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Simple Summary Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent skin cancer globally. Immunosuppression raises cSCC incidence rates, while high immunogenicity of the cutaneous tissue enables topical immunotherapy. Intriguingly, expanded applications of programmed death-1 (PD-1) blockade therapies have revealed cSCC to be one of the most amenable targets. These clinical observations prompted us to redefine cSCC biology and review current knowledge about cSCC from multiple viewpoints that involve epidemiology, clinicopathology, molecular genetics, molecular immunology, and developmental biology. This synthesis reinforces the following hypothesis: PD-1 blockade effectively restores the immunity specially allowed to exist within the fully cornified squamous epithelium, that is, the epidermis. Abstract Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent skin cancer globally. Because most cSCC cases are manageable by local excision/radiotherapy and hardly become life-threatening, they are often excluded from cancer registries in most countries. Compared with cutaneous melanoma that originates from the melanin-producing, neural crest-derived epidermal resident, keratinocyte (KC)-derived cancers are influenced by the immune system with regards to their pathogenetic behaviour. Congenital or acquired immunosurveillance impairments compromise tumoricidal activity and raises cSCC incidence rates. Intriguingly, expanded applications of programmed death-1 (PD-1) blockade therapies have revealed cSCC to be one of the most amenable targets, particularly when compared with the mucosal counterparts arisen in the esophagus or the cervix. The clinical observation reminds us that cutaneous tissue has a peculiarly high immunogenicity that can evoke tumoricidal recall responses topically. Here we attempt to redefine cSCC biology and review current knowledge about cSCC from multiple viewpoints that involve epidemiology, clinicopathology, molecular genetics, molecular immunology, and developmental biology. This synthesis not only underscores the primal importance of the immune system, rather than just a mere accumulation of ultraviolet-induced mutations but also reinforces the following hypothesis: PD-1 blockade effectively restores the immunity specially allowed to exist within the fully cornified squamous epithelium, that is, the epidermis.

Simple Summary Interferons are cytokines that play key roles in the activation of cellular components of the immune response, such as dendritic cells, macrophages and T cells. Generally, these cytokines promote anti-tumor immune responses, but under some circumstances, prolonged exposure to them can lead to suppression of the immune response. This review focuses on the immunostimulatory versus immunosuppressive roles of interferons and the mechanisms mediating such effects on both malignant cells and cells of the immune system. Abstract The interferons (IFNs) are essential components of the immune response against infections and malignancies. IFNs are potent promoters of the anti-tumor response, but there is also evidence that feedback mechanisms regulated by IFNs negatively control immune responses to avoid hyper-activation and limit inflammation. This balance of responses plays an important role in cancer surveillance, immunoediting and response to anticancer therapeutic approaches. Here we review the roles of both type I and type II IFNs on the control of the immune response against malignancies in the context of effects on both malignant cells and cells of the immune system in the tumor microenvironment.

Aim: To characterize, by means of univariate and multivariate approaches, the T helper (Th)-1 and Th-2 responses during the different phases of tumor immunoediting. Materials & methods: We used a multivariate principal component analysis applied to analyze the joint behavior of serum concentrations of IFN-γ, IL-2, IL-10 and IL-4, during the different phases of tumor immunoediting, in CBi/L mice challenged with M-406 mammary adenocarcinoma. Results & conclusion: Animals in equilibrium phase showed the widest variations in values of the four cytokines. In this experimental model, the role of IFN-γ would be related to tumor growth and progression, while IL-10 would participate in the antitumor immune response., Lay abstract Breast cancer is a complex, multifactor disease that affects about 10% of women in industrialized countries. The immune system has the ability to monitor the appearance of tumors, but the tumors have the ability to escape such rejection. For this reason, in order to design different therapeutic strategies, it is important to know the different mechanisms that take place when a tumor grows or when it is rejected. Here we sought to elucidate some of these mechanisms.

