Du fait de leurs propriétés immunostimulantes, les entérotoxines de Staphylococcus aureus (SEs) sont aussi considérées comme des outils thérapeutiques anticancéreux potentiels. Cependant, leurs implications dans de nombreuses pathologies humaines limitent leurs utilisations. Récemment, un opéron dénommé enterotoxin gene cluster (egc) codant pour cinq entérotoxines (SEG, SEI, SElM, SElN et SElO) supposées être de moindre virulence pour l’organisme, a été mis en évidence. En 2004, des patients atteints de carcinome broncho-pulmonaire ont été traités par l’administration d’un surnageant de culture d’une souche de S. aureus, contenant l’opéron egc. Ce traitement a permis d’allonger la durée de survie, et n’a eu aucun effet secondaire. Dans ce cadre, l’objectif de cette thèse a été d’étudier l’activité anti-tumorale des toxines de l’egc. Nos travaux ont mis en évidence l’activité tumoricide de ces toxines, induite par l’activation du système immunitaire. Cette toxicité est médiée par la sécrétion de nombreux médiateurs solubles comme le TNF-α et le NO. Nous avons confirmé le caractère pro-inflammatoire de type Th1 des toxines de l’egc. Nos travaux ont également montré qu’hormis SEI, les toxines de l’egc induisent des sécrétions de cytokines, chimiokines, protéases matricielles (MMPs) et facteurs de croissances nettement inférieures à celle induites par le reste des SEs. Ces résultats pourraient expliquer la faible toxicité associée aux toxines de l’egc. Enfin, nous avons montré que SElO possèdent une toxicité intrinsèque vis-à-vis des lignées tumorales. Cette étude plaide en faveur de l'intérêt des toxines de l’egc dans le développement de nouvelles approches en thérapie anti-tumorale / The use of classical superantigens (e.g. SEA, SEB and SEC) for treatment of cancer has resulted in a low response rates due to serious toxicity in humans. However, in a recent clinical study, remarkable results in treating lung cancer were obtained using superantigens encoded by the enterotoxin gene cluster (egc) without causing any significant toxicity. The current study was performed to investigate how egc superantigens (i.e. SEG, SEI, SElM, SElN and SElO) have tumoricidal activity with low toxicity. Indeed, we first demonstrated that tumoricidal activity of egc-SEs is mediated by immune cell activation, in particular, by secretion of soluble mediators such as nitric oxide and TNF-α. Thus, the proteomic analysis of the PBMC supernatants, showed that SEs-egc enhance the expression of pro-inflammatory cytokines, chimokines and many other biomarkers. Interestingly, levels were significantly higher in supernatants of SEA-stimulated PBMC than those with egc superantigens suggesting that staphylococcal superantigens differs in their inflammatory proprerties. Our results suggest that the relative lower pro-inflammatory activity of egc toxins may explain the low toxicity of these toxins observed during the clinical trial. Finally, we showed that SElO have a direct cytostatic activity against tumor cells. These findings suggest that egc-SEs seems to be good candidates for the development of new drugs in cancer therapy

Le cancer du sein représente un problème de santé publique. Les efforts déployés pour définir de nouvelles stratégies thérapeutiques ont mis en lumière les mécanismes développés par la tumeur pour inhiber l'instauration d'une réponse immune adaptative efficace. La tumeur favorise la mise en place d'une immunosubversion, caractériisée par la fort infiltration d'une sous-population de lymphocytes T CD4 régulateurs (Treg). Les Treg jouent un rôle central dans les mécanismes de contrôle de réactions pro-inflammatoires et protègent contre le développement de pathologies auto-immunes. Nous avons observé que le Treg sont recrutés spécéfiquement dans l'environnement tumoral via l'axe de chimiotactisme CCR4/CCL22 suite à la détetion des cellules transformées par les NK et les macrophages. D'autre part, plusieurs publications récentes démontrent un rôle des cellules dendritiques plasmacytoïdes (pDC) dans l'indcution de Treg chez l'homme et la souris. Nos observations montrent que des facteurs solubles sécrétés dans l'environnement tumoral inhibent la fonction clef des pDC, ce qui favorise fortement leur capacité à induire l'expansion de Treg et de T CD4 prodcuteur d'IL-10. Ces cellules T immunosuppressives expriment fortement le récepteur de co-simulation ICOS, prolifèrent in situ, exercent un important pouvoir supresseur sur les autres popuylations T et leur présence est associées à un impact péjoratif sur la survie des patientes. Par l'utilisation de l'Ac bloquant anti-COS 314.8 nous avons démontré le rôle essentiel d'ICOS dans la prolifération des Treg et l'induction de T CD4 sécréteurs d'IL-10 par