Tesis por compendio / [ES] La inmunoterapia es un tratamiento prometedor para el cáncer de mama triple negativo (TNBC), pero los pacientes recaen, lo que destaca la necesidad de comprender los mecanismos de resistencia. En esta tesis doctoral hemos descubierto que, en el tumor primario de cáncer de mama, las células tumorales que resisten el ataque de los linfocitos T son quiescentes. Las células cancerosas quiescentes (QCC) forman nichos con baja infiltración inmune. Estas células QCC exhiben mayor capacidad de regenerar tumores 2 y tienen un perfil de expresión génica relacionado con resistencia a quimioterapia y pluripotencia. Adaptamos la secuenciación de ARN unicelular para obtener también una resolución espacial precisa que nos permitiese analizar los infiltrados dentro y fuera del nicho de QCC. Este análisis transcriptómico reveló la inducción de programas relacionados con la hipoxia e identificó células T más agotadas, fibroblastos supresores y células dendríticas disfuncionales dentro de las áreas de QCC. Esto pone de manifiesto los fenotipos diferenciales en las células infiltrantes según su ubicación intratumoral. Fuimos capaces además de identificar la activación HIF1a específicamente en las QCC como el responsable del fenotipo de exclusión y disfuncionalidad inmune. La activación forzada de HIF1a en células tumorales era suficiente para recapitular el fenotipo observado en las áreas con QCC. Por todo esto, hemos demostrado que las QCC constituyen reservorios resistentes a la inmunoterapia al orquestar un medio inmunosupresor hipóxico localizado que bloquea la función de las células dendríticas y por tanto de los linfocitos T. La eliminación de las QCC es la clave que promete contrarrestar la resistencia a la inmunoterapia y prevenir la recurrencia de la enfermedad en el TNBC. / [CA] La immunoteràpia és un tractament prometedor per al càncer de mama triple negatiu (TNBC), però els pacients recauen, fent destacar la necessitat de comprendre els mecanismes de resistència. En aquesta tesi doctoral hem descobert que al tumor primari de càncer de mama, les cèl·lules tumorals que resisteixen l'atac dels limfòcits T són quiescents. Les cèl·lules canceroses quiescents (QCC) formen nínxols amb baixa infiltració immune. Aquestes cèl·lules QCC exhibeixen més capacitat de regenerar tumors i tenen un perfil d'expressió gènica relacionat amb resistència a quimioteràpia i pluripotència. Hem adaptat la sequ¿enciació d'ARN unicel·lular per obtenir també una resolució espacial precisa que ens permetés analitzar els infiltrats dins i fora del nínxol de QCC. Aquesta anàlisi transcriptòmica va revelar la inducció de programes relacionats 3 amb la hipòxia i va identificar cèl·lules T més esgotades, fibroblasts supressors i cèl·lules dendrítiques disfuncionals dins de les àrees de QCC. Això posa de manifest els fenotips diferencials a les cèl·lules infiltrants segons la seva ubicació intratumoral. Vam ser capaços a més d'identificar l'activació de HIF1a específicament a les QCC com a responsable del fenotip d'exclusió i disfuncionalitat immune. L'activació forçada de HIF1a en cèl·lules tumorals era suficient per recapitular el fenotip observat a les àrees amb QCC. Per tot això, hem demostrat que les QCC constitueixen reservoris resistents a la immunoteràpia en orquestrar un micro-ambient immunosupressor hipòxic localitzat que bloqueja la funció de les cèl·lules dendrítiques i per tant dels limfòcits T. L'eliminació de les QCC és la clau que promet contrarestar la resistència a la immunoteràpia i prevenir la recurrència de la malaltia al TNBC. / [EN] Immunotherapy is a promising treatment for Triple-Negative Breast Cancer (TNBC), but many patients relapse or do not respond, highlighting the need to understand mechanisms of resistance. In this doctoral thesis we discovered that in primary breast cancer, tumor cells that resist T cell attack are quiescent. These Quiescent Cancer Cells (QCCs) form clusters with reduced immune infiltration. They also display superior tumorigenic capacity and higher expression of chemotherapy resistance and stemness genes. We adapted single-cell-RNA-sequencing with precise