Clinical Significance of Circulating CD33+CD11b+HLA-DR- Myeloid Cells in Patients with Stage IV Melanoma Treated with Ipilimumab.

PURPOSE: High levels of circulating myeloid-derived suppressor cells (MDSCs) in various cancer types, including melanoma, were shown to correlate with poor survival. We investigated whether frequencies of circulating CD33+CD11b+HLA-DR- MDSCs could be used as immune system monitoring biomarkers to predict response and survival of patients with stage IV melanoma treated with anti-CTLA4 (ipilimumab) therapy. EXPERIMENTAL DESIGN: Peripheral blood samples from 56 patients and 50 healthy donors (HDs) were analyzed for CD33+CD11b+HLA-DR- MDSC percentage, NO-, and hROS levels by flow cytometry. We determined whether MDSC levels and suppressive features detected before anti-CTLA4 therapy correlate with the patients' response and overall survival (OS). RESULTS: Patients with melanoma had significantly higher levels of circulating CD33+CD11b+HLA-DR- MDSCs with suppressive phenotype when compared with HDs. Low levels of MDSCs before CTLA-4 therapy correlated with an objective clinical response, long-term survival, increased CD247 expression in T cells, and an improved clinical status. No predictive impact was observed for lactate dehydrogenase (LDH). Kaplan-Meier and log-rank tests performed on the 56 patients showed that the presence of more than 55.5% of circulating CD33+CD11b+ out of the HLA-DR- cells, were associated with significant short OS (P < 0.003), a median of 6.5 months, in comparison with the group showing lower MDSC frequencies, with a median survival of 15.6 months. CONCLUSIONS: Our study suggests the use of CD33+CD11b+HLA-DR- cells as a predictive and prognostic biomarker in patients with stage IV melanoma treated with anti-CTLA4 therapy. This monitoring system may aid in the development of combinatorial modalities, targeting the suppressive environment in conjunction with iplimumab, toward facilitating better disease outcomes. Clin Cancer Res; 22(23); 5661-72. ©2016 AACR.

RNF20 Links Histone H2B Ubiquitylation with Inflammation and Inflammation-Associated Cancer.

Factors linking inflammation and cancer are of great interest. We now report that the chromatin-targeting E3 ubiquitin ligase RNF20/RNF40, driving histone H2B monoubiquitylation (H2Bub1), modulates inflammation and inflammation-associated cancer in mice and humans. Downregulation of RNF20 and H2Bub1 favors recruitment of p65-containing nuclear factor κB (NF-κB) dimers over repressive p50 homodimers and decreases the heterochromatin mark H3K9me3 on a subset of NF-κB target genes to augment their transcription. Concordantly, RNF20(+/-) mice are predisposed to acute and chronic colonic inflammation and inflammation-associated colorectal cancer, with excessive myeloid-derived suppressor cells (MDSCs) that may quench antitumoral T cell activity. Notably, colons of human ulcerative colitis patients, as well as colorectal tumors, reveal downregulation of RNF20/RNF40 and H2Bub1 in both epithelium and stroma, supporting the clinical relevance of our tissue culture and mouse model findings.

Impaired SNX9 Expression in Immune Cells during Chronic Inflammation: Prognostic and Diagnostic Implications.

