<div>Cancer is responsible for the second highest cause of death in the United States, and lung cancer accounts for 13% of new cancer diagnoses, with the highest rate of cancer death at 24%. Almost 85% of these cases represent non-small cell lung cancer (NSCLC), which includes lung adenocarcinoma, the most common NSCLC subtype. Traditional cancer treatments often only temporarily stop the spread of the disease, but immunotherapies, which are becoming a standard of care, are much more promising. Natural killer (NK) cells are powerful effectors of innate immunity, and genetically engineered NK cells as immunotherapies have had encouraging clinical responses in the treatment of various cancers. However, more progress is needed for solid tumor treatment, especially for lung adenocarcinoma. The activation of cancer-associated ectoenzymes, CD39 and CD73 catalyze the phosphorylation of ATP to AMP to produce extracellular adenosine (ADO), which is a highly immunosuppressive mechanism contributing to the pathogenesis of solid tumors. Understanding adenosine effects on NK cells will help develop more robust immunotherapeutic treatments to improve cytotoxicity against solid tumors. Here, we established that tumor microenvironment ADO results in impaired metabolic and anti-tumor functions of cytokine-primed NK cells. Specifically, peripheral blood-derived NK cells stimulated with IL-2, IL-15, or a combination of IL-12 and IL-15 showed suppressed anti-tumor immunity due to ADO. This was observed by the downregulation of activation receptor expression, cytotoxicity inhibition, impairment of metabolic activity, and alterations in gene expression. To target ADO-producing CD73 on cancer cells, we redirected NK cells by fusing CD73 ScFv with intracellular and transmembrane regions of NK cell specific signaling components derived from FCyRIIIa (CD16). Engineered NK cells were shown to be cytotoxic against lung adenocarcinoma <i>in vitro</i> and impede tumor growth in a lung adenocarcinoma mouse model <i>in vivo</i>. Engineered cells also had higher levels of degranulation and cytokine release, as well as more infiltration into tumors and longer survival time in mice. In summary, the microenvironment of solid tumors is highly immunosupressive, and redirecting NK cell function using a NK-specific anti-CD73 targeting construct will help to promote anti-tumor immunity and</div><div>inhibit cancer growth for a potentially powerful new immunotherapy against solid tumors.</div>

Nierenzellkarzinome (NZKs) gelten als stark immunogene Tumore. Dies ist insbesondere auf die Infiltration durch verschiedene Immunzellpopulationen, wie T-Lymphozyten und Natürliche Killer (NK)-Zellen, sowie das klinische Ansprechen auf immuntherapeutische Strategien zurückzuführen. Bisher existieren jedoch nur sehr wenige Studien zur Rolle von humanen nativen dendritischen Zellen (DCs) in NZK-Geweben und über die Tumor-vermittelte Modulation dieser DCs. DCs nehmen als professionelle Antigen-präsentierende Zellen eine zentrale Schlüsselrolle bei der Induktion und Aufrechterhaltung der angeborenen sowie adaptiven Immunantwort ein. Daher wurde im Rahmen dieser Arbeit erstmals der Effekt von klarzelligen NZKs auf den Phänotyp sowie die immunmodulatorischen Fähigkeiten von 6-sulfo LacNAc+ (slan)DCs evaluiert. SlanDCs, welche eine große Subpopulation humaner Blut-DCs darstellen, sind neben der Sekretion großer Mengen proinflammatorischer Zytokine dazu befähigt, Tumorzellen direkt zu lysieren. Des Weiteren sind slanDCs in der Lage, die antitumoralen Effekte von NK-Zellen zu fördern und CD4+ T-Helfer-Zellen sowie Tumor-reaktive CD8+ T-Lymphozyten effizient zu stimulieren. Angesichts dieser proinflammatorischen Eigenschaften können slanDCs wesentlich an einer Tumor-gerichteten Immunantwort beteiligt sein. Auf dieser Grundlage erfolgte im Rahmen der vorliegenden Arbeit der immunhistochemische Nachweis von slanDCs in klarzelligen NZK-Geweben. Im Vergleich zu Tumor-freiem Nierengewebe trat in den primären Tumorgeweben eine erhöhte Zahl infiltrierender slanDCs auf. Zudem wurde die Präsenz von slanDCs in Lymphknoten- sowie Fernmetastasen von NZK-Patienten beobachtet. Weiterführende Untersuchungen an frischen klarzelligen NZK-Geweben demonstrierten, dass NZK-infiltrierende slanDCs einen unreifen Phänotyp ausprägen und Interleukin-10 produzieren. Ausgehend von diesen Erkenntnissen erfolgten funktionelle Analysen, bei denen