Role Of Tumor-associated Neutrophils Research Articles

A key regulator of tumor-associated neutrophils: the CXCR2 chemokine receptor.

In recent years, with the advance of research, the role of tumor-associated neutrophils (TANs) in tumors has become a research hotspot. As important effector cells in the innate immune system, neutrophils play a key role in the immune and inflammatory responses of the body. As the first line of defense against bacterial and fungal infections, neutrophils have the ability to kill invading pathogens. In the pathological state of malignant tumors, the phenotype of neutrophils is altered and has an important regulatory function in tumor development. The C-X-C motif chemokine receptor 2(CXCR2) is a key molecule that mediates the migration and aggregation signaling pathway of immune cells, especially neutrophils. This review focuses on the regulation of CXCR2 on TANs in the process of tumorigenesis and development, and emphasizes the application significance of CXCR2 inhibitors in blocking the migration of TANs to tumors.

Role of tumor-associated neutrophils in lung cancer (Review).

Lung cancer is the most common malignancy and the leading cause of cancer mortality worldwide; therefore, it is very important to understand the mechanism of its occurrence and progression. It has reported that inflammation is linked to the incidence of various malignancies. Neutrophils not only participate in the inflammatory response, but are also involved in the composition of the tumor microenvironment. Tumor-associated neutrophils (TANs) are infiltrating neutrophils in tumors that directly promote tumor development and progression. Moreover, they regulate the immune microenvironment and affect the therapeutic efficacy and prognosis of lung cancer. In the present review, the role of TANs in lung cancer development/progression and the underlying molecular signaling are evaluated, as well as the possibility of TANs as a potential therapeutic target for lung cancer intervention.

Properties and functions of myeloperoxidase and its role in ovarian cancer

Background: Elevated levels of myeloperoxidase in body fluids are increasingly being used as an indicator for the diagnosis of cancer. Aim of the study: The aim of this study was to review the literature on the physical and chemical properties of myeloperoxidase, its role in carcinogenesis, the role of tumor-associated neutrophils in cancer, and the role of myeloperoxidase in ovarian cancer. Material and methods: The research literature published between January 1999 and December 2019 was reviewed. The properties and role of myeloperoxidase in the development of ovarian cancer were selected from publications available in selected online databases, including MEDLINE, PubMed, Scopus, and Web of Science. Searches were performed using the following word combinations: “myeloperoxidase”, “ovarian cancer”, “reactive oxygen species”, “expression”, “polymorphism”, and “tumor-associated neutrophils”. Results: Thirty-five scientific articles were included in the final review. Of the 35 articles, 11 discussed the role of myeloperoxidase in carcinogenesis, and five discussed its role in the development of ovarian cancer. Conclusions: Elevated myeloperoxidase levels are associated with many types of cancer, including ovarian cancer. In the studied group of invasive ovarian tumors, up to 65% exhibited elevated levels of myeloperoxidase. Continued research on myeloperoxidase expression in ovarian cancer cells is vital and warranted.

Prognostic significance of CD8+ tumor-infiltrating lymphocytes and CD66b+ tumor-associated neutrophils in the invasive margins of stages I-III colorectal cancer.

Tumor-infiltrating immune cells play an essential role in cancer progression and may help supplement the Tumor, Node, Metastasis (TNM) classification for cancer prognosis. Currently, there are numerous conflicting reports discussing the significance of tumor-associated neutrophils (TANs) in colorectal cancer (CRC). In particular, the role of TANs in the invasive margin is unclear. The present study investigated the prognostic significance of CD66+ TANs and CD8+ tumor-infiltrating lymphocytes (TILs) in the invasive margin of 103 patients with CRC. By using immunohistochemistry, survival analysis was performed on CD8+ TILs and CD66+ TANs individually, as well as models including TILs and TANs simultaneously. The findings indicated that the densities of CD8+ TILs and CD66b+ TANs in the invasive margin may provide significant prognostic value for predicting survival. Moreover, the combined evaluation of CD8+ TILs and CD66b+ TANs in the invasive margin could further improve the validity for the prediction of oncological outcomes. In addition, multivariate analysis revealed that simultaneous low tumor infiltration by CD8+ TILs and CD66b+ was an independent predictive factor for overall survival (HR=4.17, 95% CI, 1.55-12.5; P=0.004) and disease-free survival (HR=2.75, 95% CI, 1.27-6.12; P=0.01). Given the importance of CD8+ TILs and CD66b+ TANs in the tumor microenvironment, the assessment of their densities in the invasive margin may serve as a valuable prognostic marker for CRC.