spatial resolution to profile infiltrating cells (stromal and immune cells) inside and outside the QCC niche. This transcriptomic analysis revealed hypoxia-induced programs and identified the presence of more abundant exhausted T-cells, tumor-protective fibroblasts, and dysfunctional dendritic cells inside clusters of QCCs. This uncovered differential phenotypes in infiltrating cells based on their intra-tumor location with respect to QCCs. We were also able to identify HIF1a expression in QCC as the driver of immune exclusion and dysfunction. Forced activation of a HIF1a program in cancer cells recapitulated the immune phenotype observed in the QCCs' niche. Thus, QCCs constitute immunotherapyresistant reservoirs by orchestrating a local immune-suppressive milieu that blocks DC activation impairing T-cell function. Eliminating QCCs holds the promise to counteract immunotherapy resistance and prevent disease recurrence in TNBC. / Baldominos Flores, P. (2024). Study of immune resistant mechanisms in mouse models of breast cancer [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203657 / Compendio

Yes / With an escalating incidence of breast cancer cases all over the world and the deleterious psychological impact that mastectomy has on patients along with several limitations of the currently applied modalities, it's plausible to seek unconventional approaches to encounter such a burgeoning issue. Breast tissue engineering may allow that chance via providing more personalized solutions which are able to regenerate, mimicking natural tissues also facing the witnessed limitations. This review is dedicated to explore the utilization of adipose tissue-derived mesenchymal stem cells for breast tissue regeneration among postmastectomy cases focusing on biomaterials and cellular aspects in terms of harvesting, isolation, differentiation and new tissue formation as well as scaffolds types, properties, material–host interaction and an in vitro breast tissue modeling.

Advances in multiplex fluorescence imaging now allow the measurement of more than 50protein markers in whole tissue sections at single-cell resolution. This promises to reveal tumor biology at an unprecedented level of detail, both in undisturbed growth and in therapy. However, to quantitatively analyze these images, the images must be broken down into the basic units of tumor biology: single cells and their types. In this study, we applied a graph-based unsupervised clustering method, Leiden, to perform cell type identification in highly multiplexed fluorescence images, and based on the annotated images, we ran the tumor microenvironment niches analysis in order to resolve the recurring patterns of tumor microarchitecture. This thesis first introduces several potentially feasible clustering methods selected based on the structure of the datasets studied. The performance and stability of these clustering methods were compared. The project involved benchmarking different dimensionality reduction and clustering techniques on manually annotated reference datasets and healthy tissue with known cellular composition. It was ultimately determined that appropriate data transformations combined with Leiden clustering methods with proper parameters could automatically identify cells in a way coherent with established marker profiles. The results imply that Leiden clustering can also identify clusters of cells with novel marker combinations. Careful examination of the multiplex images shows that the markers are indeed found in the tumor, leading to new hypotheses regarding tumor biology. Tumor microenvironment niches analysis found several archetypal niches with specific cellular composition, indicating active accumulation of immune cells after radiotherapy, and the less vascularized feature of rebound glioblastomas after treatment. We hope to further validate our analysis to provide new insights into the pathological process of glioblastoma. In future research, the analysis pipeline is planned to be improved so that it can be robustly used to analyze the growing data of multiplexed tumor images, both in mouse cancer models or patient samples.

