Prostate cancer (PC) is the most common cancer in Sweden. Most patients have slow growing tumors that will not cause them any harm within their lifetime, but some have aggressive tumors and will die from their disease. The ability of current clinical practice to predict tumor behavior and disease outcome is limited leading to both over- and undertreatment of PC patients. The men who die from their disease are those that develop metastases. It is therefore of great value to find better and more sensitive prognostic techniques, so that metastatic spread can be detected (or predicted) at an early time point, and so that appropriate treatment can be offered to each subgroup of patients. The aim of this thesis was to investigate if, and by what means, highly aggressive prostate tumors influence extratumoral tissues such as the non-malignant parts of the prostate and regional lymph nodes (LN), and also if any of our findings could be of prognostic importance. Gene- and protein expression analysis were the main methods used to address these questions. Our research group has previously introduced the expression Tumor Instructed (Indicating) Normal Tissue (TINT), and we use the term TINT-changes when referring to alterations in non-malignant tissue due to the growth of a tumor nearby or elsewhere in the body. In the Dunning rat PC-model we found that MatLyLu (MLL)-tumors, having a high metastatic ability, caused pre-metastatic TINT-changes that differ from those caused by AT1-tumors who have low metastatic ability. Prostate-TINT surrounding MLL-tumors had elevated immune cell infiltration, and gene ontology enrichment analysis suggested that biological functions promoting tumor growth and metastasis were activated in MLL- while inhibited in AT1-prostate-TINT. In the regional LNs we found signs of impaired antigen presentation, and decreased quantity of T cells in the MLL-model. One of the downregulated genes in the MLL-LNs was Siglec1 (also known as Cd169), expressed by LN resident macrophages that are important for antigen presentation. When examining metastasis-free LN tissue from PC patients we found CD169 expression to be a prognostic factor for PC-specific survival, and reduced expression was linked to an increased risk of PC-specific death. Some of our findings in prostate- and LN-TINT could be seen already when the tumors were very small suggesting that differences in TINT-changes between tumors with different metastatic capability can be detected early in tumor progression. However, before coming of use in the clinic more research is needed to better define a suitable panel of prognostic TINT-factors as well as the right time window of when to use them. / Populärvetenskaplig sammanfattning Prostatacancer är den i särklass vanligaste cancerformen hos män i Sverige. De flesta patienter har en mycket långsamt växande tumör som inte orsakar dem några större besvär under deras livstid, men enbart i Sverige dör ca 2500 patienter/år av sjukdomen. Det är först vid uppkomst av metastaser som sjukdomen blir dödlig. Befintliga diagnos- och prognosmetoder är otillräckliga när det gäller att uppskatta och förutse tumörens aggressivitet och risk för att bilda metastaser. Detta gör att vissa patienter inte får tillräcklig behandling eller behandlas försent medan andra behandlas i onödan. Behovet av förbättrad diagnostik är därför stort. Om vi kan hitta markörer för potentiellt metastaserande sjukdom, och i bästa fall också behandla innan metastaser uppstår, skulle det förbättra chansen för överlevnad markant. För att kunna växa och spridas behöver en tumör inte bara förbereda närliggande vävnader utan förmodligen hela kroppen. Vår hypotes är att potentiell dödliga tumörer sannolikt är bättre på detta än mer ofarliga. Man vet från studier av andra cancerformer att farliga tumörer orsakar förändringar i det organ dit cancern senare sprids. Dessa förändringar sker för att de tumörceller som senare anländer ska kunna överleva, och processen har fått namnet pre-metastatisk nisch. Bl.a. har man sett att immunsystemet hämmas och nybildning av kärl ökar. Det är vanligt att metastaser uppstår i närliggande lymfkörtlar innan uppkomst av metastaser i andra organ. Dock är väldigt lite känt om pre-metastatiska förändringar i lymfkörtlar eftersom den forskning som hittills är gjord främst har tittat på andra organ. Inom prostatacancer finns det förvånande få studier