The immune system plays dual roles in response to cancer. The host immune system protects against tumor formation via immunosurveillance; however, recognition of the tumor by immune cells also induces sculpting mechanisms leading to a Darwinian selection of tumor cell variants with reduced immunogenicity. Cancer immunoediting is the concept used to describe the complex interplay between tumor cells and the immune system. This concept, commonly referred to as the three E’s, is encompassed by 3 distinct phases of elimination, equilibrium, and escape. Despite impressive results in the clinic, cancer immunotherapy still has room for improvement as many patients remain unresponsive to therapy. Moreover, many of the preclinical results obtained in the widely used mouse models of cancer are lost in translation to human patients.To improve the success rate of immuno-oncology research and preclinical testing of immune-based anticancer therapies, using alternative animal models more closely related to humans is a promising approach. Here, we describe 2 of the major alternative model systems: canine (spontaneous) and porcine (experimental) cancer models. Although dogs display a high rate of spontaneous tumor formation, an increased number of genetically modified porcine models exist. We suggest that the optimal immuno-oncology model may depend on the stage of cancer immunoediting in question. In particular, the spontaneous canine tumor models provide a unique platform for evaluating therapies aimed at the escape phase of cancer, while genetically engineered swine allow for elucidation of tumor-immune cell interactions especially during the phases of elimination and equilibrium.

Although metabolic modulation in the tumor microenvironment (TME) is one of the key mechanisms of cancer immune escape, there is a lack of understanding of the comprehensive immune landscape of the TME and its association with tumor metabolism based on clinical evidence. We aimed to investigate the relationship between the immune landscape in the TME and tumor glucose metabolism in lung adenocarcinoma. Methods: Using RNA sequencing and image data, we developed a transcriptome-based tumor metabolism estimation model. The comprehensive TME cell types enrichment scores and overall immune cell enrichment (ImmuneScore) were estimated. Subjects were clustered by cellular heterogeneity in the TME and the clusters were characterized by tumor glucose metabolism and immune cell composition. Moreover, the prognostic value of ImmuneScore, tumor metabolism and the cell type-based clusters was also evaluated. Results: Four clusters were identified based on the cellular heterogeneity in the TME. They showed distinct immune cell composition, different tumor metabolism, and close relationship with overall survival. A cluster with high regulatory T cells showed relative hypermetabolism and poor prognosis. Another cluster with high mast cells and CD4+ central memory T cells showed relative hypometabolism and favorable prognosis. A cluster with high ImmuneScore showed favorable prognosis. Multivariate Cox analysis demonstrated that ImmuneScore was a predictive prognostic factor independent of other clinical features. Conclusions: Our results showed the association between predicted tumor metabolism and immune cell composition in the TME. Our studies also suggest that tumor glucose metabolism and immune cell infiltration in the TME can be clinically applicable for developing a prognostic stratification model.

