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<p>NK cells play a pivotal role in tumor rejection; however, once present in the tumor microenvironment, they are characterized by decreased cytotoxicity and reduced expression of activating receptors. The mechanisms governing the inactivation of NK cells within tumors remain poorly understood. Since tumor associated macrophages (TAMs) are a highly abundant and suppressive cell type within tumors, we hypothesized that they are capable of altering the function of NK cells. Following the co-culture of alternatively activated macrophages (M2) or TAMs with NK cells we observed that the expression of the cytotoxic marker CD27 on NK cells was down-regulated as well as the ability of these cells to kill YAC-1 cells in a killing assay. We have demonstrated that the mechanism by which M2 cells inhibit NK cells is TGF-β dependent. Notably, the developmental stage of NK cells after interaction with TAMs was altered and the NK cells became phenoytpically mature and potentially exhausted (CD27^lowCD11b^high). This prompted our interest in examining the developmental stage of NK cells from polyoma MT antigen (pyMT) transgenic mouse (MMTV-pMT) breast tumors. Interestingly, in contrast to the <em>in vitro</em> results, we have shown that NK cells isolated from pyMT tumors are developmentally immature; however maintain their maturity within the spleen. Their immature phenotype correlates well with their decreased expression of perforin, granzyme, and NKp46. Both our <em>in vitro</em> studies with TAMs and our <em>in vivo</em> developmental studies using the pyMT model demonstrate that NK cells are altered by their surroundings. A better understanding of how NK cells are modified by the tumor microenvironment will help to develop strategies aimed at bolstering immune responses against tumors.</p> / Master of Science (MSc)

<p>Pre-clinical and clinical data strongly support the use of immunotherapies for cancer treatment. Cancer vaccines offer a promising approach, however, the outcomes of clinical vaccine trials have been largely disappointing, prompting a need for further investigation. Using the B16F10 murine melanoma, we have investigated the local events within growing tumors following recombinant adenovirus immunization. In chapter 2, we investigated the ability of a pre-clinical vaccine to elicit only transient tumor growth suppression. We observed that tumors were initially infiltrated by a small number of highly functional tumor-specific CD8+ T cells following vaccination that instigated a rapid adaptive response in the tumor that suppressed local immune activity. In chapter 3, we questioned whether increasing the rate and magnitude of early immune attack would result in more robust tumor attack prior to tumor adaptation. Increasing the rate of tumor-specific CD8+ T cell expansion following vaccination resulted in tumor regression and durable cures in approximately 65% of treated mice. Further analysis revealed that tumor regression correlated with an early burst in immune attack that outpaced tumor adaptation. In chapter 4, we explored whether the same vaccine could be improved when combined with immunomodulatory antibodies. Vaccination combined with anti 4-1BB and anti PD-1 resulted in complete tumor regression and durable cure of >70% of treated animals and was associated with increased local immune activity. Gene expression profiling revealed a unique gene signature associated with the curative treatment, which was also associated with positive outcome in human melanoma patients. The described research sheds new light on mechanisms that limit the efficacy of therapeutic cancer vaccines. Namely, rapid tumor adaptation, triggered by early vaccine-induced CD8+ T cells, acts to suppress the local immune response prior to maximal immune attack. Strategies to overcome these adaptive processes should therefore be considered in future vaccine design.</p> / Doctor of Philosophy (Medical Science)

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

Advanced stage tumors can acquire the ability to divide uncontrollably, invade the surrounding matrix, and circulate through the bloodstream or lymphatic system to distant organs in a process known as metastasis. The brain, which is shielded from the environment by the blood brain barrier, offers an immunocompetent lodging spot for the circulating cancer cells. Therefore, it is a "popular" destination for metastasized cancers which even surpass the incidents of primary brain tumors. It is hypothesized that the disseminated cancer cells engage with the host cells of the perivascular neural niche in a poorly understood crosstalk of molecular factors, that in turn augment the metastatic colonization of cancer cells. A better understanding of this crosstalk is indispensable to apprehending the complexity of the metastasis process, and to facilitating the discovery of biomarkers that predict metastatic potential and improve patient prognosis. The larger goal of this study was to adopt a mass spectrometry-based systems biology approach to investigate the molecular mechanisms and regulatory networks that underlie the complex phenomenon of breast cancer propagation at the brain metastatic site. To achieve this, the study was divided in three sub-projects designed around the following objectives, i.e., (a) to comprehensively characterize the protein landscape of the neural niche or the brain microenvironment comprised of astrocytes, microglia and endothelial cells, (b) to explore the immunological protein networks activated in microglia cells upon stimulation with anti-inflammatory cytokines released by tumor cells in the brain, and (c) to investigate the protein-level changes elicited in HER2+ breast cancer cells when grown under conditions that simulate the brain microenvironment in-vitro. Detailed characterization of the neural niche enabled us to propose molecular mechanisms that allow for the seeding and outgrowth of metastasized cancer cells in the brain. The study further provided novel insights into the signaling networks that regulate the immune functions of the microglia and their role during cancer development. Lastly, an in-depth investigation of breast cancer cells cultured in the presence of neural niche factors revealed potential novel mechanisms of cancer cell dormancy during metastasis. Altogether, large-scale proteomics data generated in this work will help clarify the mechanisms of metastatic cancer development, and will lay the groundwork for future studies that aim at the discovery of novel biomarkers and druggable targets for the treatment of brain metastatic cancers. / Doctor of Philosophy / In the US, the incidence of breast cancer ranks only second to lung cancer, and the primary cause for almost all cancer related deaths is the development of cancer metastasis in patients. The process of metastasis involves cancer cells leaving the primary tumor, entering the bloodstream or the lymphatic system, and spreading to other parts of the body during advanced stages of the disease. The brain is a common metastatic site for cancer cells to form a secondary tumor. Previous research has found that it is the brain cells which support the progression of secondary tumors in the central nervous system by releasing protein molecules that favor the survival and growth of disseminated cancer cells. Brain cells, including endothelial cells (that form the blood vessels), astrocytes (that regulate brain development), and microglia (that are the immune cells of the brain), are the first to respond to metastasized cancer cells. A better understanding of the behavior of these cells and of their signaling molecules which support the process of cancer metastasis can help us discover new drug targets to treat cancer patients. This study had three main objectives, i.e., (a) to profile the protein make-up of brain cells and identify specific proteins that support cancer development in the brain, (b) to investigate the changes in microglial proteins when these cells are exposed to conditions that simulate cancerous growth in the brain, and (c) to explore the proteins that become active in breast cancer cells when they are subjected to conditions that simulate the brain microenvironment. To accomplish this, we used a high-throughput technology called mass spectrometry, that allows for the identification of thousands of proteins in a sample at any given time. Overall, the study provided novel insights into the biological mechanisms of secondary tumor formation, and into the early response of breast cancer cells when they encounter unfamiliar conditions in the brain. The work further supports the discovery of specific proteins that can be targeted in anti-cancer therapies.

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.