av premetastatiska nischer överhuvudtaget, och man vet därför inte om de alls förekommer eller vilka förändringar som i så fall sker. Vår grupp har tidigare myntat uttrycket TINT som står för Tumor Instructed (Indicating) Normal Tissue (TINT är ett engelskt verb som betyder färga) och syftar på förändringar i normal vävnad som inducerats av tumören, dvs. att tumörer färgar av sig på omgivningen. Det kan vara förändringar i normal vävnad nära tumören, som i det här fallet resten av prostatan, eller i vävnad långt ifrån tumören som till exempel regionala lymfkörtlar, lungor och benmärg. Syftet med det här avhandlingsarbetet var att undersöka TINT-förändringar inducerade av aggressiv cancer och se om dessa skiljer sig från TINT-förändringar inducerade av mindre farliga tumörer, samt att utvärdera om någon TINT-förändring skulle kunna användas för att prognostisera vilka patienter som har hög risk att få metastaser. Vi har använt oss av en prostatacancer-modell i råtta där vi analyserat genoch proteinuttryck i pre-metastatiska regionala lymfkörtlar, tumörer och prostata-TINT (dvs. prostatavävnad utanför tumören). TINT-förändringar inducerade av MatLyLu (MLL), en tumör med hög metastaserande förmåga, jämfördes mot TINT-förändringar inducerade av AT1, en snabbväxande tumör men med låg förmåga att bilda metastaser. Vi kunde vi se flera skillnader mellan modellerna. Genuttrycket i MLL-prostata-TINT indikerade en aktivering av cellulära funktioner som visat sig stimulera tumörväxt och spridning såsom celldelning, viabilitet, migration, invasion, och angiogenes (nybildning av kärl). I AT1-prostata-TINT var genuttrycket kopplat till samma funktioner men verkade istället inhibera dessa. Genom att titta på vävnaderna i mikroskop kunde vi se att MLL-tumörer rekryterade färre T-celler (som har en viktig funktion i immunsvaret mot tumören), men istället fler makrofager och granulocyter till både tumören och prostata-TINT (dessa typer av immunceller har visats kunna hjälpa tumörer att växa och sprida sig). MLL-tumörer hade också fler blodkärl och lymfkärl strax utanför tumören. I de regionala lymfkörtlarna från djur med MLL-tumörer visade genuttrycket tecken på försämrad antigenpresentation, samt immunhämning och/eller induktion av immuntolerans. Immuntolerans innebär att immuncellen inte längre reagerar mot det specifika antigen den blivit tolerant emot. Detta är vanligt förekommande hos individer med cancer och är ett sätt för tumören att undkomma immunförsvaret. I vävnadsprover av lymfkörtlarna kunde vi se färre antigenpresenterande celler, och liksom i tumörerna fanns det färre T-celler i MLL-modellen, något vi kunde se redan när tumörerna var väldigt små. CD169 är ett protein som bl.a. uttrycks av sinus-makrofager i lymfkörtlar. Dessa makrofager har en central funktion i att aktivera ett tumör-specifikt immunsvar. I råttmodellen kunde vi se att regionala lymfkörtlar från djur med MLL-tumörer hade lägre nivåer av CD169 än regionala lymfkörtlar från djur med AT1-tumörer, och då antalet sinus-makrofager visat sig ha prognostiskt värde i t.ex. tjocktarmscancer, ville vi se om det kunde vara så även i prostatacancer. Därför kvantifierade vi uttrycket av CD169 i metastasfria regionala lymfkörtlar från prostatacancerpatienter och såg att låga nivåer av CD169 medförde en ökad risk för att dö i prostatacancer. Sammantaget tyder resultaten på att MLL-tumören jämfört med AT1- tumören bättre lyckas förbereda omgivande vävnad för att gynna tumörväxt och spridning, både lokalt i prostatan men också längre bort från tumören i de regionala lymfkörtlarna. Våra fynd stämmer väl överens med aktuell tumörbiologisk forskning om hur tumörer påverkar sin omgivning. Något som inte visats tidigare är att miljön utanför tumören verkar skilja sig drastiskt beroende på tumörens metastaserande förmåga, samt att dessa skillnader går att se relativt tidigt under sjukdomsförloppet och förmodligen även långt bort från tumören. Vi har också visat att särskilt aggressiv prostatacancer verkar inducera en pre-metastatisk nisch i tumördränerande lymfkörtlar likt det som beskrivits i andra modellsystem och i andra cancertyper, men hittills inte i prostatacancer. Fler studier behövs för att bättre karaktärisera de förändringar som en potentiellt dödlig prostatacancer orsakar i andra vävnader, och för att ta reda på hur denna kunskap kan användas för att förbättra diagnostik och behandling.