les pDC dasn les tumeurs, offrant la perspective d'une nouvelle immunothérapie visant à éradqiuer les Treg intra-tumoraux. / Tumor immunosbversion favors disease progression and is mediated by increased IL-10 secretion, reduced type-I IFN production and regulatory T cell (Treg) accumulation among CD4+ T cell in breast tumor. We showed that the presence of high number of both Treg and/or plasmacytoid DC (pDC) a subpopulation of antigen presenting celles correlates with poor prognosis in breast carcinoma. We previoously demonstrated that CCR4+ Treg are recruited from the periphery trough CCL22 production by breast tumor cells. Tumor-asssociated Treg (Ta-Treg) are highly activated (GITRhighHDLA-DRhighCD39high), show a selective expression of high levels of ICOS and proliferate in situ (Ki-67+). Tumor associated (Ta-) pDC express a partially activated phenotype but their type-1 interferon (IFN) production is strongly impaired in human tumors. pDc secretion of type-I IFN is linked to their capacity to induce anti-viral and anti-tumor immunity in mice models. We shown that 1) Ta-Treg and Ta-pDC colocalize in breast tumor section and 2) TapDC favor Ta-Treg proliferation and IL-10 secretion by CD4+ T cells in absence of type-I IFN. Ogf importance, targeting ICOS with a neutralizing antibody suppresses Ta-Treg proliferation as well as IL-10 secretion inpDC/CD4+ T cell co-culture, demonstrationg a riole of ICOS-ICOS-L interaction in Ta-Treg proliferation mediated by Ta-pDC. At the end, we report that high ICOS expression in breast tumor sections is associated with reduced patient's overall and disease free survival. Altogether these observations suggest that ICOS in breat cancer may represent a therapeutic target to restore anti-tumor immunity

Orientadores: Simone Cristina Olenscki Gilli, Sara Teresinha Olalla Saad / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T07:27:16Z (GMT). No. of bitstreams: 1 Simoneti_GiseledaSilva_M.pdf: 1770601 bytes, checksum: 967e921fb162c153636ac91afdd1f138 (MD5) Previous issue date: 2013 / Resumo: Células dendríticas (DCs) são as principais células apresentadoras de antígeno do sistema imune, capazes de estimular o linfócito T a iniciar resposta imune especifica. Vacinas de DCs vêm sendo utilizadas como forma de tratamento imunoterápico adjuvante para várias neoplasias. Protocolos para geração dessas células têm sido desenvolvidos e o método ideal de produção para uso clínico ainda necessita ser definido. É fundamental a definição de protocolos e reagentes que ofereçam, a partir de células mononucleares do sangue periférico, células dendríticas seguras e funcionais para uso clínico. A suplementação de meios de cultura com soro de origem animal e humano leva á riscos de xenosensibilização e transmissão de doenças. O uso do soro autólogo parece oferecer menos riscos ao paciente, porém a presença de fatores imunossupressores nesse soro poderia interferir na qualidade das DCs produzidas. Vários tipos de meios livres de soro, baseados nas boas práticas de produção - "good manufacture practice" (GMP), têm sido utilizados recentemente e parecem ser uma opção viável. O objetivo desse estudo foi avaliar os resultados da diferenciação, maturação e funcionalidade de DCs de pacientes com LMA, produzidas em meios livres de soro e em meio suplementado com soro autólogo. Concluímos que os meios de cultura livres de soro foram eficientes na produção de DCs para fins imunoterápicos em pacientes com LMA. Em contrapartida, o uso de soro autólogo parece interferir na capacidade funcional das DCs geradas / Abstract: Dendritic cells (DCs) are the main antigen-presenting cells of the immune system, capable of stimulating T lymphocytes to initiate specific immune responses. Vaccines based on DCs have been used as a treatment adjuvant immunotherapy for various malignancies. Protocols for generating these cells have been developed and the optimal method of production for clinical use remains to be defined. There is a great interest in the definition of protocols and reagents providing from peripheral blood mononuclear cells, functional and safe dendritic cells for clinical use. Supplementation of culture media with serum from animal and human leads to reactions due the animal proteins and transmission of disease. The use of autologous serum seems to offer less risk to the patient, but the presence of immunosuppressive factors may affect the quality of the DCs produced. Several types of serum-free media, based on "good manufacture practice" (GMP), have been used recently and seem to be a viable option. The aim of this study was to evaluate the results of the differentiation, maturation and function of DCs from AML patients, generated in serum-free media and media supplemented with autologous serum. We concluded that the serum-free media were efficient in the production of DCs for immunotherapy in AML patients. However, the use of autologous serum appears to interfere with the functional capacity of generated DCs / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestra em Fisiopatologia Médica