Early detection of breast cancer is critical for increasing survival rates. However, currently available screening strategies provide ambiguous results, leaving invasive tissue biopsy procedures necessary for definitive diagnosis. Considerable efforts have investigated using nipple aspirate fluid (NAF), a liquid biopsy rich in proteins representative of the breast microenvironment, as a non-invasive source of early detection biomarkers. However, by using traditional two-dimensional discovery proteomic approaches, many technical challenges of using NAF have limited analysis of large sample sizing: such as low expressed volume (<10µL) or insufficient analytical material (<200µg protein). Following non-invasive collection by manual massage, we developed a one-dimensional sample preparation workflow that reduced sample handling steps, minimised sample losses and increased sample throughput to 96-samples by using a PVDF-membrane based system, which was ideally suited to the NAF proteome. Samples were prepared within a single working day, and results correlated significantly with conventional in-solution protocols. ​Proteins typically associated with the dysregulation of innate immune response and haemostatic pathways had a significantly altered proteome profile in response to breast cancer. Overall, our new workflow will allow future studies to take a more high-throughput approach, revealing biomarkers for breast cancer early detection, and providing a real impact.

High-frequency irreversible electroporation (H-FIRE) is a novel tumor ablation therapeutic that applies bipolar, high-frequency pulsed electric fields to tumors, triggering the formation of irreversible membrane pores and to induce tumor cell death. H-FIRE has demonstrated pre-clinical and clinical utility as a therapeutic for brain tumors, including gliomas. H-FIRE has been shown to induce precise, uniform ablation within the tumor tissue, as well as local changes to the tumor microenvironment and systemic changes to the immune landscape. Namely, disruption of the peritumoral blood-brain barrier (BBB) following H-FIRE ablation of brain tumors, and infiltration and activation of the innate immune system are clinically observed following H-FIRE tumor ablation. Such effects persist long after death of the treated tumor, and therefore an understanding of the mechanisms underlying these local and systemic changes are critical for the development of H-FIRE. Using in vitro models of glioma and lung carcinoma-derived brain metastases, we investigate the interactions between cancer cells that have been ablated with H-FIRE and the brain tumor microenvironments. Specifically, we demonstrate that H-FIRE-treated cancer cells can recover treatment-induced damage and proliferative capacity after treatment with specific electric field doses, while higher doses inhibit such recovery. This suggests that after H-FIRE ablation of brain tumors, tumor cells can still secrete factors to trigger alterations in their local and systemic environments. We then specifically investigate the role of tumor-derived extracellular vesicles (TDEVs) in mediating these changes, namely pBBB disruption and changes in innate immunity. We find that, following H-FIRE ablation of brain cancer cells, treated cells immediately release TDEVs that disrupt the blood-brain barrier (BBB) endothelium in vitro, and are uniquely internalized by cerebral endothelial cells in vitro, despite reduced release of TDEVs after H-FIRE. We further demonstrate that H-FIRE significantly alters the proteomic payloads of TDEVs. When TDEVs released by sham- and H-FIRE-treated glioma cells are delivered to healthy rats, only TDEVs released by H-FIRE-ablated cells are retained in the brain, suggesting changes to TDEV organotropism after H-FIRE ablation of glioma. Further, once retained in the brain, these post-H-FIRE TDEVs cluster near cerebral endothelial cells, similarly to in vitro. Although the TDEVs released by H-FIRE ablated glioma cells do not disrupt the BBB in vivo, Iba1+ cells were increased in the brains of rats that