Background: Peptide antigens from a pathogen that are harmful to the body is presented on the cell surface of molecules named the major histocompatibility complex (MHC), or human leukocyte antigens (HLA) in humans. Tumor cells may be recognized in a similar way by the presentation of tumor antigens. The HLA complex consists of three classes: class I, class II and class III. The class I antigens are subdivided into the classical HLA class Ia, which consists of HLA-A, -B and -C, and the non-classical HLA class Ib, which consists of HLA-E, -F and -G. HLA-G, which is expressed in the thymus, the pancreas, and the extravillous cytotrophoblasts among others, has an important tolerance-inducing function, where it confers protection to the fetus from destruction by the immune system of the mother. The immunoinhibitory function of HLA-G is achieved by binding to the inhibitory receptors on T cells, natural killer cells, dendritic cells and macrophages. This inhibitory function of HLA-G is believed to be exploited by tumors. However, studies suggest that expression of HLA-G is restricted in tumor cells. Methods: In this Master’s thesis, expression of HLA-A, -B, -C, -DR, -E and -G was investigated in MDA-MB-231 and MCF-7 breast cancer cell lines after they were treated with interferon-gamma (IFN-γ) for 2 days, and 5’Aza-2’-Deoxycytidine (5-Aza-2’dC) for 3 and 6 days. Experiments with transfection of MDA-MB-231 cells with a vector construct containing the HLA-G gene were also conducted.Results: It was found that for both cell lines IFN-γ significantly upregulated the classical HLA class I antigens, while no significant regulation was observed for HLA-G. Treatment with 5-Aza-2’dC revealed that expression of the classical HLA class Ia was upregulated in both cell lines at day 3 and 6. After treatment with 5-Aza-2’dC, MDA-MB-231 cells only showed a significant HLA-G expression on day 3, while in MCF-7 cells HLA-G expression was significantly upregulated at day 3 and 6. Transfection of HLA-G in MDA-MB-231 did not show a significantly upregulated cell surface HLA-G expression, however, it was observed that MDA-MB-231 contains endogenous HLA-G.Conclusions: Overall these results reveal that expression of HLA-G can be upregulated when treating MDA-MB-231 and MCF-7 cells with the demethylating agent 5-Aza-2’dC. This discovery in part helps in clarifying how expression of HLA-G is regulated in tumor cells. The interaction of immune cells with cells expressing both HLA class Ia and HLA-G molecules based on transfection versus cells only expressing HLA class Ia will help to clarify the importance of HLA-G in induction of tolerance.

The driver mutations in exon 9 of the calreticulin protein have only been identified in patients with myeloid cancers. We recently demonstrated that healthy individuals display strong and frequent T-cell responses towards this mutation. This memory T-cell response is likely evidence of the elimination of mutated cells in healthy individuals.

The cancer stem cell (CSC) paradigm posits that specific cells within a tumor, so-called CSC-like cells, have differing levels of tumorigenicity and chemoresistance. Original studies of CSCs identified them in human cancers and utilized mouse xenograft models to define the cancer initiating properties of these cells, thereby hampering the understanding of how immunity could affect CSCs. Indeed, few studies have characterized CSCs in the context of cancer immunoediting, and it is currently not clear how immunity could impact on the levels or stem-like behavior of CSCs. Using the well-studied 3′methylcholanthrene (MCA) model of primary sarcoma formation, we have defined a CSC-like population within MCA-induced sarcomas as expressing high levels of stem cell antigen-1 (Sca-1) and low levels of CD90. These Sca-1(+)CD90(−) CSC-like cells had higher tumor initiating ability, could spontaneously give rise to Sca-1-negative cells, and formed more sarcospheres than corresponding non-CSC-like cells. Moreover, when examining MCA-induced sarcomas that were in the equilibrium phase of cancer growth, higher levels of CSC-like cells were found compared to MCA-induced sarcomas in the escape phase of cancer progression. Notably, CSC-like cells also emerged during escape from anti-PD-1 or anti-CTLA4 therapy, thus suggesting that CSC-like cells could evade immune therapy. Finally, we demonstrate that paradoxically, interferon (IFN)-γ produced in vivo by immune cells could promote the emergence of CSC-like cells. Our findings define the existence of a Sca1(+)CD90(−) CSC-like population in the MCA-sarcoma model capable of differentiation, tumorsphere formation, and increased tumor initiation in vivo. These cells may also act as mediators of immune resistance during cancer immunoediting and immune therapy.

Recent progress in tumor immunology has revealed that tumors generate immunologically restrained milieu during the process of their growth, which facilitates the escape of tumors from host immune systems. Immune checkpoint molecules, which transduce co-inhibitory signals to immuno-competent cells, are one of the most important components conferring the immunosuppressive capacity in the tumor microenvironment. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death-1 (PD-1) are typical immune checkpoint molecules intimately involved in the suppression of anti-tumor immunity. Antibodies against those molecules have been developed, such as ipilimumab (anti-CTLA-4 antibody), nivolumab and pembrolizumab (anti-PD-1 antibody), and have been approved by regulatory agencies and used in some countries. Treatment with these antibodies demonstrates previously unobserved clinical efficacies superior to the conventional therapies. In this review, we first discuss the escape mechanisms of cancer from host immune systems, and then focus on the recent advances in immune checkpoint blockade therapy and on the new findings of related immune reactions, aiming to provide a better understanding of the novel cancer immunotherapies.

