Melanoma prognosis is dictated by tumor-infiltrating lymphocytes, the migratory and functional behavior of which is guided by chemokine or cytokine gradients. Here, we retrospectively analyzed the ...expression patterns of 9 homing receptors (CCR/CXCR) in naive and memory CD4+ and CD8+ T lymphocytes in 57 patients with metastatic melanoma (MMel) with various sites of metastases to evaluate whether T cell CCR/CXCR expression correlates with intratumoral accumulation, metastatic progression, and/or overall survival (OS). Homing receptor expression on lymphocytes strongly correlated with MMel dissemination. Loss of CCR6 or CXCR3, but not cutaneous lymphocyte antigen (CLA), on circulating T cell subsets was associated with skin or lymph node metastases, loss of CXCR4, CXCR5, and CCR9 corresponded with lung involvement, and a rise in CCR10 or CD103 was associated with widespread dissemination. High frequencies of CD8+CCR9+ naive T cells correlated with prolonged OS, while neutralizing the CCR9/CCL25 axis in mice stimulated tumor progression. The expansion of CLA-expressing effector memory CD8+ T cells in response to a single administration of CTLA4 blockade predicted disease control at 3 months in 47 patients with MMel. Thus, specific CCR/CXCR expression patterns on circulating T lymphocytes may guide potential diagnostic and therapeutic approaches.
Solid tumors are composed of malignant cells surrounded by a tumor-conditioned stroma that contains extracellular matrix and a variety of nonmalignant populations, including myeloid cells, ...lymphocytes, fibroblasts, and endothelial cells. These stromal elements form a local immunoregulatory network that must be overcome to achieve eradication of established tumors by immunotherapy. On March 21-22, 2012, a symposium was held in Pamplona, Spain, to share the recent advances regarding the molecules and cells that create and sustain this immune-hostile tumor microenvironment. Excellent targets for immunotherapeutic intervention were identified, and a number of therapeutic strategies under translation from mouse to human were presented.
Although the treatment of advanced melanoma patients with immune checkpoint inhibitors (ICI) significantly increased the therapeutic efficiency, many patients remain resistant to ICI that could be ...due to immunosuppression mediated by myeloid-derived suppressor cells (MDSC). These cells are enriched and activated in melanoma patients and could be considered as therapeutic targets. Here we studied dynamic changes in immunosuppressive pattern and activity of circulating MDSC from melanoma patients treated with ICI.
MDSC frequency, immunosuppressive markers and function were evaluated in freshly isolated peripheral blood mononuclear cells (PBMC) from 29 melanoma patients receiving ICI. Blood samples were taken prior and during the treatment and analyzed by flow cytometry and bio-plex assay.
MDSC frequency was significantly increased before the therapy and through three months of treatment in non-responders as compared to responders. Prior to the ICI therapy, MDSC from non-responders displayed high levels of immunosuppression measured by the inhibition of T cell proliferation assay, whereas MDSC from responding patients failed to inhibit T cells. Patients without visible metastasis were characterized by the absence of MDSC immunosuppressive activity during the ICI treatment. Moreover, non-responders showed significantly higher IL-6 and IL-8 concentrations before therapy and after the first ICI application as compared to responders.
Our findings highlight the role of MDSC during melanoma progression and suggest that frequency and immunosuppressive activity of circulating MDSC before and during the ICI treatment of melanoma patients could be used as biomarkers of response to ICI therapy.
BackgroundPredicting metastasis in melanoma patients is important for disease management and could help to identify those who might benefit from adjuvant treatment. The aim of this study was to ...investigate whether the tumor microenvironment-derived protein S100A8/A9 qualifies as prognostic marker for melanoma patients, also in the setting of immunotherapy.MethodsS100A8/A9 gene and protein expression were analyzed on melanocytic nevi, primary melanomas and metastases using a cDNA library and three independent tissue-microarrays (TMA). Serum levels of S100A8/A9 were measured using a specific ELISA in two independent cohorts of 354 stage III and stage IV melanoma patients as well as in two independent cohorts of patients treated with the PD-1 antibody pembrolizumab.ResultscDNA analysis revealed an upregulation of S100A8 and S100A9 gene expression in melanoma metastases compared to primary melanomas. Significantly higher numbers of infiltrating S100A8/A9 positive cells were found in tissue samples of metastasizing primary melanomas compared to non-metastasizing melanomas (P < .0001) and in melanomas of short-term survivors compared to long-term survivors (P < .0001). Serum S100A8/A9 levels > 5.5 mg/l were associated with impaired overall survival in two independent cohorts (both P < .0001). Importantly, patients with serum elevated S100A8/A9 treated with pembrolizumab showed significantly impaired survival compared to patients with lower S100A8/A9 levels (cohort 1: P = .0051; cohort 2: P < .0001).ConclusionsThe tumor microenvironment-associated protein S100A8/A9 serves as a novel prognostic marker for metastasis and survival of metastatic melanoma patients and predicts response to immunotherapy with pembrolizumab. These data underscore the significance of tumor microenvironment-derived factors as suitable biomarkers for melanoma.
