigated melanoma EV-associated microRNAs (miRs) using the RET transgenic melanoma mouse model and simulated their transfer to normal myeloid cells by transfecting immature mouse myeloid cells and human monocytes. We observed elevated levels of miR-125a-5p, -125b-5p, and let-7e-5p in mouse melanoma-infiltrating MDSCs. In addition, miR-125a-5p levels in the tumor microenvironment correlated with mouse melanoma progression. The delivery of miR-125a-5p, alone or in combination with let-7e-5p and miR-99b-5p from the same genomic cluster, to normal myeloid cells resulted in their conversion to MDSC-like cells. Our findings indicate that miR-125a-5p could modulate myeloid cell activation in the melanoma microenvironment via a NF-κB-dependent mechanism.

SOX2 in development and cancer biology.

The transcription factor SOX2 is essential for embryonic development and plays a crucial role in maintaining the stemness of embryonic cells and various adult stem cell populations. On the other hand, dysregulation of SOX2 expression is associated with a multitude of cancer types and it has been shown that SOX2 positively affects cancer cell traits such as the capacity to proliferate, migrate, invade and metastasize. Moreover, there is growing evidence that SOX2 mediates resistance towards established cancer therapies and that it is expressed in cancer stem cells. These findings indicate that studying the role of SOX2 in the context of cancer progression could lead to the development of new therapeutic options. In this review, the current knowledge about the role of SOX2 in development, maintenance of stemness, cancer progression and the resistance towards cancer therapies is summarized.

Tumor promoting capacity of polymorphonuclear myeloid-derived suppressor cells and their neutralization.

Myeloid-derived suppressor cells (MDSC) represent a highly immunosuppressive population that expands in tumor bearing hosts and inhibits both T and NK cell antitumor effector functions. Among MDSC subpopulations, the polymorphonuclear (PMN) one is gaining increasing interest since it is a predominant MDSC subset in most cancer entities and inherits unique properties to facilitate metastatic spread. In addition, further improvement in distinguishing PMN-MDSC from neutrophils has contributed to the design of novel therapeutic approaches. In this review, we summarize the current view on the origin of PMN-MDSC and their relation to classical neutrophils. Furthermore, we outline the metastasis promoting features of these cells and promising strategies of their targeting to improve the efficacy of cancer immunotherapy.

FOXD1 promotes dedifferentiation and targeted therapy resistance in melanoma by regulating the expression of connective tissue growth factor.

Metastatic melanoma is an aggressive skin cancer and associated with a poor prognosis. In clinical terms, targeted therapy is one of the most important treatments for patients with BRAFV600E -mutated advanced melanoma. However, the development of resistance to this treatment compromises its therapeutic success. We previously demonstrated that forkhead box D1 (FOXD1) regulates melanoma migration and invasion. Here, we found that FOXD1 was highly expressed in melanoma cells and was associated with a poor survival of patients with metastatic melanoma. Upregulation of FOXD1 expression enhanced melanoma cells' resistance to vemurafenib (BRAF inhibitor [BRAFi]) or vemurafenib and cobimetinib (MEK inhibitor) combination treatment whereas loss of FOXD1 increased the sensitivity to treatment. By comparing gene expression levels between FOXD1 knockdown (KD) and overexpressing (OE) cells, we identified the connective tissue growth factor (CTGF) as a downstream factor of FOXD1. Chromatin immunoprecipitation and luciferase assay demonstrated the direct binding of FOXD1 to the CTGF promoter. Similar to FOXD1, knockdown of CTGF increased the sensitivity of BRAFi-resistant cells to vemurafenib. FOXD1 KD cells treated with recombinant CTGF protein were less sensitive towards vemurafenib compared to untreated FOXD1 KD cells. Based on these findings, we conclude that FOXD1 might be a promising new diagnostic marker and a therapeutic target for the treatment of targeted therapy resistant melanoma.

IL-6 as a major regulator of MDSC activity and possible target for cancer immunotherapy.

Myeloid-derived suppressor cells (MDSC) are generated during tumor progression and suppress the anti-tumor functions of T and natural killer (NK) cells. Their enrichment is associated with a bad prognosis and a worse outcome of immunotherapy in cancer patients. The cytokine interleukin (IL)-6 was found to be a crucial regulator of MDSC accumulation and activation as well as a factor, stimulating tumor cell proliferation, survival, invasiveness and metastasis. Accordingly, IL-6 can serve as a negative prognostic marker in cancer. On the other hand, this cytokine is also involved in T cell activation. This review discusses the pleiotropic effects of IL-6 on immune cell populations that are critical for tumor development, such as MDSC and T cells, and summarizes the data on targeting IL-6 or IL-6 receptor (IL-6R) for tumor immunotherapy to block MDSC-mediated immunosuppression in cancer patients.