Lung Adenocarcinoma Cells Promote Self-Migration and Self-Invasion by Activating Neutrophils to Upregulate Notch3 Expression of Cancer Cells.

Background: Invasion and migration of cancer cells play a key role in lung cancer progression and metastasis. Tumor-associated neutrophils (TANs) are related to poor prognosis in many types of cancer. However, the role of TANs in lung cancer is controversial. In this study, we investigated the effect of TANs on the invasion and migration of lung adenocarcinoma. Methods: Immunohistochemistry was performed to detect the density of infiltrating TANs and the expression of Notch3 in 100 lung adenocarcinoma tissues. Flow cytometry was used to observe the viability of neutrophils, which were isolated from healthy peripheral blood and then exposed to the supernatant of cultured lung adenocarcinoma cell lines. After treating with tumor-associated neutrophils culture supernatant, NeuCS (supernatant of cultured neutrophils), tumor cells culture supernatant, Medium (serum-free medium), respectively, the migration and invasion of the lung cancer cells before and after transfected by si-Notch3 were detected by transwell assay and wound healing assay. Kaplan-Meier plotter (http://kmplot.com/analysis/index.php?p) was used to analyze the prognostic role of the density of TANs on lung adenocarcinoma and TIMER ((http://cistrome.dfci.harvard.edu/TIMER/) was used to detect the expression of Notch3 on lung adenocarcinoma. Results: The infiltration of TANs was observed in the parenchyma and stroma of the lung adenocarcinoma, the density of TANs was positively related to the TNM stage and negatively related to the differentiation and prognosis. Notch3 expression of cancer cells was negatively related to the tumor differentiation and prognosis. Compared to quiescent neutrophils, the viability of TCCS-activated neutrophils was enhanced. Both migration and invasion of A549 and PC9 cells were significantly promoted by TANs, while after knocking down Notch3, the migration and invasion of the cancer cells were not affected by TANs. Bioinformatics analysis showed that the density of TANs and the expression of Notch3 were related to the poor prognosis. Conclusion: The results indicated that lung adenocarcinoma cells promote self-invasion and self-migration by activating neutrophils to upregulate the Notch3 expression of cancer cells. The density of infiltrating TANs may be a novel marker for the poor prognosis of lung adenocarcinoma. Targeting TANs might be a potential therapeutic strategy for lung cancer treatment.

Role of Tumor-Associated Neutrophils in the Molecular Carcinogenesis of the Lung.

Simple SummaryThis review of the literature aims at giving a concise overview of the impact of tumor-associated neutrophils (TANs) on lung carcinogenesis. In the first part of this manuscript, the general action mode of TANs in cancer is depicted, listing studies on several cancer entities and on mouse models. The latter part of this work focuses specifically on TANs in lung cancer, giving an outlook on future therapeutic implications of cancer immunity, using, for example, anti-cancer vaccines.Tumorigenesis is largely influenced by accompanying inflammation. Myeloid cells account for a significant proportion of pro-inflammatory cells within the tumor microenvironment. All steps of tumor formation and progression, such as the suppression of adaptive immune response, angio- and lymphangiogenesis, and the remodeling of the tumor stroma, are to some degree influenced by tumor-associated immune cells. Tumor-associated neutrophils (TANs), together with tumor-associated macrophages and myeloid-derived suppressor cells, count among tumor-associated myeloid cells. Still, the exact molecular mechanisms underlying the tumorigenic effects of TANs have not been investigated in detail. With this review of the literature, we aim to give an overview of the current data on TANs, with a special focus on lung cancer.

Tumor-Associated Neutrophils in Hepatocellular Carcinoma Pathogenesis, Prognosis, and Therapy.