Nesta tese, foi apresentado um estudo sistemático para o desenvolvimento de uma nova formulação do veneno de abelha (BV) encapsulado em microesferas (MS) de PLGA para VIT. Estudou-se a estabilidade das proteínas do BV durante o processo de microencapsulação. Houve uma correlação entre as diferentes soluções salinas e as alterações na solubilidade e nas conformações estruturais do BV. Foram preparadas formulações BV-MS-PLGA estáveis com atividade biológica preservada. As MS tiveram eficiência de encapsulação entre 49-75% e perfis trifásicos de liberação do BV. Houve mudanças estruturais no BV liberado, mas seu reconhecimento imunológico foi mantido. Ensaios ex vivo e in vivo mostraram que as formulações evitaram as reações inflamatórias causadas pelo contato direto do BV com o organismo, aumentando a fagocitose das partículas com BV, o entregando diretamente ao meio intracelular. As formulações BV-MS-PLGAs estimularam a produção de anticorpos anti-BV equivalentes à VIT tradicional com a redução do número de injeções necessárias para se produzir o mesmo efeito imunológico. Portanto, as MS-PLGA contendo BV são seguras e eficazmente superiores à VIT tradicional. / In this thesis, it was presented a systematic study to develop a new formulation of bee venom (BV) encapsulated within PLGA-MS for VIT. It was studied the stability of BV proteins during the microencapsulation process. There was a correlation between the different salt solutions and changes in the solubility and structural conformations of BV. It was prepared stable BV-PLGA-MS formulations with preserved biological activity. The MS showed encapsulation efficiencies between 49-75%, and a triphasic release profiles of BV. Although structural changes were observed in the released BV, its immunological recognition was maintained. Ex vivo and in vivo assays showed that formulations avoided the inflammatory reactions caused by direct contact of BV and organism leading to increases in phagocytosis rate of particles containing BV, providing its delivery directly to the intracellular environment. These formulations could stimulate the production of anti-BV antibodies comparable to traditional VIT with reduced number of injections needed to produce the same immunological effect. Therefore, PLGA-MS containing BV are safe and effectively superior to the VIT traditional.

O câncer cervical é considerado um grande problema de saúde pública e um dos maiores causadores de mortes relacionadas a tumores em mulheres. O principal objetivo desta tese foi aumentar a eficácia antitumoral terapêutica da proteína gDE7 por meio da associação de adjuvantes vacinais em formulações testadas em condições experimentais com a linhagem celular tumoral TC-1. A proteína gDE7 foi produzida a partir de uma linhagem de E. coli e associada a diferentes adjuvantes. A proteína gDE7 coadministrada ao poly(I:C) conferiu proteção antitumoral completa aos camundongos previamente desafiados e induziu ativação de linfócitos T CD8+ E7-específicos polifuncionais, citotóxicos e de fenótipo de memória efetora/efetor. Foi demonstrado que a proteína gDE7 ativa de forma específica a subpopulação de células dendríticas especializada na apresentação cruzada de antígenos para linfócitos T CD8+, tanto em camundongos como em seres humanos. Esses resultados abrem perspectivas para o emprego da proteína gD como plataforma vacinal para o controle de tumores induzidos pelo HPV-16. / Cervical cancer is considered a major public health problem and one of the leading causes of cancer death in women. The main goal of this thesis was the improvement of a therapeutic antitumor vaccine based on gDE7 protein in formulations admixed with adjuvants under experimental conditions with the tumor cell line TC-1. The gDE7 protein was expressed and purified from E. coli, and then tested in combination with different vaccine adjuvants. The gDE7 protein admixed with poly(I:C) conferred complete therapeutic antitumor protection to mice previously challenged with TC-1 cells and induced polyfunctional, cytotoxic E7-specific CD8+ T cells with effector/effector memory phenotype. It was also demonstrated that the gDE7 protein activated a specialized dendritic cell subset involved in specific antigen cross-presentation to CD8+ T cells, both in mice and humans. These results open perspectives for the use of the gD protein use as a vaccine platform for the control of HPV-16-induced tumors.