In the Sp6 mouse plasmacytoma model, a whole-cell vaccination with Sp6 cells expressing de novo B7-1 (Sp6/B7) induced anatomically localized and cytotoxic T cell (CTL)-mediated protection against wild-type (WT) Sp6. Both WT Sp6 and Sp6/B7 showed down-regulated expression of MHC H-2 L(d). Increase of H-2 L(d) expression by cDNA transfection (Sp6/B7/L(d)) raised tumour immune protection and shifted most CTL responses towards H-2 L(d)-restricted antigenic epitopes. The tumour-protective responses were not specific for the H-2 L(d)-restricted immunodominant AH1 epitope of the gp70 common mouse tumour antigen, although WT Sp6 and transfectants were able to present it to specific T cells in vitro. Gp70 transcripts, absent in secondary lymphoid organs of naive mice, were detected in immunized mice as well as in splenocytes from naive mice incubated in vitro with supernatants of CTL-lysed Sp6 cell cultures, containing damage-associated molecular patterns (DAMPs). It has been shown that Toll-like receptor triggering induces gp70 expression. Damage-associated molecular patterns are released by CTL-mediated killing of Sp6/B7-Sp6/B7/L(d) cells migrated to draining lymph nodes during immunization and may activate gp70 expression and presentation in most resident antigen-presenting cells. The same could also apply for Mus musculus endogenous ecotropic murine leukaemia virus 1 particles present in Sp6-cytosol, discharged by dying cells and superinfecting antigen-presenting cells. The outcome of such a massive gp70 cross-presentation would probably be tolerogenic for the high-affinity AH1-gp70-specific CTL clones. In this scenario, autologous whole-tumour-cell vaccines rescue tumour-specific immunoprotection by amplification of subdominant tumour antigen responses when those against the immune dominant antigens are lost.

Background: Immunotherapy is at the forefront of modern oncologic care. Various novel therapies have targeted all three layers of tumor biology: tumor, niche, and immune system with a range of promising results. One emerging class in both primary and salvage therapy is oncolytic viruses. This therapy offers a multimodal approach to specifically and effectively target and destroy malignant cells, though a barrier oncoviral therapies have faced is a limited therapeutic response to currently delivery techniques. Main body: The ability to deliver therapy tailored to specific cellular targets at the precise locus in which it would have its greatest impact is a profound development in anti-cancer treatment. Although immune checkpoint inhibitors have an improved tolerability profile relative to cytotoxic chemotherapy and whole beam radiation, severe immune-related adverse events have emerged as a potential limitation. These include pneumonitis, pancreatitis, and colitis, which are relatively infrequent but can limit therapeutic options for some patients. Intratumor injection of oncolytic viruses, in contrast, has a markedly lower rate of serious adverse effects and perhaps greater specificity to target tumor cells. Early stage clinical trials using oncolytic viruses show induction of effector anti-tumor immune responses and suggest that such therapies could also morph and redefine both the local target cells' niche as well as impart distant effects on remote cells with a similar molecular profile. Conclusion: It is imperative for the modern immuno-oncologist to understand the biological processes underlying the immune dysregulation in cancer as well as the effects, uses, and limitations of oncolytic viruses. It will be with this foundational understanding that the future of oncolytic viral therapies and their delivery can be refined to forge future horizons in the direct modulation of the tumor bed. CA extern