received TDEVs released by H-FIRE-ablated glioma cells. Together, these data suggest that H-FIRE immediately alters the secretion and proteome of TDEVs, facilitating changes in TDEV organotropism and cellular tropism and immune cell recruitment to the tumor microenvironment. Together, this research indicates mechanisms by which tumor cells continue to modulate their local and systemic environments via the action of TDEVs, which is critical information for the continued development of H-FIRE and its optimization with adjuvant therapeutics for the treatment of malignant brain tumors. / Doctor of Philosophy / All cells secrete extracellular vesicles, which are packets of information that function as communication highways between cells. In cancer, tumor-derived extracellular vesicles (TDEVs) reprogram local and distant cells to support tumor growth. However, they have also been shown to change local and systemic functions, such as blood vessel function and immune response, after tumors are treated with therapeutics. Therefore, a full understanding of the role of TDEVs in how tumors communicate with the body after cancer treatment is necessary when developing new anti-cancer therapeutics. Here, in developing high-frequency irreversible electroporation (H-FIRE), a novel anti-tumor therapeutic for the treatment of malignant brain tumors, we explore how TDEVs released by brain cancer cells treated with H-FIRE interact with various cell types and structures in the body, and how these interactions may affect the response to treatment. Using a glioma model of primary brain cancer, and a lung carcinoma model of brain metastases, we first explore how tumor cells may be able to recover from damage after treatment with H-FIRE. We discover that brain cancer cells treated with specific doses of H-FIRE recover cell damage and continue to proliferate, but cells treated with higher doses of H-FIRE cannot recover these functions. The fact that tumor cells may be able to recover after H-FIRE suggests that cancer cells may still secrete factors, such as TDEVs, that interact with cells in the microenvironment after tumor treatment. We investigated the role of TDEVs released by brain cancer cells treated with H-FIRE to determine whether they cause changes in surrounding cells and structures in the brain cancer microenvironment. We determined that brain cancer cells treated with H-FIRE release TDEVs that carry proteins different from those carried by TDEVs routinely released by untreated cells. We further found that these TDEVs disrupt the blood-brain barrier (BBB) endothelium in vitro, and are uniquely internalized by cells of the endothelium. When these TDEVs were administered to the brains of healthy rats, they were retained in the brain, clustered near the endothelium, and recruited immune cells from circulation into the brain. Conversely, TDEVs that were routinely released from the brain cancer cells, in the absence of H-FIRE treatment, exhibited none of these functions. Taken together, these results show that H-FIRE changes TDEVs in numerous ways: after H-FIRE, the TDEVs may gravitate toward particular organs and cell types, and recruit immune cells. All of these changes can impact the overall therapeutic response after H-FIRE, and may also be specifically optimized and targeted with additional therapeutics to make H-FIRE more effective for brain cancer.

Le microenvironnement tumoral et les cellules et molécules signal (cytokines et chimiokines) qu’ils contiennent sont reconnus comme jouant un rôle prépondérant dans la progression des tumeurs. Il devient donc nécessaire d’étudier la relation entre les molécules signal, les cellules infiltrantes et les cellules tumorales. Le TGF-β est une puissante cytokine immunosuppressive et suppressive de la croissance cellulaire, dont le rôle dans la formation du microenvironnement tumoral leucémique est mal connu. Dans cette étude, nous avons étudié le modèle injectable de leucémie lymphoïde T EL4 (cellules tumorales produisant du TGF-β) de souche C57BL/6. Nous avons caractérisé l’infiltration de cellules