Chronic inflammation is associated with immunosuppression and downregulated expression of the TCR CD247. In searching for new biomarkers that could validate the impaired host immune status under chronic inflammatory conditions, we discovered that sorting nexin 9 (SNX9), a protein that participates in early stages of clathrin-mediated endocytosis, is downregulated as well under such conditions. SNX9 expression was affected earlier than CD247 by the generated harmful environment, suggesting that it is a potential marker sensing the generated immunosuppressive condition. We found that myeloid-derived suppressor cells, which are elevated in the course of chronic inflammation, are responsible for the observed SNX9 reduced expression. Moreover, SNX9 downregulation is reversible, as its expression levels return to normal and immune functions are restored when the inflammatory response and/or myeloid-derived suppressor cells are neutralized. SNX9 downregulation was detected in numerous mouse models for pathologies characterized by chronic inflammation such as chronic infection (Leishmania donovani), cancer (melanoma and colorectal carcinoma), and an autoimmune disease (rheumatoid arthritis). Interestingly, reduced levels of SNX9 were also observed in blood samples from colorectal cancer patients, emphasizing the feasibility of its use as a diagnostic and prognostic biomarker sensing the host's immune status and inflammatory stage. Our new discovery of SNX9 as being regulated by chronic inflammation and its association with immunosuppression, in addition to the CD247 regulation under such conditions, show the global impact of chronic inflammation and the generated immune environment on different cellular pathways in a diverse spectrum of diseases.

Paving the Road to Tumor Development and Spreading: Myeloid-Derived Suppressor Cells are Ruling the Fate.

Cancer development is dependent on intrinsic cellular changes as well as inflammatory factors in the tumor macro and microenvironment. The inflammatory milieu nourishes the tumor and contributes to cancer progression. Numerous studies, including ours, have demonstrated that the tumor microenvironment is immunosuppressive, impairing the anticancer immune responses. Chronic inflammation was identified as the key process responsible for this immunosuppression via induction of immature myeloid-derived suppressor cells (MDSCs). Upon a prolonged immune response, MDSCs are polarized toward immunosuppressive cells meant to control the exacerbated immune response. In cancer, the chronic inflammatory response renders the MDSCs harmful. Polarized MDSCs suppress T-cells and natural killer cells, as well as antigen-presenting cells, abrogating the beneficial immune response. These changes in the immunological milieu could also lead to high frequency of mutations, enhanced cancer cell stemness, and angiogenesis, directly supporting tumor initiation, growth, and spreading. The presence of MDSCs in cancer poses a serious obstacle in a variety of immune-based therapies, which rely on the stimulation of antitumor immune responses. Cumulative data, including our own, suggest that the selection of an appropriate and effective anticancer therapy must take into consideration the host's immune status as well as tumor-related parameters. Merging biomarkers for immune monitoring into the traditional patient's categorization and follow-up can provide new predictive and diagnostic tools to the clinical practice. Chronic inflammation and MDSCs could serve as novel targets for therapeutic interventions, which can be combined with conventional cancer treatments such as chemotherapy, radiotherapy, and cancer cell-targeted and immune-based therapies. Intervention in environmental and tumor-specific inflammatory mechanisms will allow better clinical management of cancer toward more efficient treatment.

Adverse immunoregulatory effects of 5FU and CPT11 chemotherapy on myeloid-derived suppressor cells and colorectal cancer outcomes.

Colorectal cancer is associated with chronic inflammation and immunosuppression mediated by myeloid-derived suppressor cells (MDSC). Although chemotherapy reduces tumor burden at early stages, it tends to have limited effect on a progressive disease, possibly due to adverse effects on the immune system in dictating disease outcome. Here, we show that patients with advanced colorectal cancer display enhanced MDSC levels and reduced CD247 expression and that some conventional colorectal cancer chemotherapy supports the immunosuppressive tumor microenvironment. A FOLFOX combined therapy reduced immunosuppression, whereas a FOLFIRI combined therapy enhanced immunosuppression. Mechanistic studies in a colorectal cancer mouse model revealed that FOLFIRI-like therapy including the drugs CPT11 and 5-fluorouracil (5FU) damaged host immunocompetence in a manner that limits treatment outcomes. CPT11 blocked MDSC apoptosis and myeloid cell differentiation, increasing MDSC immunosuppressive features and mouse mortality. In contrast, 5FU promoted immune recovery and tumor regression. Thus, CPT11 exhibited detrimental immunoregulatory effects that offset 5FU benefits when administered in combination. Our results highlight the importance of developing therapeutic regimens that can target both the immune system and tumor towards improved personalized treatments for colorectal cancer.

Chronic inflammation and cancer: suppressing the suppressors.