der Einfluss der kommerziell erhältlichen klarzelligen NZK-Linien ACHN und Caki-1 sowie der primären klarzelligen NZK-Linien MZ1257RC und MZ2877RC auf bedeutende immunmodulatorische Fähigkeiten von slanDCs untersucht wurde. In diesem Zusammenhang zeigte sich, dass NZK-Zellen effektiv in der Lage sind, sowohl die slanDC-vermittelte Proliferation von CD4+ und CD8+ T-Lymphozyten, als auch die slanDC-induzierte Differenzierung naïver CD4+ T-Lymphozyten in proinflammatorische T-Helfer 1-Zellen zu inhibieren. Darüber hinaus wurde demonstriert, dass NZK-Zellen das Potenzial von slanDCs zur Aktivierung von NK-Zellen hemmen. Untersuchungen der zugrunde liegenden Mechanismen zeigten, dass die funktionelle Inhibition von slanDCs durch klarzellige NZK-Zellen über membranständige Moleküle vermittelt wird. Die im Rahmen dieser Dissertation gewonnenen Erkenntnisse weisen darauf hin, dass NZKs die Ausreifung sowie wesentliche funktionelle Eigenschaften von DCs inhibieren. Dies deutet auf einen neuen Immunescape-Mechanismus klarzelliger NZKs hin, welcher auf einer Tumorzell-vermittelten Generierung von tolerogenen slanDCs basiert und eine unzureichende Aktivierung der angeborenen sowie adaptiven Tumor-gerichteten Immunantwort zur Folge hat. Diese neuen Erkenntnisse können einen Beitrag zu einem besseren Verständnis der Interaktion von NZKs mit nativen humanen DCs leisten und die Konzeption neuer therapeutischer Strategien ermöglichen, welche auf einer Verstärkung der antitumoralen Eigenschaften von DCs beruhen.

<p>  </p> <p>Metastatic breast cancer (MBC) is an extremely recalcitrant disease capable of overcoming targeted therapies and evading immune surveillance via the engagement of complicated signaling networks. Resistance to targeted therapies and therapeutic failure of immune checkpoint blockade (ICB) are two major challenges in treating MBC. To survive in the dynamic tumor microenvironment (TME) during metastatic progression, shared signaling nodes are required for MBC cells to regulate the signaling networks efficiently, which are potential multifunctional therapeutic targets. SH2 containing protein tyrosine phosphatase-2 (SHP2) is a druggable oncogenic phosphatase that is a key shared node in both tumor cells and immune cells. How tumor-cell autonomous SHP2 manages its signaling inputs and outputs to facilitate the growth of tumor cells, drug resistance, immunosuppression, and the limited response of ICB in MBC is not fully understood. Herein, we used inducible genetic depletion and two distinct types of pharmacological inhibitors to investigate anti-tumor effects with immune reprogramming during SHP2 targeting. </p> <p>We first focus on the signaling inputs and outputs of SHP2. We find that phosphorylation of SHP2 at Y542 predicts the survival rates of breast cancer patients and their immune profiles. Phosphorylation of SHP2 at Y542 is elevated with differential activation mechanisms under a growth-factor-induced and extracellular matrix (ECM)-rich culture environment. Phosphorylation of SHP2 at Y542 is also elevated in HER2 positive MBC cells upon acquired resistance to the HER2 kinase inhibitor, neratinib. The resistant cells can be targeted by SHP2 inhibitors. SHP2 inhibitors block ERK1/2 and AKT signaling and readily prevented MBC cell growth induced by multiple growth factors. Inhibition of SHP2 also blocks these signaling events generated from the ECM signaling. In fact, the inhibitory effects of SHP2 blockade are actually enhanced in the ECM-rich culture environment. We utilize the <em>in vitro</em> T-cell killing assays and demonstrate that pretreatment of tumor cells with FGF2 and PDGF reduces the cytotoxicity of CD8+ T cells in a SHP2-dependent manner. Both growth factors and ECM-rich culture environment transcriptionally induce PD-L1 via SHP2. SHP2 inhibition balances MAPK signaling and STAT1 signaling, which prevents growth factor-mediated suppression of INF-γ-induced expression of MHC class I. </p> <p>Next, we evaluate the efficacy of SHP2 inhibitors. Blockade of SHP2 in the adjuvant setting decreased pulmonary metastasis <em>in vivo</em> and extended the survival of systemic tumor-bearing mice. Tumor-cell autonomous depletion of SHP2 reduces pulmonary metastasis and relieves exhaustion markers on CD8+ and CD4+ cells. Meanwhile, both systemic SHP2 inhibition and tumor-cell autonomous SHP2 depletion reduce tumor-infiltrated CD4+ T cells and M2-polarized