myéloïdes et lymphoïdes au niveau des tumeurs par cytométrie en flux et par microscopie à fluorescence. L’analyse des cellules infiltrant les tumeurs EL4 nous a permis de montrer la forte présence de lymphocytes T et de cellules myéloïdes CD11b+. Nous avons donc poursuivi l’étude afin de mieux caractériser ces cellules. Nous avons montré que ces cellules se retrouvent en périphérie de la tumeur et en périphérie des vaisseaux sanguins de la tumeur. Ces cellules ont des phénotypes nous laissant croire qu’elles appartiennent à la famille des cellules dite myéloïdes suppressives. Ces cellules ont de forts niveaux de transcrits de VEGF et de MMP9 au niveau de la tumeur ainsi qu’au niveau systémique, mais ne semblent pas avoir une forte capacité inhibitrice in vitro. Afin de déterminer si la production tumorale de TGF-β influe le recrutement de ces cellules, nous avons transformé des cellules EL4 à l’aide d’un shRNA afin de diminuer la production de TGF-β (shRNA-TGF-β) et, comparé l’infiltration myéloïde et lymphoïde de tumeurs formées avec des cellules EL4 contrôles (shRNA-Luc). Une diminution de 50% dans les niveaux de transcrits de TGF-β n’affecte pas la croissance tumorale mais semble diminuer l’infiltration par des cellules myéloïdes. La présente étude nous a permis de mieux comprendre le modèle de leucémie EL4 et le rôle des populations cellulaires myéloïdes dans le microenvironnement tumoral leucémique. La diminution du TGF-β produit par les cellules tumorales réduit l’infiltration de ces populations myéloïdes dans la tumeur EL4. Le rôle précis de ces cellules est encore à déterminer. Ces résultats sont en accord avec le fait qu’une thérapie anti-TGF-β n’est pas suffisante pour contrer la progression tumorale, mais pourrait influer sur le résultat post-chimiothérapie et l’immunothérapie en altérant la composition du microenvironnement. / The cells and signal molecules (cytokines and chemokines) making up the tumoral microenvironnement are known to play an essential role in tumor progression. It seems to be necessary to study the relationship between infiltrating cells, tumor cells and signal molecules. TGF-β is a potent immunosuppressive and growth suppressive cytokine whose role in the formation of the leukemia microenvironnement remains unclear. In this study, we investigated the injectable T lymphocyte leukemia EL4 model (tumor cells producing TGF-β) of C57BL/6 strain. We characterised the myeloid and lymphoid infiltration in EL4 tumors using flow cytometry and fluorescence microscopy. Our analysis of EL4 tumor infiltrating cells showed a high concentration of T lymphocytes and myeloid cells CD11b+. We have undertaken our study to better characterize these cells. We showed that these cells are present at the periphery of the tumor and are surrounding blood vessels in the tumor. These cells have phenotypes leading us to believe that they belong to the family of so-called myeloid suppressor cells. They have high levels of transcripts of VEGF and MMP9 in the tumor and the systemic level, but do not seem to have a strong inhibitory capacity in vitro. To determine whether the tumor production of TGF-β affects the recruitment of these cells, we transformed EL4 cells using a shRNA to reduce the production of TGF-β (TGF-β shRNA ) and compared the myeloid and lymphoid infiltration of tumors formed with EL4 cell controls ( shRNA-Luc ) . A 50% decrease in transcript levels of TGF-β does not affect tumor growth but appears to decrease infiltration by myeloid cells. This study allowed us to better understand the pattern of EL4 leukemia and the role of myeloid leukemia cell populations in the tumor microenvironment. The decrease of TGF-β produced by tumor cells reduces the infiltration of these myeloid populations within the EL4 tumor. The precise role of these cells still needs to be determined. These results are in agreement with the fact that anti-TGF-β therapy is not sufficient to counteract tumor progression, but may affect the post-chemotherapy and immunotherapy results by altering the composition of the microenvironment.