Serous epithelial ovarian cancer (EOC) is a potent suppressor of the immune defense. Here, we studied interactions between EOC and the immune system that lead to escape from tumor immune surveillance. We explored: 1) tumor escape from cytotoxicity by exosome-mediated modulation of the NK-cell receptors NKG2D and DNAM-1; 2) cytokine mRNA profiles in the EOC microenvironment and peripheral blood and their role in the suppression of the anti-tumor immune responses; 3) expression of long non-coding (lnc) RNAs in EOC tumors and exosomes. We found that EOC-secreted exosomes carried MICA/B and ULBP1-3, ligands of NKG2D, and could downregulate the NKG2D receptor and impair NKG2D-mediated cytotoxicity. In contrast, the DNAM-1 receptor ligands PVR and nectin-2 were seldom found in exosomes and were not associated with the exosomal membrane leaving the DNAM-1 receptor-mediated cytotoxicity intact. We compared cytokine mRNA expression in the tumor microenvironment and in immune cells of peripheral blood in EOC patients and patients with benign ovarian conditions. EOC patients were unable to mount an IFN-gamma mRNA response needed for tumor cell elimination. Instead, there was a significant up-regulation of inflammation and immune suppression i.e. responses promoting tumorigenesis and T-regulatory cell priming that suppress anti-tumor immunity. In addition, we studied lncRNAs in tissues and sera exosomes from EOC and benign ovarian conditions aiming to assess the lncRNA(s) expression profile and look for lncRNA(s) as possible marker(s) for early diagnosis. We found a deregulated lncRNAs expression in EOC tissues that correlated well with the lncRNAs expression in exosomes. Candidate lncRNAs with the highest expression and abundance were suggested for evaluation as EOC diagnostic markers in a future large cohort study. Our studies of EOC tissue and EOC exosomes highlight the immunosuppressive tumor microenvironment and the complex tumor exosome-mediated network of immunosuppressive mechanisms, and provide a mechanistic explanation of the observation that NKG2D-mediated cytotoxicity does not function in EOC patients and is partially replaced by the accessory DNAM-1 dependent cytotoxic pathway. The deregulated lncRNAs expression in EOC tissues and exosomes might serve for diagnostic purposes but could also be a potential risk of spreading tumor-derived lncRNAs in EOC exosomes to recipient cells throughout the body.

Neuroblastoma is a childhood cancer of the peripheral sympathetic nervous system, emerging from cells of the neural crest. In Sweden, neuroblastoma accounts for 20 cases out of all, 300-350, pediatric cancer cases each year (Barncancerfonden 2019, Turup on behalf of Cancer Centrum 2019). This cancer often appears in the sympathetic ganglia and/or the adrenal gland and has a high rate of metastasis that often results in morbidity (Matthay et al. 2016). Recent findings implicating a mutation in the Rho/Rac signaling pathway, a pathway involved in neural crest differentiation and migration, were found in every fourth neuroblastoma patient (Dyberg et al. 2017) These mutations tend to shift Rho to a more active state which is believed to lead to more downstream Rho-associated Kinase (ROCK) activation. While inhibition of ROCK has been seen to promote MYCN protein degradation, induce neuroblastoma cell differentiation and repress neuroblastoma growth in vitro and in vivo (Dyberg et al. 2017). Rho/ROCK signaling pathway effects on cytoskeletal arrangement and cell shape have also been suggested to be involved in tumor promoted changes of the TME (Johan and Samuel, 2018). In this master’s thesis project, we explore the effects of the Rho/ROCK pathway on the tumor microenvironment (TME) and immune response (IR) in neuroblastoma. More specifically we are focusing on populations of T cells, macrophages and fibroblasts in tumors, and looking into tumor vascular structure (such as blood vessel) and extracellular matrix (ECM) formation after ROCK inhibitor treatment within neuroblastoma tumors from transgenic mice model TH-MYCN and multi-cellular tumor spheroids (MCTS), a three-dimensional (3D) in vitro model simulating TME in neuroblastoma cell lines. Through our studies we hope to find insights into the Rho/ROCK signaling pathway and involvement of the tumor microenvironment in cancer therapy, while elucidating potential new drugs and drug targets for improving outcomes in neuroblastoma treatment.