Les modèles animaux ont largement contribué à notre compréhension de l’immunologie humaine et des mécanismes pathologiques associés au développement des maladies. Cependant, les modèles murins ne permettent pas de reproduire toute la complexité des pathologies humaines. Les souris à système immunitaire humain (HIS), par leur capacité à récapituler l’hématopoïèse humaine et à être infectées par des pathogènes humains, constituent une solution de choix pour combler ce fossé inter-espèce. Après greffe de cellules souches hématopoïétiques humaines, des souris hôtes sévèrement immunodéprimées permettent un haut niveau de développement du système hémato-lymphoïde humain tout au long de leur vie. Cependant, certains types cellulaires, comme les cellules lymphoïdes innées, ne parviennent pas à se différencier et à fonctionner normalement dans les modèles murins HIS actuels. Ici, nous décrivons le développement d’un modèle souris HIS original, nommé BRGSF, montrant une amélioration de la maturation, de la fonction et de l’homéostasie des cellules natural killer (NK) humaines et des autres ILCs. De plus, en récapitulant les différentes étapes du développement des ILCs humaines, ce modèle souris BRGSF nous a permis d’identifier pour la première fois un précurseur d’ILC (ILCP) présent à la fois dans notre modèle HIS ainsi que dans le sang périphérique et plusieurs organes lymphoïdes et non-lymphoïdes humains. Cette population circulante d’ILCPs pourrait constituer un substrat pour la production d’ILCs matures dans les tissus périphériques en réponse à des stress environnementaux, inflammatoires et/ou infectieux. Dans une seconde partie de ce travail de thèse, nous avons utilisé ces souris BRGS afin de tester l’efficacité de deux immunothérapies reposant sur les lymphocytes innés pour le traitement d’un carcinome colorectal exprimant EGFR et muté pour KRAS. La première approche a consisté en la co-administration des cellules NK dérivées de sang de cordon ombilical et d'anticorps monoclonal cetuximab afin de promouvoir le mécanisme de cytotoxicité cellulaire dépendante des anticorps (ADCC) contre la tumeur. La seconde stratégie a reposé sur l’injection de nanobodies VHH combinant l’inhibition de l’EGFR et l’activation spécifique du récepteur Vγ9Vδ2 des cellules T effectrices. Les résultats de cette étude soulignent l’importance des modèles murins HIS pour la compréhension du développement des lymphocytes innés humains et pour mieux les mettre à profit dans les thérapies anti-tumeurs / Animal models have extensively contributed to our understanding of human immunobiology and to uncover the underlying pathological mechanisms occurring in the development of the disease. However, mouse models do not always reproduce the genetic complexity inherent in human disease conditions. Human immune system (HIS) mouse models that are susceptible to human pathogens and can recapitulate human hematopoiesis provide one means to bridge the interspecies gap. Severely immunodeficient host mice support life-long, high level human hematolymphoid development after engraftment with human hematopoietic stem cells (HSC). However, the differentiation and function of some blood cell types, including innate lymphoid cells (ILCs), is poorly characterized in current HIS mice. Here we describe the development of a novel HIS mouse model, named BRGSF, which demonstrate enhanced maturation, function and homeostasis of human natural killer (NK) cells and other ILCs. Furthermore, the BRGSF-based HIS mouse model recapitulated the developmental stages of human ILCs. We could identify for the first time an ILC precursor (ILCP) population that is present both in HIS mice and in human peripheral blood as well as in several lymphoid and non-lymphoid human tissues. This circulating human ILCP population may provide a substrate to generate mature ILCs in tissues in response to environmental stressors, inflammation and infection. In a second part of the thesis we used BRGS immunodeficient mice to assess two innate lymphocyte-based immunotherapeutic approaches for treating EGFR-expressing KRAS-mutated colorectal carcinoma in vivo. The first model used a combination of umbilical cord blood (UCB)-derived NK cells and the monoclonal antibody cetuximab to promote antibody dependent cell cytotoxicity (ADCC) against the tumors. In a second model, we evaluated the therapeutic suitability of novel bispecific VHH constructs that combine inhibition of the EGFR with the target-specific activation of effector Vγ9Vδ2-T cells. These studies highlight the utility for HIS-based mouse models to understand human innate lymphocyte development and to harness these potent effectors for anti-tumor therapies.