Cancer diseases represent second most frequent cause of death after cardiovascular diseases in Europe. Nowadays used medical treatments like chemotherapy and radiotherapy are nonspecific and cause huge side effects. Various systems to deliver therapy directly inside the tumour microenvironment and reduce side effects are under development. Protein nanoparticles seem to be very promising strategy to achieve that goal. Our group in cooperation with CNR in Rome tested nanoparticles based on heavy chain of human ferritine. These constructs, modified to expose the tumor targeting molecule, were able to be specifically internalised by B16F10 melanoma cells in vitro. They also specifically target and localise at the sites of primary melanoma and lung metastases of different size in mouse in vivo model. These nanoparticles can carry either therapeutic or diagnostic molecules. Thus they represent a suitable candidate for further studies for potential use in clinical praxis as a diagnostic and/or therapeutic agents (theranostics). Powered by TCPDF (www.tcpdf.org)

Hodgkin lymphoma (HL) is a heterogeneous condition varying from engaging one single lymph node site to a widespread condition. The prognosis with contemporary treatment is excellent for the vast majority. However, the treatment might cause severe late adverse effects in a proportion of the affected individuals. We evaluated all children and adolescents diagnosed in Sweden and registered in the Swedish Childhood Cancer Register over a period of 25 years. The incidence has been stable and the overall survival (OS) is very good, comparable to the best results in the world. Approximately ten percent encountered a relapse, but even after relapse the chances of survival were good. During the study period there were no detectable changes in survival estimates. The use of radiotherapy has decreased. Epstein Barr virus (EBV) and numbers of eosinophils, mast cells and macrophages in the tumors were investigated in 98 cases. Young children were more likely to express EBV. In patients with advanced disease the mast cell and macrophage counts were higher and they also had more affected laboratory parameters. Patients with Nodular Lymphocyte Predominant Hodgkin Lymphoma did not express EBV in the tumor, had significantly lower numbers of eosinophils, mast cells and macrophages and less affected laboratory parameters compared to classical HL. Outcome and clinical presentation were investigated in a cohort of children, adolescents and young adults in Sweden and Denmark and treatment in pediatric and adult departments was compared. OS and event-free survival (EFS) did not differ between the three age groups nor between pediatric and adult treatment. However, the Danish pediatric patients had lower EFS, which corresponded to less use of radiotherapy. Adolescents and young adults shared similar characteristics, while children presented differently with less advanced disease and male preponderance. Hospitalization rates and outpatient visits after end of treatment were evaluated to see whether the excess need of resources described in the literature is evenly distributed among the survivors or whether it is limited to a smaller group. Most of the patients had a low burden of health care use and the relapsing patients were the main drivers of the excess need.

O melanoma é o mais agressivo tumor da pele, cuja resistência aos tratamentos quimioterápicos tem promovido a crescente utilização de imunoquimioterapia, como é o caso da utilização de agonistas dos receptores Toll-Like (TLRs). Nesse contexto, os compostos abreviados por P-MAPA e seu sintético estrutural MRB-CFI-1 com reconhecidas propriedades antitumorais e imunológicas, são fortes candidatos na terapia e prevenção desse tipo de câncer. Esse estudo visa determinar o potencial anticâncer do P-MAPA e de MRB-CFI-1 contra o melanoma murino em consequência ao padrão de resposta microambiental semelhante ao de morte imunogênica, em regimes de tratamento terapêutico ou vacinal, na vigência de quimioterapia com cisplatina e/ou em associação com antígenos de células tumorais totais. Após avaliação In vivo do crescimento de tumores B16F10 implantados em modelos murinos selvagem e nocaute para o gene Myd88, na vigência ou não do tratamento com cisplatina e/ou P-MAPA, nossos resultados mostraram que o P-MAPA apresentou atividade pró-tumoral e antagonizou a ação da cisplatina em inibir o crescimento