BackgroundMyeloid-derived suppressor cells (MDSCs) represent a negative prognostic factor in malignant melanoma. These cells are generated under chronic inflammatory conditions typical of cancer. The ...transcription factor signal transducer and activator of transcription 3 (STAT3) orchestrates MDSC accumulation and acquisition of immunosuppressive properties. Here we studied STAT3 inhibition by Napabucasin as a way to block MDSC accumulation and activity and its potential to treat malignant melanoma.MethodsIn vitro generated murine MDSC and primary MDSC from melanoma-bearing mice were used to investigate the effects of Napabucasin on MDSC in vitro. The RET transgenic mouse model of malignant melanoma was used to examine Napabucasin therapy efficiency and its underlying mechanisms in vivo. Furthermore, STAT3 activation and its correlation with survival were explored in MDSC from 19 patients with malignant melanoma and human in vitro generated monocytic myeloid-derived suppressor cell (M-MDSC) were used to evaluate the effects of Napabucasin.ResultsNapabucasin was able to abrogate the capacity of murine MDSC to suppress CD8+ T-cell proliferation. The STAT3 inhibitor induced apoptosis in murine MDSC, significantly increased expression of molecules associated with antigen processing and presentation, as well as slightly decreased expression of immunosuppressive factors on these cells. RET transgenic mice treated with Napabucasin showed prolonged survival accompanied by a strong accumulation of tumor-infiltrating antigen-presenting cells and activation of CD8+ and CD4+ T cells. Interestingly, patients with malignant melanoma with high expression of activated STAT3 in circulating M-MDSC showed significantly worse progression-free survival (PFS) than patients with low levels of activated STAT3. In addition, Napabucasin was able to abrogate suppressive capacity of human in vitro generated M-MDSC.ConclusionOur findings demonstrate that STAT3 inhibitor Napabucasin completely abrogated the immunosuppressive capacity of murine MDSC and human M-MDSC and improved melanoma-bearing mouse survival. Moreover, patients with malignant melanoma with high expression levels of activated STAT3 in M-MDSC displayed shorter PFS, indicating its role as a promising therapeutic target in patients with malignant melanoma and a predictive marker for their clinical outcome.
Neutrophils play a key role in cancer biology. In contrast to circulating normal-density neutrophils (NDN), the amount of low-density neutrophils (LDN) significantly increases with tumor progression. ...The correlation between these neutrophil subpopulations and intratumoral neutrophils (TANs) is still under debate. Using 4T1 (breast) and AB12 (mesothelioma) tumor models, we aimed to elucidate the source of TANs and to assess the mechanisms driving neutrophils’ plasticity in cancer. Both NDN and LDN were found to migrate in response to CXCL1 and CXCL2 exposure, and co-infiltrate the tumor site ex vivo and in vivo, although LDN migration into the tumor was higher than NDN. Tumor-derived factors and chemokines, particularly CXCL1, were found to drive neutrophil phenotypical plasticity, inducing NDN to transition towards a low-density state (LD-NDN). LD-NDN appeared to differ from NDN by displaying a phenotypical profile similar to LDN in terms of nuclear morphology, surface receptor markers, decreased phagocytic abilities, and increased ROS production. Interestingly, all three subpopulations displayed comparable cytotoxic abilities towards tumor cells. Our data suggest that TANs originate from both LDN and NDN, and that a portion of LDN derives from NDN undergoing phenotypical changes. NDN plasticity resulted in a change in surface marker expression and functional activity, gaining characteristics of LDN.
Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest cancers in the world. PDAC cells activate tumor-specific immune responses but simultaneously trigger a strong ...immunosuppression. We showed that PDAC cells produce high amount of chronic inflammatory mediators and PDAC tumors build an immunosuppressive cytokine milieu, which correlates with tumor progression. We observed a low frequency of dendritic cells (DC) and a pronounced accumulation of macrophages and myeloid-derived suppressor cells (MDSC) in murine PDAC tumors. A strong accumulation of MDSC has also been demonstrated in the peripheral blood of resected PDAC patients. While DC and macrophages seem not to play a significant role in this PDAC model in the context of immunosuppression, MDSC are highly suppressive, and their accumulation is associated with an increase in intratumoral VEGF concentration during the PDAC progression. Application of the phosphodiesterase-5 inhibitor sildenafil led to a prolonged survival of PDAC-bearing female mice, which was due to the decrease in MDSC frequencies and in the systemic VEGF level. This led to a restoration of anticancer immune responses, manifested in the recovery of T lymphocyte functions and in an increase in the frequency of conventional CD4
+
T cells in tumors and IFNγ level in serum of PDAC-bearing mice. Thus, MDSC are strongly involved in the PDAC-associated immunosuppression and that their depletion could create new approaches for therapy of PDAC.