Potential therapeutic effect of low-dose paclitaxel in melanoma patients resistant to immune checkpoint blockade: A pilot study.

The low dose application of chemotherapeutic agents such as paclitaxel was previously shown to initiate anti-tumor activity by neutralizing myeloid-derived suppressor cells (MDSCs) in melanoma mouse models. Here, we investigated immunomodulating effects of low-dose paclitaxel in 9 metastatic melanoma patients resistant to prior treatments. Three patients showed response to therapy (two partial responses and one stable disease). In responding patients, paclitaxel decreased the frequency and immunosuppressive pattern of MDSCs in the peripheral blood and skin metastases. Furthermore, paclitaxel modulated levels of inflammatory mediators in the serum. In addition, responders displayed enhanced frequencies of tumor-infiltrating CD8+ T cells and their activity indicated by the upregulation of CD25 and TCR ζ-chain expression. Our study suggests that low-dose paclitaxel treatment could improve clinical outcome of some advanced melanoma patients by enhancing anti-tumor immunity and might be proposed for combined melanoma immunotherapy.

Neutrophils in Tumorigenesis: Missing Targets for Successful Next Generation Cancer Therapies?

Neutrophils-once considered as simple killers of pathogens and unexciting for cancer research-are now acknowledged for their role in the process of tumorigenesis. Neutrophils are recruited to the tumor microenvironment where they turn into tumor-associated neutrophils (TANs), and are able to initiate and promote tumor progression and metastasis. Conversely, anti-tumorigenic properties of neutrophils have been documented, highlighting the versatile nature and high pleiotropic plasticity of these polymorphonuclear leukocytes (PMN-L). Here, we dissect the ambivalent roles of TANs in cancer and focus on selected functional aspects that could be therapeutic targets. Indeed, the critical point of targeting TAN functions lies in the fact that an immunosuppressive state could be induced, resulting in unwanted side effects. A deeper knowledge of the mechanisms linked to diverse TAN functions in different cancer types is necessary to define appropriate therapeutic strategies that are able to induce and maintain an anti-tumor microenvironment.

T-type calcium channel inhibition restores sensitivity to MAPK inhibitors in de-differentiated and adaptive melanoma cells.

BACKGROUND: Drug resistance remains as one of the major challenges in melanoma therapy. It is well known that tumour cells undergo phenotypic switching during melanoma progression, increasing melanoma plasticity and resistance to mitogen-activated protein kinase inhibitors (MAPKi). METHODS: We investigated the melanoma phenotype switching using a partial reprogramming model to de-differentiate murine melanoma cells and target melanoma therapy adaptation against MAPKi. RESULTS: Here, we show that partially reprogrammed cells are a less proliferative and more de-differentiated cell population, expressing a gene signature for stemness and suppressing melanocyte-specific markers. To investigate adaptation to MAPKi, cells were exposed to B-Raf Proto-Oncogene (BRAF) and mitogen-activated protein kinase kinase (MEK) inhibitors. De-differentiated cells became less sensitive to MAPKi, showed increased cell viability and decreased apoptosis. Furthermore, T-type calcium channels expression increased in adaptive murine cells and in human adaptive melanoma cells. Treatment with the calcium channel blocker mibefradil induced cell death, differentiation and susceptibility to MAPKi in vitro and in vivo. CONCLUSION: In summary, we show that partial reprogramming of melanoma cells induces de-differentiation and adaptation to MAPKi. Moreover, we postulated a calcium channel blocker such as mibefradil, as a potential candidate to restore sensitivity to MAPKi in adaptive melanoma cells.

Myeloid Cell Modulation by Tumor-Derived Extracellular Vesicles.