dos tumores, de forma dependente de Myd88. Além disso, através de análises qualitativa e quantitativa pelo software ImageJ em fotomicrografias de secções tumorais coradas histologicamente, observamos que o P-MAPA promoveu mudanças microambientais nos tumores que podem impactar negativamente em seu desempenho. Como monoterapia em esquema de vacinação com lisado tumoral total em combinação com quimioterapia, o P-MAPA em dose baixa falhou em suprimir o crescimento de tumores B16F10, mas o seu sintético MRB-CFI-1 foi capaz de prevenir o crescimento desse tipo de melanoma num regime de vacinação profilática. Apesar do sucesso terapêutico desse imunomodulador em diversos modelos de câncer e de doenças infecciosas, o P-MAPA não foi eficaz em produz respostas microambientais contra o melanoma murino, dados esses que limitam a aplicabilidade clínica do composto. De outro modo, o composto fosfato inorgânico MRB-CFI-1 foi protetivo em retardar o aparecimento desse tipo de doença. Assim, o presente estudo foi importante por ampliar o entendimento funcional do P-MAPA numa abordagem imunoquimioterápica em modelos biológicos de tumores de melanoma, e representa uma importante mudança na utilização de constituintes individuais similares ao P-MAPA que sejam mais eficazes, de fácil obtenção, e de produção controlada e garantida / Melanoma is the most aggressive skin cancer, whose resistance to chemotherapeutic treatments has promoted the increasing use of immunochemotherapy, as is the case for the use of Toll-Like receptor agonists (TLRs). In this context, the compounds abbreviated by P-MAPA and its structural synthetic MRB-CFI-1 with recognized antitumor and immunological properties are strong candidates in the therapy and prevention of this type of cancer. This study aims to determine the anti-cancer potential of P-MAPA and MRB-CFI-1 against murine melanoma as a consequence of the microenvironmental response pattern similar to that of immunogenic death in therapeutic or vaccine treatment regimens when using chemotherapy with cisplatin alone or in combination with whole tumor cell antigens. After In vivo evaluation of the growth of B16F10 tumors implanted in wild-type and Myd88 gene knockout mice, under treatment or not with cisplatin and / or P-MAPA, our results showed that P-MAPA showed pro-tumor activity and antagonized the action of cisplatin in inhibiting the growth of tumors in a Myd88-dependent manner. In addition, using qualitative and quantitative analysis by ImageJ software in histological images of tumor sections, we observed that P-MAPA promoted microenvironmental changes in tumors that may negatively impact its performance. As monotherapy in vaccination schedule with total tumor lysate in combination with chemotherapy, low dose P-MAPA failed to suppress the growth of B16F10 tumors, but its synthetic MRB-CFI-1 was able to prevent the growth of this type of melanoma in prophylactic vaccination regimen. Despite the therapeutic success of this immunomodulator in various cancer models and infectious diseases, P-MAPA has not been effective in producing microenvironmental responses against murine melanoma, data that limit the clinical applicability of the compound. Otherwise, the inorganic phosphate compound MRB-CFI-1 was protective in delaying the onset of this type of disease. Thus, the present study was important because it broadened the functional understanding of P-MAPA in an immuno-chemotherapeutic approach in biological models of melanoma tumors and represents an important change in the use of individual constituents similar to P-MAPA that are more efficient, easily obtainable, and controlled and guaranteed production

Introdução: Indivíduos sobreviventes à sepse apresentam maior mortalidade à longo prazo e maior risco de apresentar infecções oportunistas. Existem evidências clínicas e experimentais de desregulação imune no estado pós-sepse. Essas alterações apresentam semelhança com aquelas encontradas no microambiente tumoral, estando relacionadas à imunossupressão. O presente trabalho avaliou o papel de macrófagos associados ao tumor (TAM) em modelo de progressão tumoral em camundongos sobreviventes à sepse. Materiais e Métodos: Camundongos C57/BL6 foram submetidos a ligadura e punção cecal (CLP) e tratados com ertapenem (20 mg/kg, i.p., 6 horas após CLP e 12/12 h por 3 dias). Os animais sobreviventes de sepse eram inoculados com células de melanoma B16-F10 (30 mil, s.c., 