tumor associated macrophages. </p> <p>Finally, we investigate potential combination therapies with SHP2 inhibitors. The combination of SHP2 inhibitors and FGFR-targeted kinase inhibitors synergistically blocks the growth of MBC cells. Pharmacological inhibition SHP2 sensitizes MBC cells growing in the lung to α-PD-L1 antibody treatment via relieving T cell exhaustion induced by ICB. </p> <p>Overall, our findings support the conclusion that MBC cells are capable of simultaneously engaging several survival pathways and immune-suppressive mechanisms via SHP2 in response to multiple growth factors and ECM signaling. Inhibition of SHP2, potentially in combination with other targeted agents and ICB, holds promise for the therapeutic management of MBC.</p>

No description available.

[ES] La mayoría de las terapias contra el cáncer de mama que se utilizan actualmente en la práctica clínica se centran en atacar las células tumorales. Sin embargo, los nuevos avances en el campo de la inmunología han resaltado el papel principal del microambiente tumoral en la modulación tumoral. Específicamente, los fibroblastos asociados al cáncer desempeñan un papel importante en la progresión tumoral, la modulación de la inmunidad tumoral y la resistencia a la terapia. Por ello, esta tesis doctoral titulada "Nanopartículas de sílice mesoporosas dirigidas al microambiente tumoral como herramienta para el tratamiento del cáncer de mama" se centra en el diseño de un nanodispositivo dirigido a los fibroblastos asociados al cáncer y en la evaluación de su potencial como nueva estrategia terapéutica para el tratamiento del cáncer de mama. Se diseñó y sintetizó una nanopartícula utilizando nanopartículas mesoporosas de sílice como soporte, cargadas con doxorrubicina y funcionalizadas con un péptido ligando de FAP-¿ (NP-FAP-DOX). La caracterización de NP-FAP-DOX mostró una liberación controlada de la carga y un perfil no tóxico in vitro. Los estudios in vitro evaluaron la eficacia de las nanopartículas para dirigirse a FAP-¿, la citotoxicidad celular y la penetrabilidad tumoral en las líneas celulares de cáncer de mama, en los fibroblastos asociados al cáncer derivados de biopsias de pacientes con cáncer de mama triple negativo y en los organoides derivados de pacientes con cáncer de mama. Estos estudios demostraron que NP-FAP-DOX se dirigió eficazmente y produjo un efecto citotóxico en células de cáncer de mama con expresión positiva de FAP-¿, así como en fibroblastos asociados al cáncer. Además, la NP-FAP-DOX presentó una buena eficiencia de penetración en los organoides derivados de paciente, manteniendo así la acción dirigida y el efecto citotóxico en este modelo tridimensional. Finalmente, se evaluó la eficacia de NP-FAP-DOX in vivo en un modelo murino de cáncer de mama triple negativo. La NP-FAP-DOX mostró una buena capacidad para atacar tumores y una administración eficaz de fármacos, lo que dio como resultado un efecto antitumoral in vivo. Además, el tratamiento in vivo con NP-FAP-DOX se dirigió eficazmente a los fibroblastos asociados al cáncer y los eliminó, lo que llevó a la remodulación del microambiente tumoral y a la activación de la respuesta inmunitaria del tumor. Específicamente, este tratamiento promovió la infiltración de linfocitos, aumentó el porcentaje de células asesinas naturales y disminuyó los macrófagos M2, lo que llevó a un aumento de la proporción M1/M2 en los tumores. Además, las nanopartículas mejoraron el perfil terapéutico y de seguridad del fármaco libre, previniendo la toxicidad cardíaca y sistémica inducida por doxorrubicina. Con todo, estos resultados demostraron el potencial de los nanodispositivos diseñados como un nuevo sistema de administración de fármacos dirigido para el tratamiento del cáncer de mama. Estas nanopartículas pueden mejorar la eficacia de la administración de fármacos, superar los efectos secundarios adversos y mejorar la eficacia de la terapia mediante la modulación del microambiente tumoral. / [CA] La majoria de les teràpies contra el càncer de mama que s'utilitzen actualment en la pràctica clínica, es centren en atacar les cèl·lules tumorals. Tanmateix, els nous avanços en el camp de la immunologia han ressaltat el paper principal del microambient tumoral en la modulació tumoral. Específicament, els fibroblasts associats al càncer tenen un paper important en la progressió tumoral, la modulació de la immunitat tumoral i la