Simple SummaryHepatocellular carcinoma is the most prevalent primary liver cancer, accounting for >80% of primary liver cancers worldwide. Inflammation has come to light as a hallmark of cancer development, and it has become increasingly apparent over the past decade that tumor-associated inflammation drives the involvement of neutrophils in disease progression and metastasis. Infiltrating TANs exhibit either anti-tumorigenic (N1) or protumorigenic (N2) phenotypes. In the vast majority of solid human tumors, high infiltration with tumor-associated neutrophils (TANs) has been correlated with increased tumor growth, lymph node metastasis, and poor prognosis overall, whereas evidence from other studies advocate that under certain conditions, TANs exert cytotoxic and inhibitory activity towards tumor cells, halting the progression of cancer. In this review, we summarize current evidence on the role of neutrophils in the pathogenesis and progression of HCC and we highlight their potential utilization in HCC prognosis and therapy.Hepatocellular carcinoma represents the most prevalent primary liver cancer worldwide, and it is either caused by intrinsic genetic mutations or by a multitude of extrinsic risk factors. Even though the interplay between chronic inflammatory changes and hepatocarcinogenesis has been at the forefront of clinical investigation for the past few decades, the role of tumor-associated neutrophils (TANs) in HCC development still remains ambiguous. On the one hand, N1 TANs exhibit an anti-tumorigenic activity, mediated by direct or indirect tumor cell lysis, whereas on the other hand, N2 TANs have been correlated with increased HCC growth, invasiveness, and metastasis. The association of an elevated Neutrophil-to-Lymphocyte Ratio (NLR) with poor prognosis in patients with HCC, has been recently brought into spotlight, consolidating its widespread use as a reliable biomarker. Due to the decisive involvement of TANs in HCC pathogenesis and development, the utilization of various neutrophil-centered anticancer treatment modalities has been under clinical experimentation, selectively targeting and modulating the processes of neutrophil recruitment, activation, and migration. This review summarizes current evidence on the role of TANs in HCC pathogenesis and progression, as well as in their potential involvement in tumor therapy, shedding light on emerging anticancer treatment methods targeting neutrophils.

Role of Neutrophils and Myeloid-Derived Suppressor Cells in Glioma Progression and Treatment Resistance.

Recent efforts in brain tumor research have been directed towards the modulation of the immune system for therapeutic interventions. Several human cancers, including gliomas, are infiltrated with immune cell types—including neutrophils and myeloid-derived suppressor cells—that contribute to tumor progression, invasiveness, and treatment resistance. The role of tumor-associated neutrophils and myeloid-derived suppressor cells in cancer biology remains elusive, as these cells can exert a multitude of pro-tumor and antitumor effects. In this review, we provide the current understanding and novel insights on the role of neutrophils and myeloid-derived suppressor cells in glioma progression and treatment resistance, as well as the mechanisms of pleiotropic behaviors in these cells during disease progression, with an emphasis on possible strategies to reprogram these cells towards their antitumor actions.

Neutrophil Heterogeneity in Cancer: From Biology to Therapies.

Neutrophils have been extensively described in the pathophysiology of autoimmune and infectious diseases. Accumulating evidence also suggests the important role of neutrophils in cancer progression through their interaction with cancer and immune cells in blood and in the tumor microenvironment (TME). Most studies have described neutrophils as key drivers of cancer progression, due to their involvement in various tumor promoting functions including proliferation, aggressiveness, and dissemination, as well as in immune suppression. However, such studies were focusing on late-stages of tumorigenesis, in which chronic inflammation had already developed. The role of tumor-associated neutrophils (TANs) at early stages of tumor development remains poorly described, though recent findings indicate that early-stage TANs may display anti-tumor properties. Beyond their role at tumor site, evidence supported by NLR retrospective studies and functional analyses suggest that blood neutrophils could also actively contribute to tumorigenesis. Hence, it appears that the phenotype and functions of neutrophils vary greatly during tumor progression, highlighting their heterogeneity. The origin of pro- or anti-tumor neutrophils is generally believed to arise following a change in cell state, from resting to activated. Moreover, the fate of neutrophils may also involve distinct differentiation programs yielding various subsets of pro or anti-tumor neutrophils. In this review, we will discuss the current knowledge on neutrophils heterogeneity across different tissues and their impact on tumorigenesis, as well as neutrophil-based therapeutic strategies that have shown promising results in pre-clinical studies, paving the way for the design of neutrophil-based next generation immunotherapy.