15 dias após a CLP). Animais naïve foram usados como controle. Foram avaliadas a progressão tumoral, sobrevida e formação de metástases espontâneas à distância. No D+14, animais foram sacrificados para mensuração do acúmulo de TAM por citometria de fluxo (CD45+F4/80+CD206+) e de citocinas no soro e no tumor por ELISA (IFN-?, IL-10, TNF-?, TGF-?, CCL2, CXCL12). Macrófagos derivados de medula óssea de animais pós-CLP ou naïve foram coinoculados com células B16 para avaliação de progressão tumoral e sobrevida. TAM de animais naïve ou pós-CLP foram isolados através de gradiente de Percoll seguido de adesão seletiva e o RNA foi isolado para análise diferencial de expressão gênica por microarray. Para avaliação da participação da via CXCL12/CXCR4 foi realizada sua inibição com o AMD3100, antagonista de CXCR4 (5 mg/kg, i.p., D+10 e D+14). Foi avaliada a progressão tumoral, sobrevida, acúmulo de TAM e proliferação extramedular de TAM no D+14. Resultados: Animais sobreviventes de sepse apresentaram aumento de progressão tumoral (após 15, 30 e 60 dias da CLP), aumento da carga de metástases (após 15 dias da CLP) e redução de sobrevida. Foi detectado o aumento de TAM nos animais pós-CLP, associado a maior marcação de Ki67, em comparação com animais naïve no D+14. Verificamos aumento das concentrações séricas de TGF-?, CXCL12, CCL2 e TNF-?. Camundongos naïve que coinoculados com macrófagos derivados de medula óssea de animais pós-CLP apresentaram aumento de progressão tumoral e redução de sobrevida em comparação com o grupo controle. TAM de animais pós-CLP apresentaram menor expressão de genes relacionados ao MHC-II e genes relacionados à ativação leucocitária. A inibição de CXCL12/CXCR4 preveniu a progressão tumoral induzida por sepse, com menor acúmulo de TAM e menor presença de TAM Ki67+. Conclusões: O estado pós-sepse promove a progressão tumoral de melanoma B16 em camundongos, o qual foi associado a aumento de 12 TAM. A via CXCL12/CXCR4 participa do processo de acúmulo de TAM nesse modelo experimental. / Background: Survivors from sepsis present higher long-term mortality and increased risk of opportunistic infections. There is clinical and experimental evidence for an immunosuppressive immune dysregulation in post-sepsis. These alterations are similar to those found in tumor microenvironment. The present work assessed the role of tumorassociated macrophage (TAM) in a model of tumor progression in sepsis-surviving mice. Materials and Methods: C57/BL6 mice were submitted to cecal ligation and puncture (CLP) and treated with ertapenem (20 mg/kg, ip. - 6 h after CLP and then each 12 h for 3 days). Sepsis surviving mice were inoculated with B16-F10 melanoma cells (30,000, sc., 15 days after CLP). Naïve mice were used as controls. Tumor progression, survival and distant spontaneous metastasis were evaluated. Mice were killed at D+14 for TAM measurement through flow cytometry (CD45+F4/80+CD206+) and for cytokines (IFN-?, IL-10, TNF-?, TGF-?, CCL2, CXCL12) quantification by ELISA. Bone marrow-derived macrophage (BMDM) were isolated and co-inoculated together with B16 melanoma cells for tumor progression and survival evaluation. TAM from naïve or post-sepsis mice were isolated through Percoll gradient (70/30) followed by selective adhesion. The RNA was isolated for gene expression analysis using microarray assay. To evaluate the role of CXCL12/CXCR4, we used the specific antagonist AMD3100 (5 mg/kg, ip., at D+10 and D+14) and assessed tumor progression, survival and TAM accumulation at D+14. Results: Sepsis-surviving mice showed increased tumor progression (15, 30 or 60 days after CLP), higher metastatic burden (15 days after CLP), and less overall survival. TAM were increased in post-sepsis mice at D+14. We found increased serum levels of TGF-?, CXCL12, CCL2 e TNF-?. Naïve mice inoculated with BMDM from post-sepsis and B16 cells showed higher tumoral progression and less survival, when compared to the control group. TAM from post-sepsis showed decreased expression of MHC-II related genes and genes related to leukocyte activation. The inhibition of CXCL12/CXCR4 prevented the post-sepsis-induced tumor progression, with less TAM accumulation and reduced expression of Ki67 in TAM. Conclusions: The post-sepsis state promotes the progression of B16 melanoma in mice, which was associated with an increase in TAM accumulation. CXCL12/CXCR4 mediates TAM accumulation in this experimental model.