resistència a teràpia. Per això, aquesta tesi doctoral titulada "Nanopartícules de sílice mesoporoses dirigides al microambient tumoral com a ferramenta per al tractament del càncer de mama" es centra en el disseny d'un nanodispositiu dirigit als fibroblasts associats al càncer i en l'avaluació del seu potencial com a nova estratègia terapèutica per al tractament del càncer de mama. Es va dissenyar i sintetitzar una nanopartícula utilitzant nanopartícules mesoporoses de sílice com a suport, carregades amb doxorubicina i funcionalitzades amb un pèptid lligand de FAP-¿ (NP-FAP-DOX). La caracterització de NP-FAP-DOX va mostrar un alliberament controlat de la càrrega i un perfil no tòxic in vitro. Els estudis in vitro van avaluar la eficàcia de les nanopartícules en l'acció dirigida a FAP-¿, la citotoxicitat cel·lular i la penetrabilitat tumoral en les línies cel·lulars de càncer de mama, en els fibroblasts associats al càncer derivats de biòpsies de pacients amb càncer de mama triple negatiu i en els organoides derivats de pacients amb càncer de mama. Aquests estudis van demostrar que NP-FAP-DOX es dirigia eficaçment i produïa un efecte citotòxic en les cèl·lules de càncer de mama amb expressió positiva de FAP-¿, així com en fibroblasts associats al càncer. A més, la NP-FAP-DOX va presentar una bona eficiència de penetració en els organoides derivats de pacient, mantenint així l'acció dirigida i l'efecte citotòxic en aquest model tridimensional. Finalment, es va avaluar la eficàcia de NP-FAP-DOX in vivo en un model murí de càncer de mama triple negatiu. La NP-FAP-DOX va mostrar una bona capacitat per a atacar tumors i una administració eficaç de fàrmacs, el que va donar com a resultat un efecte antitumoral in vivo. Addicionalment, el tractament in vivo amb NP-FAP-DOX es va dirigir eficaçment als fibroblasts associats al càncer generant la seua depleció i així, la remodelació del microambient tumoral i l'activació de la resposta immunitària del tumor. Específicament, aquest tractament va promoure la infiltració de limfòcits, va augmentar el percentatge de cèl·lules citocides naturals i va disminuir els macròfags M2, el que va conduir a un augment en la proporció M1/M2 en els tumors. A més, les nanopartícules van millorar el perfil terapèutic i de seguretat del fàrmac lliure, prevenint la toxicitat cardíaca i sistèmica induïda per la doxorubicina. Amb tot, aquests resultats demostraren el potencial dels nanodispositius dissenyats com un nou sistema d'administració de fàrmacs dirigits per al tractament del càncer de mama. Aquestes nanopartícules poden millorar l'eficàcia de l'administració de fàrmacs, reduir els efectes secundaris adversos i millorar l'eficàcia de la teràpia mitjançant la modulació del microambient tumoral. / [EN] Most of the breast cancer therapies currently used in the clinical practice are focused on targeting tumor cells. Nevertheless, new advances in the immunology field uncovered the main role of the tumor microenvironment in tumor modulation. Specifically, cancer-associated fibroblasts play an important role in tumor progression, tumor immunity modulation, and therapy resistance. Hence, this Ph.D. thesis entitled "Mesoporous silica nanoparticles targeting tumor microenvironment as a tool for breast cancer treatment" is focused on the design of a nanodevice targeting cancer-associated fibroblasts and on the evaluation of its potential as a new therapeutic strategy for breast cancer treatment. A nanoparticle was designed and synthesized using mesoporous silica nanoparticles as support, loaded with doxorubicin, and functionalized with a FAP-¿ ligand peptide (NP-FAP-DOX). NP-FAP-DOX's characterization showed controlled cargo release and an in vitro nontoxic profile. The in vitro studies evaluated the nanoparticle efficacy to target FAP-¿, cellular cytotoxicity, and tumor penetration in breast cancer cell lines, cancer-associated fibroblasts derived from triple-negative breast cancer patient biopsies, and breast cancer patient-derived organoids. These studies probed that the NP-FAP-DOX efficiently targeted and produced a cytotoxic effect in breast cancer cells with FAP-¿ positive expression as well as in cancer-associated fibroblasts. Moreover, the NP-FAP-DOX presented good penetration efficiency in patient-derived organoids, while maintaining the targeting and cytotoxic effect in this 3D model. Finally, the NP-FAP-DOX's in vivo efficacy was