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2017 Submitted by Repositório FFUL (repositorio@ff.ul.pt) on 2018-12-21T15:40:46Z No. of bitstreams: 1 MICF_Sofia_Martins.pdf: 1979845 bytes, checksum: d2684615e876b1ee0e2e8db9bf2e3d37 (MD5) Made available in DSpace on 2018-12-21T15:50:11Z (GMT). No. of bitstreams: 1 MICF_Sofia_Martins.pdf: 1979845 bytes, checksum: d2684615e876b1ee0e2e8db9bf2e3d37 (MD5) Previous issue date: 2017-12-28

Since it is known that the immune system affects tumor growth, it has been studied if immunotherapy can be developed to combat cancer. While some successes have been claimed, the increasing knowledge on tumor-immune interactions has, however, also shown the limitations of this approach. Tumors may show selective outgrowth of cells escaped from immune control. Escape variants arise spontaneously due to the genetically instable nature of tumor cells. This is one of the most obvious limitations of cancer immunotherapy. However, new therapies are becoming available, designed to respond to tumor-immune escape.

Interactions between the immune system and tumors have been among the highlights of present immunological research. An extensive body of new knowledge recently substantiated the long-presumed concept of cancer immunosurveillance. Immune system searches the organism for cells expressing tumor antigens or cellular stress signals and destroys them. T-cells, NK-cells and dendritic cells, as well as cytokine signaling and direct cell cytotoxicity play dominant role in this process. However, a fraction of nascent tumors can evade these mechanisms and create a dynamic equilibrium, gradually sculpting its phenotype by clonal selection. Eventually, tumor cells escape immune control by concealing themselves from recognition or by actively subjugating local immune response. This immunosubversion results in formation of immunosuppressive tumor microenvironment by recruiting protumorigenic cell populations, such as Treg cells, macrophages and myeloid derived suppressor cells. Soluble signaling molecules, as well as surface- expressed immune checkpoint molecules are exploited by tumor cells for inhibition of anti-tumor immunity. Highly effective therapeutic antibodies blocking these checkpoints have been developed for clinical use, with many more in current trials. Several other promising immunotherapeutic...

Despite the promising clinical outcome, the primary challenge of the curative cancer immunotherapy is to overcome the dichotomy of the immune response: tumor-evoked immunostimulatory versus tumor-induced immunosuppressive. The goal needs to be two-fold, to re-establish sustainable antitumor-cancer immunity and to eliminate immunosuppression. The successful elimination of cancer cells by immunosurveillance requires the antigenic presentation of the tumor cells or tumor-associated antigens and the expression of immunostimulatory cytokines and chemokines by cancer and immune cells. Tumors are heterogeneous and as such, some of the tumor cells are thought to have stem cell characteristics that enable them to suppress or desensitize the host immunity due to acquired epigenetic changes. A central mechanism underlying tumor epigenetic instability is the increased histone deacetylase (HDAC)-mediated repression of HDAC-target genes regulating homeostasis and differentiation. It was noted that pharmacological HDAC inhibitors are not effective in eliminating tumor cells partly because they may induce immunosuppression. We have shown that epithelial-specific tumor suppressor maspin, an ovalbumin-like non-inhibitory serine protease inhibitor, reprograms tumor cells toward better differentiated phenotypes by inhibiting HDAC1. Recently, we uncovered a novel function of maspin in directing host immunity towards tumor elimination. In this review, we discuss the maspin and maspin/HDAC1 interplay in tumor biology and immunology. We propose that maspin based therapies may eradicate cancer.