Abstract 1084: The role of tumor-associated neutrophils (TAN) and CD8 positive T lymphocyte in the progression of hepatocellular carcinoma (HCC)

Abstract [Background]Neutrophils are known as immune cells involved in biological defense against foreign substances. On the other hand, in the tumor microenvironment, it is said that neutrophils affect on tumor progression as a tumor-associated neutrophil (TAN). Although, TAN and other immune cells in the progression of HCC is unclear. [Method]One hundred twenty eight patients who underwent curative hepatectomy for HCC in our institution were enrolled. Immunohistochemistry was performed with CD66b and CD8 antibody to evaluate TAN and CD8+ T lymphocytes, respectively. Expression high or low was determined by cut-off value of each number of immune cells. We counted the number of each immune cells in both tumor (intra-tumor) and the tumor marginal area (extra-tumor). The relationship between the infiltration of each immune cell and clinicopathological factors was examined. [Result]Comparing the number of immune cells of intra-tumor and extra-tumor, there were significantly more cells in extra-tumor area (p<0.001). There was no difference in between background clinicopathological factors and the number of TAN or CD8+ T lymphocytes in either intra-tumor or extra-tumor areas. Overall survival was significantly better in the group with high CD8+ T lymphocytes in extra-tumor area compared to the low group (p = 0.0491), intriguingly though CD8+ T lymphocytes in intra-tumor area was not associated with prognosis. In the contrast, overall survival was significantly poor in the group with high TAN in extra-tumor area compared to the low group (p = 0.0296), though TAN in intra-tumor area was not associated with prognosis. [Conclusion]Infiltration of TAN and CD8+ T lymphocytes not in intra-tumor but in the extra-tumor area may be involved in the progression of HCC. Citation Format: Toshihiko Yusa, Hirohisa Okabe, Yo-ichi Yamashita, Takanobu Yamao, Naoki Umezaki, Tatsunori Miyata, Shigeki Nakagawa, Hiromitsu Hayashi, Katsunori Imai, Akira Chikamoto, Takatoshi Ishiko, Hideo Baba. The role of tumor-associated neutrophils (TAN) and CD8 positive T lymphocyte in the progression of hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1084.

Abstract 2821: Gut commensal bacteria modulate functions of tumor-associated neutrophils in human colorectal cancer

Abstract Tumor infiltration by immune cells critically impacts on clinical outcome of human colorectal cancer (CRC). While high densities of CD8+ T cells and T-helper type 1 cells are associated with prolonged patient survival, the role of tumor-associated neutrophils (TANs) is debated. CRC arise in an environment heavily populated by microrganisms. Upon CRC oncogenesis, gut commensal bacteria translocate into the submucosa where they directly interact with residing immune cells. Neutrophils represent a front-line arm of the immune system in the response to bacteria. However, little is known about their interaction with commensal bacteria within CRC tissues. We investigated the interplay between neutrophils and commensal bacterial species present within the CRC microenvironment. Using an orthotopic CRC xenograft model, we found that commensal bacteria stimulate CRC cells to produce neutrophil recruiting chemokines. We then compared in vitro chemokine induction capacity of Fusobacterium nucleatum (Fn) and Bacteroides fragilis (Bf), two most abundant bacterial species of CRC microenvironment. Fninduced significantly higher levels of IL-8 by CRC cells than Bf, thus more effectively promoting neutrophil recruitment. Accordingly, in human CRC samples, abundance of Fn, but not Bf, significantly correlated with high infiltration by CD66b+ cells. Functional studies indicate that neutrophils cultured in the presence of Fn, but not Bf, lose their ability to enhance proliferation and cytokine release by CD8+ T cells undergoing antigenic stimulation. Moreover, neutrophils exposed to Fn, but not Bf, stimulate release of IL-6 by tumor-associated stromal cells, leading to enhanced tumor cell proliferation. Our data cumulatively suggest that distinct bacterial components of the human gut flora might differentially modulate functions of CRC infiltrating neutrophils, thus ultimately influencing their prognostic significance. Note: This abstract was not presented at the meeting. Citation Format: Valeria Governa, Eleonora Cremonesi, Diego Calabrese, Valentina Mele, Emanuele Trella, Raoul A. Droeser, Martin Bolli, Serenella Eppenberger-Castori, Salvatore Piscuoglio, Charlotte K. y. Ng, Jesus Francisco Glaus Garzon, Lubor Borsig, Klaus-Peter Janssen, Luigi M. Terracciano, Giulio Cesare Spagnoli, Dimitri Christoforidis, Pietro Edoardo Majno-Hurst, Giandomenica Iezzi. Gut commensal bacteria modulate functions of tumor-associated neutrophils in human colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2821.