La sensibilité des cellules de mélanomes aux molécules de thérapies ciblées dépend du microenvironnement tumoral (interactions cellule-cellule et cellule-matrice extracellulaire). Les systèmes tridimensionnels (3D) de culture in vitro reflètent mieux l’architecture structurelle native des tissus et sont attrayants pour l’étude des interactions cellulaires. Nous avons développé et comparé plusieurs modèles de mélanome métastatique : les cellules de mélanomes (SK-MEL-28 et SK-MEL-3, mutées BRAF V600E et SK-MEL-2, BRAF sauvages) cultivées en monocouche (2D) et co-cultivées en 3D sur des équivalents de derme avec des fibroblastes, afin de mieux comprendre les facteurs modulant la sensibilité cellulaire à un inhibiteur de BRAF (BRAFi, Vémurafenib) et au Vémurafenib associé à un inhibiteur de MEK (MEKi, Cobimetinib). La sensibilité cellulaire aux traitements a été évaluée sous différents aspects : prolifération cellulaire (numération cellulaire, incorporation d'EdU, test MTS), analyse des voies de signalisation MAPK et PKB / Akt (Western-blot), apoptose (TUNEL), libération de cytokines et de facteurs de croissance (ELISA) et histologie (modèles 3D). Un effet cytostatique de BRAFi a été observé sur les cellules SK-MEL-28 et SK-MEL-3 cultivées dans les modèles 2D et 3D. La lignée cellulaire SK-MEL-2 était résistante au BRAFi lorsqu'elle a été cultivée en monocouche, mais sensible lorsqu'elle a été co-cultivée avec des fibroblastes incorporés dans une matrice de collagène de type I. Les milieux conditionnés par les fibroblastes 3D (équivalents de derme) ont sensibilisé les cellules SK-MEL-2 (2D) au BRAFi. L'analyse des surnageants de culture cellulaire a révélé que les équivalents de derme libéraient certains facteurs solubles (IL-6, IL-8, HGF, TGF-β) : ces sécrétions ont été modifiées au cours du traitement par Vémurafenib. La combinaison du traitement avec MEKi a renforcé l'action du Vémurafenib sur les cellules de mélanomes métastatiques tout en diminuant la capacité de prolifération des fibroblastes. Des populations de cellules contenant des cellules de mélanomes ou des fibroblastes associés au cancer (CAFs) ont été isolées à partir d'une biopsie de métastase cutanée provenant d'une patiente atteinte d'un mélanome métastatique. Ces cellules ont permis de réaliser des modèles de mélanome métastatique patient-spécifique afin d’étudier in vitro la sensibilité des cellules de la patiente aux traitements dans un microenvironnement tumoral (sécrétion paracrine de cellules stromales et matrice de collagène). Ces modèles prédictifs 3D patient-spécifique pourront être utilisés pour déterminer des stratégies de thérapies personnalisées, ainsi que pour comprendre les phénomènes de résistance des cellules de mélanomes aux traitements. / Melanoma cell sensitivity to targeted therapy molecules is dependent on the tumor microenvironment (cell-cell and cell-extracellular matrix interactions). Three dimensional (3D) in vitro cell culture systems better reflect the native structural architecture of tissues and are attractive to investigate cellular interactions. We have developed and compared several metastatic melanoma models: melanoma cells (SK-MEL-28 and SK-MEL-3, BRAF V600E mutant and SK-MEL-2 BRAF wt) cultured as a monolayer (2D) and co-cultured on 3D dermal equivalents with fibroblasts to better unravel factors modulating cell sensitivity to a BRAF inhibitor (BRAFi, Vemurafenib) and a BRAFi combined with a MEK inhibitor (MEKi, Cobimetinib). Cell sensitivity to treatments was evaluated under various aspects: cell proliferation (cell counting, EdU incorporation, MTS assay), MAPK and PKB/Akt signaling pathway analysis (Western-blotting), apoptosis (TUNEL), cytokine and growth factor release (ELISA) and histology (3D models). A cytostatic effect of BRAFi was observed on SK-MEL-28 and SK-MEL-3 cells in both models. SK-MEL-2 cell line