Mestrado em Biomedicina Molecular Angiogenesis is crucial for breast cancer growth, progression and dissemination, making it a promising therapeutic target. Given the dominant role of Vascular Endothelial Growth Factor (VEGF) signaling in tumor angiogenesis, the majority of clinical studies with anti-angiogenic therapies in breast cancer were based on VEGF blockade. However, lack of clinical efficacy was observed in all clinical studies with antiangiogenic therapies for breast cancer. Since angiopoietin 2 (ANG-2) has been reported to induce therapeutic resistance after VEGF-targeted therapies, breast cancer patients may benefit from a therapeutic strategy that simultaneously blocks ANG-2 and VEGF-A. Indeed, using the bispecific ANG-2/VEGF-A targeting antibody (A2V), durable inhibition of tumor progression, as well as increased survival, could be observed after dual ANG-2/VEGF-A blockade in the aggressive mouse model of breast cancer, the MMTV-PyMT transgenic mouse. Consistent with tumor inhibition, increased tumor necrosis and a drastic reduction of vascular density was observed after A2V therapy, with little evidence of tumor revascularization or resistance to therapy. Moreover, A2V led to increased perivascular coverage of the remaining tumor blood vessels, favoring vascular normalization, which is known to facilitate the development of an immunosupportive microenvironment. These findings suggested that breast cancer immunotherapy may benefit from the combination with A2V. Therefore the A2V mAb was combined with a CD40 agonistic antibody (FGK45). The combined therapeutic approach led to remarkable tumor growth inhibition in an orthotopic MMTV-PyMT model, with the achievement of cases of stable disease and further increase in tumor necrosis. Increased infiltration of CD8+ cells after FGK45 therapy, either alone or in combination with A2V, could also be observed. Of note, a correlation between the extent of tumor inhibition and increased tumor infiltration by CD8+ cells was detected, suggesting that the influx of CD8+ cells in the combination therapy-treated tumors was responsible for the remarkable anti-tumor responses observed. In summary, these results point to a significant synergistic activity of anti-ANG-2/VEGF-A and CD40 agonistic therapy in the MMTV-PyMT model of breast carcinoma. This combinatorial approach may benefit breast cancer patients, so further studies that explore the mechanistic bases of the cooperative activity of antiangiogenic and immunostimulatory therapy are warranted. A angiogénese é um processo crucial para o crescimento, progressão e disseminação do cancro da mama, tornando-se assim um alvo terapêutico promissor. A maioria dos estudos clínicos de terapias anti-angiogénicas em cancro da mama baseiam-se na neutralização do fator de crescimento endotelial vascular (VEGF), uma vez que este apresenta um papel dominante na angiogénese tumoral. No entanto, todos os estudos clínicos baseados em terapias anti-angiogénicas têm-se revelado ineficazes no tratamento de pacientes com cancro da mama. Uma vez que a Angiopoietina 2 (ANG-2), outro fator de crescimento vascular, tem sido reportada como responsável pela resistência a terapias baseadas no bloqueio de função do VEGF, é possível que pacientes com cancro da mama beneficiem de uma estratégia terapêutica que neutralize simultaneamente ANG-2 e VEGF-A. De facto, através do uso de um anticorpo ‘biespecífico’, capaz de neutralizar tanto ANG-2 como VEGF-A e denominado A2V, observou-se uma forte inibição da progressão tumoral e aumento da sobrevivência num modelo pré-clínico de cancro da mama agressivo, baseado em ratinhos transgénicos e denominado MMTV-PyMT. Consistentemente, esta terapia levou ao aumento de necrose tumoral e a uma drástica redução na densidade vascular, sem evidência de revascularização ou resistência à terapia. A administração de A2V induziu ainda o aumento da associação entre células perivasculares (‘pericytes’) e células endoteliais dos restantes vasos sanguíneos tumorais, favorecendo a maturação e normalização vascular, o que facilita o desenvolvimento de um microambiente que suporta respostas imunológicas. Estes resultados sugeriam assim que imunoterapia em cancro da mama poderia beneficiar da combinação com A2V, tendo-se por conseguinte combinado o anticorpo A2V com o anticorpo agonista de CD40 (FGK45), no modelo ortotópico de MMTV-PyMT. Esta terapia induziu uma notável inibição do crescimento tumoral, com obtenção de casos estáveis, e aumento adicional de necrose tumoral. Observou-se ainda o aumento de infiltração de células CD8+ após terapia com FGK45, quer isoladamente ou em combinação com A2V, e uma correlação entre a extensão da inibição do tumor e o aumento da infiltração tumoral por células CD8+. Isto sugere que o influxo de células CD8+ nos tumores tratados com A2V e FGK45 foi o principal responsável pelas notáveis respostas anti-tumorais observadas. Em resumo, estes resultados apontam para uma sinergia entre a neutralização simultânea de ANG-2/VEGF-A e imunoterapia baseada em agonistas do recetor CD40, no modelo pré-clínico de cancro da mama MMTV-PyMT. Esta abordagem combinatória pode beneficiar pacientes com cancro da mama, por isso, mais estudos são necessários para explorar os mecanismos subjacentes à atividade cooperativa da terapia anti-angiogénica e a imunoterapia.