Study on the influence of tumor-associated neutrophils on the biological characteristics of malignant tumors and its mechanism

The occurrence and development of malignant tumors is closely related to the tumor microenvironment. Tumor-associated neutrophils (TAN) are important inflammatory cells in the tumor microenvironment. Studies have confirmed that TAN plays an important role in tumor growth, invasion, metastasis, pathological angiogenesis, and immunity escapes. TAN can be activated as an anti-tumor N1 type and a tumor-promoting N2 type. This article reviews the biological characteristics of TAN and the role of TAN in the malignant tumors and its mechanism. Key words: Neoplasms; Neutrophil; Cell proliferation; Tumor microenvironment

Role of tumor-associated neutrophils in regulation of tumor growth in lung cancer development: A mathematical model.

Neutrophils display rapid and potent innate immune responses in various diseases. Tumor-associated neutrophils (TANs) however either induce or overcome immunosuppressive functions of the tumor microenvironment through complex tumor-stroma crosstalk. We developed a mathematical model to address the question of how phenotypic alterations between tumor suppressive N1 TANS, and tumor promoting N2 TANs affect nonlinear tumor growth in a complex tumor microenvironment. The model provides a visual display of the complex behavior of populations of TANs and tumors in response to various TGF-β and IFN-β stimuli. In addition, the effect of anti-tumor drug administration is incorporated in the model in an effort to achieve optimal anti-tumor efficacy. The simulation results from the mathematical model were in good agreement with experimental data. We found that the N2-to-N1 ratio (N21R) index is positively correlated with aggressive tumor growth, suggesting that this may be a good prognostic factor. We also found that the antitumor efficacy increases when the relative ratio (Dap) of delayed apoptotic cell death of N1 and N2 TANs is either very small or relatively large, providing a basis for therapeutically targeting prometastatic N2 TANs.

The Role of Tumor-Associated Neutrophils in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common causes of cancer deaths worldwide and the number of CRC patients is increasing progressively. Despite the improvement of the surgical techniques and chemotherapy, we have not completely overcome this disease yet due to the metastases. Therefore, understanding the mechanisms through which metastasis occurs is important for overcoming CRC. Normal host cells in the tumor microenvironment, such as macrophages and fibroblasts, have been reported to promote the growth of CRCs. Although neutrophils were originally considered to have defensive functions against tumor cells, it has been revealed that some populations of neutrophils, called as tumor-associated neutrophils (TANs), have tumor-supportive functions. The plasticity between tumor-suppressive and -supportive neutrophils are regulated by transforming growth factor (TGF)-β and Interferon-β signaling. Some studies have demonstrated that TANs promote the spread of cancer cells to distant organs. TANs contribute to the tumor invasion and angiogenesis through the production of matrix metalloproteinase-9 (MMP9), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) in the primary and metastatic sites. Neutrophils also promotes tumor cell dissemination by capturing circulating tumor cells using neutrophil extracellular traps and promote their migration to distant sites. The neutrophil-to-lymphocyte ratio is a well-defined predictive marker for CRC patients. In this review, we highlight the molecular signaling between TANs and CRC cells and the possibility of TANs as a potential target for cancer therapy.

Abstract A66: Tumor-associated neutrophils in early stage human lung cancer are not immunosuppressive, but exhibit an inflammatory phenotype and provide accessory signals for T cell activation