evaluated in a murine triple-negative breast cancer model. The NP-FAP-DOX showed a tumor-targeting ability and effective drug delivery, resulting in an in vivo antitumoral effect. Moreover, the NP-FAP-DOX in vivo treatment efficiently targeted and depleted cancer-associated fibroblasts, leading to tumor microenvironment re-modulation and activation of tumor immune response. Specifically, this treatment promoted lymphocyte infiltration, increased the percentage of natural killer cells, and decreased the M2-like macrophages leading to an increased M1/M2 ratio in tumors. Besides, the nanoparticles improved the therapeutic and safety profile of the free drug, preventing doxorubicin-induced cardio and systemic toxicity. Overall, these results demonstrated the potential of the designed nanodevices as a new targeted drug delivery system for breast cancer treatment. These nanoparticles can improve drug delivery efficacy, overcome adverse side effects, and enhance therapy efficacy through the modulation of the tumor microenvironment. / Trigo Lameirinhas, AC. (2024). Mesoporous Silica Nanoparticles Targeting Tumor Microenvironment as a Tool for Breast Cancer Treatment [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/210630

Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.

Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.

La réponse immunitaire est régulée spatialement et temporellement. Les cellules immunitaires font partie d’une plus grande communauté de populations cellulaires interconnectées qui coordonnent leurs actions par la signalisation intercellulaire. Suivant une blessure hépatique, la distribution et la composition du compartiment immunitaire évoluent rapidement au fil du temps. Par conséquent, l’information sur la position des cellules immunitaires dans le tissu hépatique est essentielle à la bonne compréhension de leurs fonctions dans la santé et la maladie. Cependant, l’organisation spatiale des cellules immunitaires en réponse à une atteinte hépatique aiguë, ainsi que les conséquences fonctionnelles de leur distribution topographique spécifique, restent mal comprises. Les macrophages hépatiques sont des cellules effectrices clés pendant l’homéostasie et en réponse à des blessures, et sont impliqués dans la pathogenèse de plusieurs maladies du foie. L’hétérogénéité et plasticité des macrophages dans le foie a été exposée avec l’émergence du séquençage de l’ARN, la cytométrie en flux et la cytométrie de masse. Ces techniques ont sensiblement contribué à la compréhension de l’origine, et fonctions des macrophages dans le foie. Cependant, ces technologies impliquent la destruction du tissu pour la préparation de suspension cellulaires ce qui entraîne une perte d’information spatiale et de contexte tissulaire. Par conséquent, la caractérisation spatiale et temporelle des macrophages dans le tissu hépatique pendant l’homéostasie tissulaire, et en réponse à une blessure, fournit une nouvelle information sur la façon dont les macrophages se rapportent aux cellules voisines et leur comportement pendant les réponses immunitaires. Dans la première partie de cette étude, nous avons conçu une stratégie pour le phénotypage spatial des cellules immunitaires hépatiques dans des échantillons de tissus. Cette stratégie combine techniques d'imagerie et l’alignement numérique des images pour surmonter les limitations actuelles du nombre de marqueurs pouvant être visualisés simultanément. En outre, nous avons généré des protocoles pour la quantification automatisée des cellules d’intérêt dans des sections de tissus pour réduire la subjectivité associée à la quantification par inspection visuelle, et pour augmenter la surface et la vitesse de l’analyse. Par conséquent, un plus grand nombre de populations de cellules immunitaires ont été visualisées, quantifiées et cartographiées, et leurs relations spatiales ont été déterminées. Dans la deuxième partie de l’étude, nous avons déterminé la cinétique et la dynamique spatiale des cellules de Kupffer (KCs) et des macrophages dérivés de monocytes (MoMFs) en réponse à une atteinte hépatique aiguë au CCl4, afin de mieux comprendre leurs rôles fonctionnels, et la répartition du travail entre eux. Nous avons constaté que les KC et les MoMFs présentent des différences au niveau de la distribution tissulaire, la morphologie, et la cinétique. En plus, seulement les KCs ont proliféré pour repeupler la population de