La inmunoedición del cáncer es un proceso dinámico compuesto de tres fases: eliminación (EL), equilibrio (EQ) y escape (ES) que abarca las posibles funciones protectoras del huésped y de esculpir el tumor del sistema inmunitario durante el desarrollo del tumor. Los modelos animales son herramientas útiles para estudiar enfermedades como el cáncer. El presente estudio fue diseñado para caracterizar la interacción entre el adenocarcinoma mamario M-406 y CBi, CBi - y CBi / L líneas de ratones endogámic Cancer immunoediting is a dynamic process composed of three phases: elimination (EL), equilibrium (EQ) and escape (ES) that encompasses the potential host-protective and tumor-sculpting functions of the immune system throughout tumor development. Animal models are useful tools for studying diseases such as cancer. The present study was designed to characterize the interaction between mammary adenocarcinoma M-406 and CBi, CBi− and CBi/L inbred mice lines. Fil: Pagura, Lucas. Facultad de Ciencias Bioquímicas y Farmacéuticas. Universidad Nacional de Rosario; Rosario; Argentina Fil: Cáceres, Juan Manuel. Facultad de Ciencias Médicas. Universidad Nacional de Rosario; Rosario; Argentina Fil: Cardinali, Albertina. Facultad de Ciencias Médicas. Universidad Nacional de Rosario; Rosario; Argentina Fil: Scharovsky, Graciela Olga. Institute of Experimental Genetics. School of Medical Sciences. National University of Rosario; Rosario; Argentina Fil: Di Masso, Ricardo José. Facultad de Ciencias Médicas. Universidad Nacional de Rosario; Rosario; Argentina Fil: Zacarias-Fluck, Mariano. Preclinical Research. Vall d’Hebron Institute of Oncology (VHIO); Barcelona; Spain Fil: Rico, María José. Institute of Experimental Genetics. School of Medical Sciences. National University of Rosario; Rosario; Argentina Fil: Rozados, Viviana R. Institute of Experimental Genetics. School of Medical Sciences. National University of Rosario; Rosario; Argentina

Upon activation, neutrophils release fibers composed of chromatin and neutrophil proteins termed neutrophil extracellular traps (NETs). NETs trap and kill microbes, activate dendritic cells and T cells, and are implicated in autoimmune and vascular diseases. Given the growing interest in the role of neutrophils in cancer immunoediting and the diverse function of NETs, we searched for NETs release by tumor-associated neutrophils (TANs). Using pediatric Ewing sarcoma (ES) as a model, we retrospectively examined histopathological material from diagnostic biopsies of eight patients (mean ± SD age of 11.5 ± 4.7 years). TANs were found in six patients and in two of those we identified NETs. These two patients presented with metastatic disease and despite entering complete remission after intensive chemotherapy had an early relapse. NETs were not identified in the diagnostic biopsies of two patients with localized disease and two with metastatic disease. This study is the first to show that TANs in ES are activated to make NETs, pointing to a possible role of NETs in cancer.