Extracellular vesicles (EV) can carry proteins, RNA and DNA, thus serving as communication tools between cells. Tumor cells secrete EV, which can be taken up by surrounding cells in the tumor microenvironment as well as by cells in distant organs. Tumor-derived EV (TEV) contain factors induced by tumor-associated hypoxia such as heat shock proteins or a variety of microRNA (miRNA). The interaction of TEV with tumor and host cells can promote cancer angiogenesis, invasion and metastasis. Myeloid cells are widely presented in tissues, comprise the majority of immune cells and play an essential role in immune reactions and tissue remodeling. However, in cancer, the differentiation of myeloid cells and their functions are impaired, resulting in tumor promotion. Such alterations are due to chronic inflammatory conditions associated with cancer and are mediated by the tumor secretome, including TEV. A high capacity of myeloid cells to clear EV from circulation put them in the central position in EV-mediated formation of pre-metastatic niches. The exposure of myeloid cells to TEV could trigger numerous signaling pathways. Progenitors of myeloid cells alter their differentiation upon the contact with TEV, resulting in the generation of myeloid-derived suppressor cells (MDSC), inhibiting anti-tumor function of T and natural killer (NK) cells and promoting thereby tumor progression. Furthermore, TEV can augment MDSC immunosuppressive capacity. Different subsets of mature myeloid cells such as monocytes, macrophages, dendritic cells (DC) and granulocytes take up TEV and acquire a protumorigenic phenotype. However, the delivery of tumor antigens to DC by TEV was shown to enhance their immunostimulatory capacity. The present review will discuss a diverse and complex EV-mediated crosstalk between tumor and myeloid cells in the context of the tumor type, TEV-associated cargo molecules and type of recipient cells.

Modern Aspects of Immunotherapy with Checkpoint Inhibitors in Melanoma.

Although melanoma is one of the most immunogenic tumors, it has an ability to evade anti-tumor immune responses by exploiting tolerance mechanisms, including negative immune checkpoint molecules. The most extensively studied checkpoints represent cytotoxic T lymphocyte-associated protein-4 (CTLA-4) and programmed cell death protein 1 (PD-1). Immune checkpoint inhibitors (ICI), which were broadly applied for melanoma treatment in the past decade, can unleash anti-tumor immune responses and result in melanoma regression. Patients responding to the ICI treatment showed long-lasting remission or disease control status. However, a large group of patients failed to respond to this therapy, indicating the development of resistance mechanisms. Among them are intrinsic tumor properties, the dysfunction of effector cells, and the generation of immunosuppressive tumor microenvironment (TME). This review discusses achievements of ICI treatment in melanoma, reasons for its failure, and promising approaches for overcoming the resistance. These methods include combinations of different ICI with each other, strategies for neutralizing the immunosuppressive TME and combining ICI with other anti-cancer therapies such as radiation, oncolytic viral, or targeted therapy. New therapeutic approaches targeting other immune checkpoint molecules are also discussed.

A universal anti-cancer vaccine: Chimeric invariant chain potentiates the inhibition of melanoma progression and the improvement of survival.

For many years, clinicians and scientists attempt to develop methods to stimulate the immune system to target malignant cells. Recent data suggest that effective cancer vaccination requires combination immunotherapies to overcome tumor immune evasion. Through presentation of both MHC-I and II molecules, DCs-based vaccine platforms are effective in generating detectable CD4 and CD8 T cell responses against tumor-associated antigens. Several platforms include DC transfection with mRNA of the desired tumor antigen. These DCs are then delivered to the host and elicit an immune response against the antigen of interest. We have recently established an mRNA genetic platform which induced specific CD8+ cytotoxic T cell response by DC vaccination against melanoma. In our study, an MHC-II mRNA DCs vaccine platform was developed to activate CD4+ T cells and to enhance the anti-tumor response. The invariant chain (Ii) was modified and the semi-peptide CLIP was replaced with an MHC-II binding peptide sequences of melanoma antigens. These chimeric MHC-II constructs are presented by DCs and induce proliferation of tumor specific CD4+ T cells. When administered in combination with the MHC-I platform into tumor bearing mice, these constructs were able to inhibit tumor growth, and improve mouse survival. Deciphering the immunological mechanism of action, we observed an efficient CTLs killing in addition to higher levels of Th1 and Th2 subsets in the groups immunized with a combination of the MHC-I and MHC-II constructs. These universal constructs can be applied in multiple combinations and offer an attractive opportunity to improve cancer treatment.

Histone methyltransferase SETDB1 contributes to melanoma tumorigenesis and serves as a new potential therapeutic target.

Alterations in histone modifications play a crucial role in the progression of various types of cancer. The histone methyltransferase SETDB1 catalyzes the addition of methyl groups to histone H3 at lysine 9. Here, we describe how overexpression of SETDB1 contributes to melanoma tumorigenesis. SETDB1 is highly amplified in melanoma cells and in the patient tumors. Increased expression of SETDB1, which correlates with SETDB1 amplification, is associated with a more aggressive phenotype in in vitro and in vivo studies. Mechanistically, SETDB1 implements its effects via regulation of thrombospondin 1, and the SET-domain of SETDB1 is essential for the maintenance of its tumorigenic activity. Inhibition of SETDB1 reduces cell growth in melanomas resistant to targeted treatments. Our results indicate that SETDB1 is a major driver of melanoma development and may serve as a potential future target for the treatment of this disease.