Abstract Tumor-recruited myeloid cells represent a significant portion of inflammatory cells within the tumor microenvironment and influence nearly all steps of tumor progression. Although tumor-associated neutrophils (TANs) are present in large numbers, the role of TANs in cancer progression remains unclear and has only been recently investigated in murine models. The goal of this study was to provide a phenotypic and functional characterization of TANs in freshly harvested surgically resected lung cancer samples. Tumors from Non-Small Cell Lung Cancer (NSCLC) patients with Stage I-II squamous cell and adenocarcinoma histology were studied. Importantly, fresh lung tumors from patients were processed within 20 minutes of removal from the patient. We developed an approach to prepare human lung tumors for immunological studies by combining gentle mechanical manipulation with an optimized cocktail of enzymes used at low doses. This approach retrieved all major tumor-associated cell populations with high cell yield and maintained the expression of cell surface markers and the functions of isolated cells. In multicolor tracings, TANs were defined as CD15hi/CD66b+/CD11b+/MPO+/Arg1+/IL-5Ra-/ cells. We found that TANs comprised 5—25% of cells isolated from all digested human lung tumors (n=42). Compared to blood neutrophils, TANs displayed an activated phenotype (CD62Llo/CD54hi) and a novel repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. They also produced substantial quantities of the pro-inflammatory factors MCP-1, IL-8, MIP-1a, and IL-6, as well as the anti-inflammatory IL-1R antagonist. TANs were not hypofunctional cells, since they were able to phagocytize bacteria and produce reactive oxygen species. Understanding the role of TANs in regulating T cell responses in cancer patients is particularly important because cytotoxic T lymphocytes are the chief effector cells mediating antigen-driven anti-tumor immunity. We found that TANs were able to stimulate antigen non-specific T cell proliferation and IFN-γ release. Crosstalk between TANs and activated T cells led to substantial up-regulation of CD54, CD86, OX40L, and 4-1BBL co-stimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive feedback loop. This stimulatory activity of TANs appeared to be related to tumor size, as TANs from the majority of the smaller tumors (< 3cm) were more stimulatory than those from larger tumors. We also identified a novel subset of TANs with a hybrid “composite” phenotype of neutrophils and antigen-presenting cells (MPO+CD66b+CD14+HLA-DR+CCR7+CD86+). We found that this hybrid subset had the ability to: 1) dramatically stimulate antigen non-specific T cell proliferation, 2) induce the proliferation of allogeneic T cells in mixed lymphocyte reaction and 3) trigger antigen-specific memory T cell response to a mixture of T cell epitopes from human CMV, Epstein-Barr, flu viruses, as well as tetanus toxoid from all the common HLA types. Thus, our data suggest that in patients with early-stage lung cancer, TANs do not significantly contribute to inhibition of T cell responses. Rather, the majority of neutrophils recruited into the tumor microenvironment undergo phenotypic and functional changes that result in the formation of cells that resemble the murine anti-tumor “N1 TANs” and could potentially augment and support T cells responses. Citation Format: Evgeniy Eruslanov, Pratik Bhojnagarwala, Jon Quatromoni, Tom Stephen, Anjana Ranganathan, Charuhas Deshpande, Tatiana Akimova, Anil Vachani, Leslie Litzky, Wayne Hancock, Jose Conejo-Garcia, Michael Feldman, Sunil Singhal, Steven Albelda. Tumor-associated neutrophils in early stage human lung cancer are not immunosuppressive, but exhibit an inflammatory phenotype and provide accessory signals for T cell activation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A66.

Prognostic and predictive significance of CD15 and neutrophil/lymphocyte ratio (NLR) in metastatic colorectal cancer (mCRC) patients (pts) undergoing to first-line chemotherapy (CT).

e22199 Background: Both local and systemic inflammatory response are known to occur in mCRC patients. The role of Tumor Associated Neutrophils (TANs) in peritumoral microenvironment as well as thei...

Abstract A82: Neutrophils recruit T-regulatory cells into the tumor via secretion of CCL17 – a new mechanism of tumor immune surveillance.