BackgroundTumor cells modulate host immunity by secreting extracellular vesicles (EV) and soluble factors. Their interactions with myeloid cells lead to the generation of myeloid-derived suppressor ...cells (MDSC), which inhibit the antitumor function of T and NK cells. We demonstrated previously that EV derived from mouse and human melanoma cells induced immunosuppressive activity via increased expression of programmed cell death ligand 1 (PD-L1) on myeloid cells that was dependent on the heat-shock protein 90α (HSP90α) in EV. Here, we investigated whether soluble HSP90α could convert monocytes into MDSC.MethodsCD14 monocytes were isolated from the peripheral blood of healthy donors, incubated with human recombinant HSP90α (rHSP90α) alone or in the presence of inhibitors of TLR4 signaling and analyzed by flow cytometry. Inhibition of T cell proliferation assay was applied to assess the immunosuppressive function of rHSP90α-treated monocytes. HSP90α levels were measured by ELISA in plasma of patients with advanced melanoma and correlated with clinical outcome.ResultsWe found that the incubation of monocytes with rHSP90α resulted in a strong upregulation of PD-L1 expression, whereas reactive oxygen species (ROS) and nitric oxide (NO) production as well as the expression of arginase-1, ectoenzymes CD39 and CD73 remained unchanged. The PD-L1 upregulation was blocked by anti-TLR4 antibodies and a nuclear factor-κB inhibitor. rHSP90α-treated monocytes displayed the downregulation of HLA-DR expression and acquired the resistance to apoptosis. Moreover, these monocytes were converted into MDSC as indicated by their capacity to inhibit T cell proliferation, which was mediated by TLR4 signaling as well as PD-L1 and indoleamine 2,3-dioxygenase (IDO) 1 expression. Higher levels of HSP90α in plasma of patients with melanoma correlated with augmented PD-L1 expression on circulating monocytic (M)-MDSC. Patients with melanoma with high levels of HSP90α displayed shorter progression-free survival (PFS) on the treatment with immune checkpoint inhibitors (ICIs).ConclusionOur findings demonstrated that soluble rHSP90α increased the resistance of normal human monocytes to apoptosis and converted them into immunosuppressive MDSC via TLR4 signaling that stimulated PD-L1 and IDO-1 expression. Furthermore, patients with melanoma with high concentrations of HSP90α displayed increased PD-L1 expression on M-MDSC and reduced PFS after ICI therapy, suggesting HSP90α as a promising therapeutic target for overcoming immunosuppression in melanoma.
Despite recent improvement in the treatment of malignant melanoma by immune-checkpoint inhibitors, the disease can progress due to an immunosuppressive tumor microenvironment (TME) mainly represented ...by myeloid-derived suppressor cells (MDSC). However, the relative contribution of the polymorphonuclear (PMN) and monocytic (M) MDSC subsets to melanoma progression is not clear. Here, we compared both subsets regarding their immunosuppressive capacity and recruitment mechanisms. Furthermore, we inhibited PMN-MDSC migration in vivo to determine its effect on tumor progression.
Using the
transgenic melanoma mouse model, we investigated the immunosuppressive function of MDSC subsets and chemokine receptor expression on these cells. The effect of CXCR2 inhibition on PMN-MDSC migration and tumor progression was studied in
transgenic mice and in C57BL/6 mice after surgical resection of primary melanomas.
Immunosuppressive capacity of intratumoral M- and PMN-MDSC was comparable in melanoma bearing mice. Anti-CXCR2 therapy prolonged survival of these mice and decreased the occurrence of distant metastasis. Furthermore, this therapy reduced the infiltration of melanoma lesions and pre-metastatic sites with PMN-MDSC that was associated with the accumulation of natural killer (NK) cells.
We provide evidence for the tumor
promoting properties of PMN-MDSC as well as for the anti-tumor effects upon their targeting in melanoma bearing mice.
Purpose: The purpose of the study was to investigate signaling molecules involved in the acquisition of tolerogenic properties by
dendritic cells (DC) in ret transgenic mice with spontaneous melanoma ...progression and to target these molecules to overcome the barrier for effective
melanoma immunotherapy.
Experimental Design: DC functions and expression patterns of p38 mitogen-activated protein kinase (MAPK) in DCs were evaluated in a ret transgenic murine cutaneous melanoma model, which shows high similarity to human cutaneous melanoma with respect to clinical
development. In contrast to transplantation melanoma models (like B16), this model allows the study of melanoma progression
under conditions of natural interactions between tumor and host cells over time.
Results: We showed a strong tumor infiltration with immature DCs and a reduction in the number of mature DCs in lymphoid organs during
melanoma progression. DCs from melanoma-bearing mice secreted significantly more interleukin 10 and less interleukin 12p70,
and showed a decreased capacity to activate T cells compared with DCs from tumor-free animals. Observed DC dysfunction was
linked to considerable activation of p38 MAPK. Inhibition of its activity in spleen DCs from tumor-bearing mice led to normalization
of their cytokine secretion pattern and T-cell stimulation capacity.
Conclusions: Our data show a critical role of constitutively activated p38 MAPK in the acquirement of tolerogenic pattern by DCs during
melanoma progression that contributes to the suppression of antitumor T-cell immune responses. We suggest that new strategies
of melanoma immunotherapy can include inhibitors of p38 MAPK activity in DCs.
PDF