was clearly resistant to BRAFi when cultured as a monolayer but not when co-cultured with 3D fibroblasts embedded in a type I collagen matrix. Conditioned media provided by 3D fibroblasts (dermal equivalents) underlined 2D SK-MEL-2 sensitivity to BRAFi. Cell culture supernatant analysis revealed that dermal equivalents released some soluble factors (IL-6, IL-8, HGF, TGF-β): these secretions were modified during vemurafenib treatment. The combination of treatment with MEKi enhances the action of Vemurafenib on metastatic melanoma cells while decreasing the proliferation capacity of fibroblasts. Cell populations containing melanoma cells or fibroblasts associated with cancer (CAFs) were isolated from a cutaneous metastasis biopsy of a patient with metastatic melanoma. These cells allowed the realization of patient-specific models of metastatic melanoma in order to study in vitro the sensitivity of the patient’s melanoma cells to treatments in a tumor microenvironment (paracrine secretion of stromal cells and collagen matrix). These 3D predictive patient-specific models could be used to determine personalized therapy strategies, as well as to understand the resistance phenomena of melanoma cells to treatments

This diploma thesis includes an experimental and a bioinformatic part. The two parts are linked together through the subject of transcription factors of 'forkhead box O' (FOXO) family. FOXO transcription factors have a key role in many cellular processes including cell cycle regulation, apoptosis and metabolism. For a long time, they have been considered strictly as the tumor-suppressors yet a growing number of evidence is pointing out to their pro-tumorigenic role. In consequence FOXO transcription factors are studied intensively as potential therapeutic targets in cancer. In the past decade, in silico prediction of protein-protein interactions has become popular in basic research as well as in drug development. Nonetheless, the predicted structures are still far from fitting to the expected behavior of the respective biomolecules. In the experimental part of this thesis, I verified the interaction of four in silico predicted protein binders based on naturally occurring PDZ domain with FOXO4 using microscale thermophoresis. Non-invasive bladder tumors represent a heterogeneous disease where reliable prediction of tumor aggressiveness is still lacking despite an intensive research. In the bioinformatic part of this thesis, I described the cellular composition of the tumor microenvironment and demonstrated...

Chemotherapy is still one of the most widely used anticancer therapies. It is mostly about inhibiting the proliferation of rapidly dividing cells, so it is not selective for tumor cells. As a result, many undesirable side effects are associated with chemotherapy. The disadvantageous properties of chemotherapeutics can be largely eliminated by using conjugates of polymers with low molecular weight drugs. An example of such a conjugate is a doxorubicin-linked HPMA polymer. In addition to the properties obtained by polymer binding, such as achieving solubility in aqueous solutions, reducing systemic toxicity, increasing the maximum tolerated dose, or passive targeting by the EPR effect, the fact that doxorubicin induces immunogenic cell death is used in therapy with this drug. It has already been shown that after complete cure of the experimental mice with polymeric conjugates of HPMA with doxorubicin, some mice develop long-term resistance to re-inoculation with a lethal dose of tumor cells. Resistance is specific to the particular line of tumor cells from which the mouse was cured, and CD8+ cytotoxic T cells and IFNγ play an important role. In this work, we monitored changes in the proportion of immune populations and their activation markers after treatment with HPMA-based polymers with doxorubicin...