Abstract Introduction: Regulatory T cells (T-regs) play an important role in immunological self-tolerance and are functionally immune-suppressive subsets of T cells. It has been repeatedly shown that T-regs are able to migrate in response to CCL22 and CCL17, and that depletion or inhibition of T-regs can enhance anti-tumor immunity. However, there is still limited data on the mechanisms of accumulation of T-regs within the tumor microenvironment. We have recently described the characteristics of “native” pro-tumorigemic N2 tumor-associated Neutrophils (TAN), and found that TAN significantly up-regulated many chemokines compared to bone-marrow (BM) naïve neutrophils. One of the highly up-regulated genes in TAN is CCL17. Aims: We aimed to study the role of TAN in the secretion of CCL17 and the recruitment of T-regs into the tumor microenvironment. We hypothesized that the secretion of CCL17 by N2 TAN has an important role in the recruitment of T-regs, thus inhibiting the T-cytotoxic cell activity in the tumor. Material and Methods: CCL17 production by N1 and N2 TAN and by BM naive Neutrophils was evaluated using ELISA, RT-PCR and IHC staining. We conducted in vivo and in vitro migration assays, and used depletion of TAN to reveal the role of CCL17 secreted from TAN in chemoattraction of T-regs. Results: N2-TAN were more capable of producing CCL17 ex-vivo than N1-TAN or BM neutrophils. The level of CCL17 secreted from TAN increased during tumor development. In vitro migration assay using CD4+CD25+ cells purified from spleens of naïve mice revealed that migration of T-cells towards TAN were 5.27±1.76 times higher compared to BM neutrophils (p<0.05). Furthermore, migration was inhibited by neutralizing anti-mouse CCL17. In vivo migration assay using the air pouch model strengthen these results, demonstrating that more CD4+CD25+ cells migrated into the pouch when injecting TAN compared to BM neutrophils, again inhibited using anti-CCL17 mAb, suggesting that TAN-induced T-regs chemoattraction is CCL17 dependent. Systemic neutrophil depletion after tumor establishment reduced T-reg migration into the tumor. Conclusions: Our results show that TAN from established tumors produced CCL17, increasing T-regs migration into the tumor. These results suggest a new indirect mechanism by which TAN may inhibit the anti-tumor immune activity, promoting tumor growth. Citation Format: Inbal Mishalian, rachel Bayuh, Lida Zolotarov, Liran Levy, Zvi Gregorio Fridlender. Neutrophils recruit T-regulatory cells into the tumor via secretion of CCL17 – a new mechanism of tumor immune surveillance. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A82.

Abstract 1912: Significant changes in chemokine genes in tumor-associated neutrophils (TANs) using a microarray approach

Abstract Background: The role of tumor-associated neutrophils (TAN) in tumor biology is unclear. We have recently published that resident TAN can acquire a protumor phenotype (similar to M2 macrophages), largely driven by TGF-beta to become N2 neutrophils. After TGF-beta blockade, neutrophils acquire an antitumor phenotype (N1 TAN (similar to M1 macrophages). In the current study, we further evaluated the characteristics of TAN using a gene microarray approach. Methods: Balb/C mice (10-20 in each group) bearing large flank tumors of the mesothelioma line AB12, where treated with either control or SM16 (a TGF-β-R1 kinase inhibitor) chow. After one week of treatment, the tumors were harvested, and neutrophils were isolated using microbeads (CD11b) and flow cytometry (Ly6G), into two groups - control TAN (N2) and SM16 TAN (N1). Neutrophils were also isolated from bone marrow of naïve mice (BM) and from spleens harvested from tumor-bearing mice (i.e., the granulocytic fraction of myeloid derived suppressor cells (G-MDSC)). mRNA from each subgroup (4-6 samples) was isolated and subjected to microarray analysis on Illumina chips. Selected results were validated by RT-PCR and neutrophils from a second non Small Cell Lung Cancer tumor, LKR, were studied. Results: After filtering, we found 16,322 informative probes. Hierarchical clustering and PCA analysis showed that the four groups were clearly separated. We found that many more genes were up-regulated than down-regulated in the N2 TAN from untreated mice compared to either BM or G-MDSC. Many of the highly up-regulated genes in TAN were chemokines, and genes encoding for inflammatory and immune responses, especially chemokines. This includes chemoattractants for T-cells (e.g. CXCL-9, CXCL10 and CXCL16), neutrophils (eg. CXCL1, CXCL2 and CCL3), B-cells (CXCL13) and macrophages (e.g. CCL2, CXCL-10 and CCL7). N1 (SM16-treated) and N2 TAN were more similar to each other than to the other groups, suggesting an intra-tumoral polarization between them. In N1 TAN, three different chemokines attracting macrophages were up-regulated - CCL2, CCL6 and CXCL10, and only one major chemokine, the T-regs chemoattractant CCL17, was downregulated. There was no significant difference in most other chemokines. Conclusion: Our data shows the major differences between naïve neutrophils, G-MDSC and N1/N2 TAN. The patterns support the hypothesis that neutrophils enter the tumor, possibly as G-MDSC, and the change between N2 and N1 is intra-tumoral (and not by differential recruitment). Our data further suggests that TAN secrete many chemokines, initiating the recruitment of other inflammatory cells of the immune system. N1 neutrophils appear to up-regulate several macrophage attracting chemokines compared to N2 TAN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1912.