The development of checkpoint immunotherapy has been a paradigm shift in the treatment of cancer, leading to dramatic improvement in treatment outcomes across a broad range of tumor types. Nevertheless, our current understanding of the tumor immune microenvironment and mediators of resistance to therapy are limited. The recent development of high-throughput single-cell RNA-Sequencing (scRNA-Seq) technology has opened up an unprecedented window into the transcriptional states of distinct tumor-infiltrating immune and stromal cells. However, even this technology has its biological limitations, with very high levels of data dropout induced by low total mRNA molecules and capture efficiency. This thesis explores the application of a transcriptional regulatory protein activity inference approach to single-cell data in order to resolve gene dropout and more deeply characterize upstream drivers of cell state within the micro-environment of several distinct tumor types. To this end, algorithms for inference of protein activity, drug sensitivity, and cell-cell interaction have been adapted to scRNA-Seq data, along with an approach for querying enrichment of single-cell-derived population marker gene sets patient-by-patient in larger bulk-RNA-Seq cohorts. By applying these tools systematically, we have identified distinct cellular sub-populations associated with clinical outcome in different tumor types, including a novel population of C1Q+/TREM2+/APOE+ macrophages associated with post-surgical tumor recurrence in clear cell renal carcinoma, a sub-population of fibroblasts associated with improved response to immunotherapy in head and neck squamous cell carcinoma, tumor cell subpopulations with distinct inferred drug sensitivities in cholangiocarcinoma and prostate cancer, as well as tumor-specific regulatory T-cells (Tregs), active as a mechanism of immunotherapy resistance across a range of tumor types. In ongoing clinical trials from both primary and metastatic prostate cancer as well as clear cell renal carcinoma, we are able to assess which of these populations are enriched in non-responders to checkpoint immunotherapy. The proteomic master regulators of each of these single-cell types have direct utility as potential biomarkers for treatment response, but they may also be therapeutically modulated as novel targets for combination immunotherapy, potentially improving treatment response rates and treatment outcomes in future clinical trials. Finally, this thesis also presents a discovery-to-validation platform to accelerate micro-environment-directed drug repurposing in the context of immunotherapy resistance and rapid CRISPRko validation of novel therapeutic targets. This platform has been developed specifically to validate newly identified master regulators of tumor-specific immunosuppressive regulatory T-cells (Tregs), resulting in discovery of low-dose gemcitabine as a tumor-specific Treg-modulating drug synergistic with anti-PD1 checkpoint immunotherapy and TRPS1 as a proteomic master regulator with clinically significant effect on tumor Treg-infiltrating and tumor growth rate. However, the platform itself may be readily extended in future work to prioritize agents against immunosuppressive macrophage and fibroblast populations for clinical development and trials. As we have discovered, different cancers have different populations of cells driving therapy response and resistance. Taken together, the analytical and validation tools presented in this thesis represent an opportunity to tailor future immuno-therapies at the single-cell level to particular tumor types and to individual patients.