macrophages résidents pendant la réparation tissulaire. Finalement, nous avons montré que le degré de colocalization de KCs et des MoMFs avec les cellules stellaires est différent. En plus, cette colocalisation varie avec la progression de la réponse immunitaire. Dans l’ensemble, nous avons montré que les KCs et les MoMFs ont des profils spatiaux et temporels différents en réponse à une atteinte hépatique aiguë. Dans l’ensemble, les observations faites dans cette étude suggèrent que le comportement spatial et temporel d’une sous-population donnée de cellules immunitaires est distinct et sous-tend sa capacité à remplir ses fonctions spécifiques pendant la réponse immunitaire. / The immune response is spatially and temporally regulated. Immune cells are part of a larger community of interconnected immune and non-immune cell populations that coordinate their actions mostly through cell-cell intercellular signaling. In the liver, the distribution pattern, and the composition of the immune compartment evolve during an immune response to injury influencing disease pathology, progression, and response to treatment. Hence, information on the location and interacting partners of immune cells in the hepatic tissue is critical for the proper understanding of their functions in health and disease. However, the spatial organization of hepatic resident and infiltrating immune cells in response to acute injury, and the functional consequences of their specific topographical distribution, remain poorly defined. Hepatic macrophages are key effector cells during homeostasis and in response to injury and are involved in the pathogenesis of several liver diseases. The heterogeneity and plasticity of the macrophage compartment in the liver have only recently started to be appreciated with the emergence of RNA sequencing, flow cytometry, and mass cytometry. Detailed transcriptomic and phenotypic profiling have deeply expanded our understanding of macrophage biology. However, these technologies involve tissue disruption with loss of spatial information and tissue context. Therefore, the spatial and temporal profiling of liver macrophages in tissue samples during the steady state, and in response to injury, provide novel information on how the macrophages relate to neighboring cells and their behavior during immune responses. In the first part of this study, we designed a strategy for the spatial phenotyping of hepatic immune cells in tissue samples. This strategy combined serial and sequential labeling, and digital tissue alignment to overcome current limitations in the number of markers that can be simultaneously visualized. In addition, we generated protocols for automated quantification of cells of interest in whole tissue sections which removed the subjectivity associated with quantification by visual inspection and greatly increased the area and the speed of the analysis. As a result, a larger number of immune cell populations were visualized, quantified, and mapped, and their spatial relations were determined in an unbiased manner. In the second part of this study, we monitored the kinetics, and spatial dynamics of resident Kupffer cells (KCs) and infiltrating monocyte-derived macrophages (MoMFs) in response to acute liver injury with CCl4, to gain insight into their functional roles, and the distribution of labor between them. KCs and MoMFs exhibited different tissue distribution patterns and cell morphology, different kinetics, and occupied neighboring but unique microanatomical tissue locations. KCs and MoMFs displayed a different capacity to replenish the macrophage pool upon acute injury, and were differentially related to hepatic stellate cells. Different kinetics and spatial profiles revealed that KCs and MoMFs have distinct spatial signatures and suggest that they perform distinct functions during the wound-healing response to acute liver injury. In summary, we optimized techniques and put together a strategy for the spatial profiling of hepatic immune cells. Then, we used this methodology to profile resident and infiltrating macrophage subpopulations to gain insight into their biology and distinct contribution to healing in response to acute liver injury. Overall, the observations made in this study suggest that the spatial and temporal behavior of a given subpopulation of immune cells underlie its ability to perform its specific functions during the immune response.