Immunosuppression mediated by myeloid-derived suppressor cells (MDSCs) during tumour progression.

Under steady-state conditions, bone marrow-derived immature myeloid cells (IMC) differentiate into granulocytes, macrophages and dendritic cells (DCs). This differentiation is impaired under chronic inflammatory conditions, which are typical for tumour progression, leading to the accumulation of IMCs. These cells are capable of inducing strong immunosuppressive effects through the expression of various cytokines and immune regulatory molecules, inhibition of lymphocyte homing, stimulation of other immunosuppressive cells, depletion of metabolites critical for T cell functions, expression of ectoenzymes regulating adenosine metabolism, and the production of reactive species. IMCs are therefore designated as myeloid-derived suppressor cells (MDSCs), and have been shown to accumulate in tumour-bearing mice and cancer patients. MDSCs are considered to be a strong contributor to the immunosuppressive tumour microenvironment and thus an obstacle for many cancer immunotherapies. Consequently, numerous studies are focused on the characterisation of MDSC origin and their relationship to other myeloid cell populations, their immunosuppressive capacity, and possible ways to inhibit MDSC function with different approaches being evaluated in clinical trials. This review analyses the current state of knowledge on the origin and function of MDSCs in cancer, with a special emphasis on the immunosuppressive pathways pursued by MDSCs to inhibit T cell functions, resulting in tumour progression. In addition, we describe therapeutic strategies and clinical benefits of MDSC targeting in cancer.

Opposing roles of eosinophils in cancer.

Eosinophils are a subset of granulocytes mostly known for their ability to combat parasites and induce allergy. Although they were described to be related to cancer more than 100 years ago, their role in tumors is still undefined. Recent observations revealed that they display regulatory functions towards other immune cell subsets in the tumor microenvironment or direct cytotoxic functions against tumor cells, leading to either antitumor or protumor effects. This paradoxical role of eosinophils was suggested to be dependent on the different factors in the TME. In addition, the clinical relevance of these cells has been recently addressed. In most cases, the accumulation of eosinophils both in the tumor tissue, called tumor-associated tissue eosinophilia, and in the peripheral blood were reported to be prognostic markers for a better outcome of cancer patients. In immunotherapy of cancer, particularly in therapy with immune checkpoint inhibitors, eosinophils were even shown to be a potential predictive marker for a beneficial clinical response. A better understanding of their role in cancer progression will help to establish them as prognostic and predictive markers and to design strategies for targeting eosinophils.

Interactions among myeloid regulatory cells in cancer.

Mounting evidence has accumulated on the critical role of the different myeloid cells in the regulation of the cancerous process, and in particular in the modulation of the immune reaction to cancer. Myeloid cells are a major component of host cells infiltrating tumors, interacting with each other, with tumor cells and other stromal cells, and demonstrating a prominent plasticity. We describe here various myeloid regulatory cells (MRCs) in mice and human as well as their relevant therapeutic targets. We first address the role of the monocytes and macrophages that can contribute to angiogenesis, immunosuppression and metastatic dissemination. Next, we discuss the differential role of neutrophil subsets in tumor development, enhancing the dual and sometimes contradicting role of these cells. A heterogeneous population of immature myeloid cells, MDSCs, was shown to be generated and accumulated during tumor progression as well as to be an important player in cancer-related immune suppression. Lastly, we discuss the role of myeloid DCs, which can either contribute to effective anti-tumor responses or play a more regulatory role. We believe that MRCs play a critical role in cancer-related immune regulation and suggest that future anti-cancer therapies will focus on these abundant cells.

Deciphering myeloid-derived suppressor cells: isolation and markers in humans, mice and non-human primates.

In cancer, infection and inflammation, the immune system's function can be dysregulated. Instead of fighting disease, immune cells may increase pathology and suppress host-protective immune responses. Myeloid cells show high plasticity and adapt to changing conditions and pathological challenges. Despite their relevance in disease pathophysiology, the identity, heterogeneity and biology of myeloid cells is still poorly understood. We will focus on phenotypical and functional markers of one of the key myeloid regulatory subtypes, the myeloid derived suppressor cells (MDSC), in humans, mice and non-human primates. Technical issues regarding the isolation of the cells from tissues and blood, timing and sample handling of MDSC will be detailed. Localization of MDSC in a tissue context is of crucial importance and immunohistochemistry approaches for this purpose are discussed. A minimal antibody panel for MDSC research is provided as part of the Mye-EUNITER COST action. Strategies for the identification of additional markers applying state of the art technologies such as mass cytometry will be highlighted. Such marker sets can be used to study MDSC phenotypes across tissues, diseases as well as species and will be crucial to accelerate MDSC research in health and disease.