L'immunothérapie basée sur le blocage des points de contrôle immunitaire (ICBs) est un traitement prometteur pour les patients atteints de mélanome ; cependant, seule une petite sous-population en tire un bénéfice à long terme. Un des défis pour améliorer l'efficacité et étendre le bénéfice des ICBs aux patients non répondeurs est de concevoir des approches innovantes permettant de transformer les tumeurs dites "froides ou désertes pour les cellules immunitaire" en tumeurs dites "chaudes ou infiltrées par les cellules immunitaires" qui sont éligibles aux ICBs. Nous avons étudié l'impact du ciblage du gène de l'autophagie Beclin1 sur le paysage immunitaire des tumeurs de mélanome B16-F10. Nos résultats ont démonté que ce ciblage inhibait significativement la croissance tumorale B16-F10 et augmentait l'infiltration des leucocytes CD45+. Le phénotypage immunitaire a révélé une augmentation de l'infiltration de cellules NK (Natural Killer) actives, de macrophages inflammatoires et résidents de type 1, de cellules dendritiques et de lymphocytes T CD8+ actifs. L’inhibition de la croissance tumorale Becn1- n'était plus observée par la déplétion des CD8+ de l'hôte, soulignant ainsi leur rôle dans le contrôle du développement de ces tumeurs. Nos résultats ont démontré que La régulation du paysage immunitaire des tumeurs Becn1- était associée à une modulation du réseau de cytokines/chémokines dans le microenvironnement tumoral (TME). Ainsi, les tumeurs Becn1- présentaient une signature de cytokines inflammatoires (comprenant CCL5, CXCL10 et IFNg) qui pourrait être responsable de l'établissement de microenvironnement inflammatoire permissif aux cellules CD8. Nous avons révélé que la surexpression de l'IFNg dans le TME des tumeurs Becn1- était responsable de l'induction de PD-L1 sur les cellules tumorales par la voie d'activation JAK/STATs. En conclusion, cette étude met en évidence Beclin1 comme une cible majeure, capable d'induire l'infiltration des cellules effectrices immunitaires dans les mélanomes en induisant une signature inflammatoire. Elle fournit également la preuve de concept pour combiner des inhibiteurs d'autophagie avec les ICBs comme une approche de pointe pour améliorer leur efficacité. / Immune Checkpoint Blockades (ICBs)-based immunotherapy has emerged as a promising treatment for melanoma patients; however only a small subset of patients reaps a long term benefit. One of the major challenges to enhance the efficacy and extend the benefit of ICBs to non-responder patients is to design innovative approaches allowing the switch of “immune desert cold tumors” to “immune infiltrated hot tumors" which are eligible for ICB-based therapies. Here, we investigated the impact of targeting the early autophagy gene Beclin1 on the immune landscape of B16-F10 melanoma tumors. We found that targeting Beclin1 (Becn1-) significantly inhibited B16-F10 tumor growth and increased the infiltration of CD45+ leukocytes into the tumor bed. Immune phenotyping revealed an increased infiltration of active Natural Killer (NK) cells, inflammatory and resident type 1 macrophages, dendritic cells, and active CD8+ T lymphocytes. The inhibition of Becn1- tumor growth was no longer observed by depleting host CD8+ T cells, thus highlighting their major role in the control of Becn1- B16-F10 tumor development. We showed that Beclin1-dependent regulation of the immune landscape was associated with profound modulation of the cytokine/chemokine network in the tumor microenvironment (TME). Importantly, we revealed that Becn1- tumors displayed an inflammatory cytokine signature (comprised, but not restricted to, CCL5, CXCL10 and IFNg) that could be responsible for the switch from cold non T-inflamed to hot T-inflamed tumors. Mechanistically, we reported that the overexpression of IFNg in Becn1- TME was responsible for the induction of Programed Death ligand-1 (PD-L1) on tumor cells through the activation of JAK/STATs pathway. Overall, this study highlights Beclin1 as a valuable target, able to drive immune effectors cells into the melanoma tumors by inducing an inflammatory signature. This study provides the proof of concept for combining drugs inhibiting early autophagy process along with ICBs as a cutting-edge approach to improve their efficacy.

No description available.

Cancer is the second leading cause of death in the Czech Republic. Breast cancer and colorectal cancer have relatively high mortality rate. One of the areas of current clinical research in oncology is the study of prognostic biomarkers, which aims to optimize the decision-making process for a patient. Immune response and processes in the tumor microenvironment have been shown to influence to a large extent the biological nature of the tumor in terms of its aggressiveness and ability to metastasize in the host's body. There are certain tumors that could induce a strong immune response, while others do not. The ability to induce an anti-tumor cell response and to attract specific lymphocyte subpopulations directly into tumor tissue has been shown to be very closely related to the prognosis of cancer patients. There is evidence and correlation of the presence of so-called tumor infiltrating lymphocytes in tumor tissue and overall patient survival. Stratification of cancer patients based on immuno-predictors both in the plasma and directly in the tumor microenvironment makes it possible to identify suitable candidates for rediscovered modern anti-tumor immunotherapy, which can already be considered a standard therapeutic modality. In our projects, we focused on the identification of biomarkers that...