Cancer cells use the interaction between immune-checkpoint receptor programmed cell death protein 1 (PD-1) on activated T cells and programmed cell death ligand 1 (PD-L1) on tumor cells and various cell types of the tumor microenvironment to evade immune surveillance. Blocking the interaction between PD-1 and PD-L1 with checkpoint inhibitors can improve T-cell reactions and mediate efficient antitumor activity in a variety of solid tumors including melanoma. However, clinical data from patients with myeloid diseases that are treated with these agents are limited to clinical trials in advanced disease. Pembrolizumab (PEM) is a humanized monoclonal antibody of the immunoglobulin G4/κ isotype designed to block PD-1/PD-L1 interactions and is approved for various solid tumors including advanced melanoma.

Les fonctions immunitaires déclinent au cours du vieillissement, un phénomène qui pourrait être lié à l'accumulation de cellules sénescentes dans les tissus. La sénescence est un état irréversible d'arrêt de croissance qui s'engage principalement en réponse à des dommages irréparables de l'ADN. Les cellules sénescentes ont un phénotype sécrétoire pro-inflammatoire (SASP) qui affecte les tissus voisins. CD73 est une enzyme qui travaille en collaboration avec CD39 pour produire de l’adénosine à partir d‘adénosine triphosphate (ATP). Il a été démontré que des concentrations plus élevées d'adénosine dans un microenvironnement tumoral nuisent aux fonctions des cellules immunitaires. L'objectif de ce projet est de déterminer si les fibroblastes sénescents ont la capacité d'induire l'expression de CD39/CD73 à la surface des cellules tueuses naturelles (NK) et d'inhiber la réponse immunitaire antitumorale. Nos résultats montrent que les cellules NK-92, NKAES (cellules tueuses naturelles amplifiées) et les cellules NK primaires expriment des niveaux plus élevés de CD39/CD73 lorsqu'elles sont cultivées avec des fibroblastes sénescents. De plus, nous avons observé que le marqueur CD73 est aussi augmenté dans les fibroblastes sénescents. L'augmentation était cependant plus prononcée lorsque la sénescence était induite en raison de la surexpression de l’oncogène hRASv12 plutôt que suite à l'exposition à des radiations ionisantes. En outre, la cytotoxicité des cellules NK diminue lorsque celles-ci sont exposées à un environnement sénescent et lorsqu'on traite les cellules avec 2-Chloro Adénosine (CADO), un analogue de l'adénosine. Nous supposons que l'augmentation de l'expression de CD39/CD73 conduira à une production accrue d'adenosine, créant ainsi un environnement immunosuppressif. La caractérisation de l'impact de la sénescence cellulaire sur les fonctions des cellules NK pourrait donner un aperçu du développement de stratégies visant à augmenter la capacité du système immunitaire à éliminer les cellules tumorales, améliorant potentiellement les résultats du traitement du cancer. / Immune functions decline during aging, a phenomenon that may be linked to the accumulation of senescent cells in tissues. Senescence is an irreversible state of cell growth arrest often in response to irreparable DNA damage. Senescent cells have a proinflammatory secretory phenotype (SASP) that affects nearby tissues. CD73 is an enzyme that works in collaboration with CD39 to produce adenosine from adenosine triphosphate (ATP). Higher concentrations of adenosine in a tumor microenvironment were shown to impair immune cell functions. The objective of this project is to determine whether senescent fibroblasts have the ability to induce CD39/CD73 expression at the surface of natural killer (NK) cells and inhibit the antitumoral immune response. Our results show that NK-92, NKAES and primary NK cells express higher levels of CD39/CD73 when grown in co-culture with senescent fibroblasts. Similarly, we also observed that the CD73 marker is increased in senescent fibroblasts. The effect was, however, more pronounced when fibroblasts were induced to senesce because of the overexpression of oncogenic hRASv12 compared to when induced to senesce following exposure to ionizing radiation. In addition, the cytotoxicity of NK cells decreases when NK cells are exposed to a senescent environment and when treated with 2- Chloroadenosine (CADO), an analog of adenosine. We hypothesize that increased CD39/CD73 expression will lead to an increased production of adenosine creating an immunosuppressive environment. Characterization of the impact of cellular senescence on the function of NK cells could provide insights into the development of strategies to increase the ability of the immune system to eliminate tumor cells, potentially improving cancer treatment outcomes.