Circulating and Tumor Myeloid-derived Suppressor Cells in Resectable Non-Small Cell Lung Cancer.

RATIONALE: Myeloid-derived suppressor cell (MDSC) expansion has been found to play a role in disease progression in patients with cancer. However, the characteristics of MDSCs in lung cancer are poorly understood. OBJECTIVES: We prospectively investigated MDSCs and inflammatory factors in tumor and peripheral blood samples from patients with resectable non-small cell lung cancer and studied their correlations with the disease prognosis. METHODS: A complex analysis of MDSC subsets and inflammatory mediators was performed using flow cytometry and a Bio-Plex assay. MEASUREMENTS AND MAIN RESULTS: A significant increase in the frequency of circulating monocytic (M)-MDSCs was observed in the patients with non-small cell lung cancer compared with the healthy donors (HDs). Moreover, the frequencies of M- and polymorphonuclear (PMN)-MDSCs were higher in tumors than in the peripheral blood of the same patients. This accumulation was associated with elevated concentrations of inflammatory mediators involved in MDSC migration to and activation in the tumor microenvironment. An analysis of the MDSC immunosuppressive pattern showed increased programmed death-ligand 1 expression on circulating cells from patients compared with HDs. Tumor PMN-MDSCs displayed higher programmed death-ligand 1 expression levels than the same cells in the peripheral blood. The frequency of CCR5 (C-C chemokine receptor 5) expression on circulating M-MDSCs was significantly higher in the patients than in the HDs. Clinical data analysis revealed negative correlations between recurrence-free survival and the frequencies of PMN-MDSCs and CCR5+ M-MDSCs in the circulation but not in tumors. CONCLUSIONS: Our findings suggest that the level of MDSCs in the peripheral blood but not in tumor tissues predicts recurrence after surgery.

Loss of neural crest-associated gene FOXD1 impairs melanoma invasion and migration via RAC1B downregulation.

Recent studies suggest that malignant melanoma heterogeneity includes subpopulations of cells with features of multipotent neural crest (NC) cells. Zebrafish and mouse models have shown that reactivation of neural crest-specific pathways during transformation determines the invasiveness of melanoma cells. In our study, we show that the neural crest-associated transcription factor FOXD1 plays a key role in the invasion and the migration capacities of metastatic melanomas both in vivo and in vitro. Gene expression profiling analysis identified both an upregulation of FOXD1 in NC and melanoma cells, as well as a downregulation of several genes related to cell invasion in FOXD1 knockdown cells, including MMP9 and RAC1B. Furthermore, we demonstrate that knockdown of RAC1B a tumor-specific isoform of RAC1, significantly impaired melanoma cell migration and invasion and could abrogate enhanced invasiveness induced by FOXD1 overexpression. We conclude that FOXD1 may influence invasion and migration via indirect regulation of MMP9 and RAC1B alternative splicing in melanoma cells.

SOX2-mediated upregulation of CD24 promotes adaptive resistance toward targeted therapy in melanoma.

Melanoma is often characterized by a constitutively active RAS-RAF-MEK-ERK pathway. For targeted therapy, BRAF inhibitors are available that are powerful in the beginning but resistance occurs rather fast. A better understanding of the mechanisms of resistance is urgently needed to increase the success of the treatment. Here, we observed that SOX2 and CD24 are upregulated upon BRAF inhibitor treatment. A similar upregulation was seen in targeted therapy-resistant, melanoma-derived induced pluripotent cancer cells (iPCCs). SOX2 and CD24 are known to promote an undifferentiated and cancer stem cell-like phenotype associated with resistance. We, therefore, elucidated the role of SOX2 and CD24 in targeted therapy resistance in more detail. We found that the upregulation of SOX2 and CD24 required activation of STAT3 and that SOX2 induced the expression of CD24 by binding to its promoter. We find that the overexpression of SOX2 or CD24 significantly increases the resistance toward BRAF inhibitors, while SOX2 knock-down rendered cells more sensitivity toward treatment. The overexpression of CD24 or SOX2 induced Src and STAT3 activity. Importantly, by either CD24 knock-down or Src/STAT3 inhibition in resistant SOX2-overexpressing cells, the sensitivity toward BRAF inhibitors was re-established. Hence, we suggest a novel mechanism of adaptive resistance whereby BRAF inhibition is circumvented via the activation of STAT3, SOX2 and CD24. Thus, to prevent adaptive resistance, it might be beneficial to combine Src/STAT3 inhibitors together with MAPK pathway inhibitors.