Chronic inflammation typical to various chronic diseases is associated with immunosuppression, mediated primarily by immature myeloid-derived suppressor cells (MDSCs). A variety of factors induce MDSC differentiation arrest, thus manipulating the host's immune function and suppressing the innate and adaptive immune systems, as reflected by their impaired status associated with down-regulated expression of the CD247 molecule. Such chronic inflammation-induced immunosuppressive features are also found in many tumors, generating tumor micro- and macro-environments that act as critical barriers to effective anti-tumor responses and therapies. This knowledge offers new and novel candidate immune targets for therapeutic interventions, in combination with more conventional approaches as chemotherapy, radiotherapy, and cancer cell targeted therapy. Therapeutic manipulation of chronic inflammation during cancer development is likely to enhance efficacy of treatments such as vaccinations, and adoptive T cell transfer, thus switching the chronic pro-cancer inflammatory environments into an anti-cancer milieu. Based on the functional relevance of immune networking in tumors, it is advantageous to merge monitoring immune biomarkers into the traditional patient's categorization and treatment regiments, which will provide new prognostic and/or predictive tools to clinical practice. A better identification of environmental and tumor-specific inflammatory mechanisms will allow directing the clinical management of cancer toward a more personalized medicine.

Tumor necrosis factor-α blocks differentiation and enhances suppressive activity of immature myeloid cells during chronic inflammation.

Elevated concentrations of tumor necrosis factor-α (TNF-α) are detected in pathologies characterized by chronic inflammation. Whether TNF-α plays a role in manipulating the host's immune system toward generating an immunosuppressive milieu, typical of ongoing chronic inflammation, is unclear. Here we showed that TNF-α exhibited a dual function during chronic inflammation: arresting differentiation of immature myeloid-derived suppressor cells (MDSCs) primarily via the S100A8 and S100A9 inflammatory proteins and their corresponding receptor (RAGE) and augmenting MDSC suppressive activity. These functions led to in vivo T and NK cell dysfunction accompanied by T cell antigen receptor ζ chain downregulation. Furthermore, administration of etanercept (TNF-α antagonist) during early chronic inflammatory stages reduced MDSCs' suppressive activity and enhanced their maturation into dendritic cells and macrophages, resulting in the restoration of in vivo immune functions and recovery of ζ chain expression. Thus, TNF has a fundamental role in promoting an immunosuppressive environment generated during chronic inflammation.

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.

New insights into chronic inflammation-induced immunosuppression.

Chronic inflammation is a common factor linking various pathologies that differ in their etiology and physiology such as cancer, autoimmune diseases, and infections. At a certain stage of each of these diseases, while the chronic inflammation proceeds, some key players of the immune system become immunosuppressed as natural killer (NK) cells and T cells. The suppressive environment induced during chronic inflammation is governed by a complex processes characterized by the accumulation and activation of immune suppressor cells, pro-inflammatory cytokines, chemokines, growth and angiogenic factors, and by the activation of several inflammatory signaling pathways mediated predominantly by NFκB and STAT3 transcription factors. A substantial body of evidence supports the notion that the development of a suppressive environment during chronic inflammation limits the success of immune-based and conventional therapies, skewing the balance in favor of a developing pathology. Thus, appropriate, well-designed and fine tuned immune interventions that could resolve inflammatory responses and associated immunosuppression could enhance disease regression and reinforce successful responses to a given therapy. This review describes the interrelationship between chronic inflammation and induced immunosuppression, and explains the current evidence linking inflammation and pathological processes, as found in cancer. We further highlight potential strategies, harnessing the immunosuppressive environment in treating autoimmune diseases and facilitating transplantation. In parallel, we emphasize the use of modalities to combat chronic inflammation-induced immunosuppression in cancer, to enhance the success of immune-based therapies leading to tumor regression. In both cases, the urgent necessity of identifying biomarkers for the evaluation of host immune status is discussed, with the goal of developing optimal personalized treatments.