No description available.

Introduction: Le mélanome est le type de cancer de la peau le plus fréquent et les métastases du système nerveux central en sont une complication fréquente et grave. Les cellules de mélanome interagissent avec une grande variété de types de cellules dans le microenvironnement tumoral (MET), ce qui peut entraîner des effets pro- ou antitumoral. Plusieurs voies immunosuppressives ont été récemment découvertes comme des cibles médicamenteuses prometteuses, notamment la voie de l'adénosine. L'adénosine extracellulaire s'accumule dans le MET suite à l'hydrolyse de l'ATP par les ectonucléotidases CD39 et CD73. Les principaux régulateurs de la voie de l'adénosine sont CD39, CD73, et les récepteurs A2a et A2b. Matériel et Méthodes: Pour caractériser spatialement le MET des métastases cérébrales du mélanome (MCM), nous avons quantifié l'expression de 35 marqueurs protéiques à l'aide du time of flight (CyTOF) Imaging Mass Cytometry (IMC) dans 21 MCM, et segmenté et classé plus de 130 000 cellules. Ensuite, pour évaluer les effets du ciblage du récepteur A2b et du CD73 dans la voie de l'adénosine sur le développement du mélanome, nous avons utilisé les tests de prolifération IncuCyte et MTS pour évaluer la prolifération des cellules de mélanome. Résultats: Dans notre ensemble de données, les caractéristiques immunitaires du MET étaient hétérogènes dans tous les échantillons et le type de cellule le plus courant après les cellules cancéreuses du mélanome était les macrophages dérivés de la moelle osseuse (MDMO). Les échantillons à propagation leptoméningée avaient significativement moins de neutrophiles, de MDMO de type M1, d'autres cellules T et plus de cellules cancéreuses dans leur microenvironnement. Nous avons observé que la stimulation du récepteur A2b a un effet antiprolifératif sur les cellules cancéreuses du mélanome. Conclusion: Cette recherche met en évidence le rôle du MET dans la progression du mélanome et l'importance du MET comme base pour le développement de nouvelles thérapies pour les patients atteints de cancer. / Background: Melanoma is the most frequent type of skin cancer and metastasis to the central nervous system is a common and serious complication of it. Melanoma cells interact with a wide variety of cell types in the tumor microenvironment (TME) which can lead to tumor-promoting or tumor suppressive effects. Several immunosuppressive pathways have emerged as promising drug targets, including the adenosine pathway. The extracellular adenosine accumulates in the TME as the result of ATP hydrolysis by the ectonucleotidases CD39 and CD73. Key regulators of the adenosine pathway are CD39, CD73, A2a and A2b receptor. Methods: To spatially characterize the TME of melanoma brain metastases (MBM), we quantified the expression of 35 protein markers using time of flight (CyTOF) Imaging Mass Cytometry (IMC) in 21 MBMs, and segmented and classified over 130 000 cells. Then, to evaluate the effects of targeting the A2b receptor and CD73 in the adenosine pathway on the development of melanoma, we used the IncuCyte and MTS proliferation assays to assess the proliferation of melanoma cells. Results: In our dataset, the immune landscape of the TME was heterogeneous across all samples and the most common cell type after melanoma cancer cells were bone marrow derived macrophages (BMDM). Samples with leptomeningeal spread had significantly less neutrophils, M1-like BMDM, T other cells and more cancer cells in their microenvironment. We observed that stimulation of the A2b receptor has an antiproliferative effect on melanoma cancer cells. Conclusion: This research highlights the role of the TME in the progression of melanoma and the importance of the TME as grounds for development of new therapies for cancer patients.