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.

Die pädiatrische Akute Lymphatische Leukämie ist heutzutage gut behandelbar. Trotzdem ist die Prognose im Falle eines Rezidives weiterhin schlecht. Die häufigsten extramedullären Orte für Rezidive sind das zentrale Nervensystem und der Hoden. Um ein Rezidiv im Hoden zu verhindern, ist es nötig die zellulären und molekularen Vorgänge zu verstehen. Dafür wurde Patientenmaterial auf seine Expression bestimmter Oberflächenmoleküle analysiert. Verglichen wurden Proben von Patienten mit verschiedenen Rezidiv-Arten. Um die funktionellen Aspekte der Hodenphysiologie auf die Leukämiezellmigration und -lokalisation zu untersuchen wurde ein PDX-ALL Mausmodel mit Hodenbeteiligung etabliert. Um potenziell involvierte Chemokin-Chemokinrezeptor-Achsen zu identifizieren, wurden 55 Knochenmarksproben von Patienten untersucht. Die Expressionsmuster der Rezeptoren wurde mittels Durchflusszytometrie analysiert. Es wurde festgestellt, dass CXCR4 meistens sehr hoch exprimiert wird. Deshalb wurde anschließend der Einfluss der CXCR4-CXCL12 Achse in in vitro Versuchen mit Hilfe primären Hodenstromas untersucht. Um verschiedene Subpopulationen zu untersuchen, wurde die Isolation von Hodenmakrophagen, Sertoli Zellen und Peritubulären Zellen aus der Maus etabliert und ihr Einfluss auf ALL Zellen untersucht. Es wurde festgestellt, dass Hodenmakrophagen bei Tumorkontakt ihren Phänotyp zugunsten einer tumorfördernden Subpopulation verändern. Zu guter Letzt wurde ein adaptives PDX-ALL Mausmodel mit Hodenbeteiligung entwickelt. Dabei wurden die Hoden von vorpubertären Mäusen bevorzugt infiltriert. Die Bedeutung der CXCR4-CXCL12 Achse für die Hodeninfiltration wurde in einem in vivo Versuch validiert. Während Knochenmark und Milz nach einer anti-CXCR4 Gabe kleine ALL Populationen aufwiesen, war der Hoden komplett Tumor-frei. Das Model kann für Versuche genutzt werden, um weitere Signalwege zu identifizieren, welche in die Hoden gerichtete Migration und das Überleben der Zellen involviert sind. / Advancing therapy strategies led to event-free-survival increase of paediatric acute lymphoblastic leukaemia (ALL). Relapses that occur are often drug resistant and come with poor prognosis. Extramedullary sites for relapse are the central nervous system (CNS) and the testis. To prevent testicular relapse, factors responsible for the infiltration and survival need to be discovered. Primary patient material was analysed with regard to differential expression of surface molecules on leukemic cells from BM. To analyse functional aspects of testis physiology on leukemic cell migration and localisation an adoptive PDX-ALL mouse model with testicular involvement was established. To identify potential chemokine-chemokine receptor axes responsible for the leukaemia cell migration towards the testis, a characterization of 55 patient BM samples was performed. The expression pattern of chemokine receptors on BM derived leukaemia cells was compared by flow cytometry. CXCR4 was identified to be highly expressed on patients cells, regardless of the subclass of relapse. Relevance of the CXCR4-CXCL12 axis was studied in vitro. Primary testicular stroma cultures were available, which provided a useful tool to examine testis directed migration. Isolation of testicular macrophages, Sertoli cells and peritubular cells was established and their impact on ALL cell survival was analysed. The macrophages were found to alter their phenotype upon ALL cell contact towards a tumour favourable subtype. Most importantly, a murine adoptive patient-derived xenograft (PDX)-ALL cell transfer model with testicular involvement was established. Testes of pre-puberty mice were preferentially infiltrated. Blocking CXCR4 with an antibody validated relevance of the CXCR4-CXCL12 signalling axis. The testis showed no infiltration in anti-CXCR4 treated animals. The mouse model will be useful to identify and validate further signalling pathways, participating in testis directed migration and survival.

DCs play a pivotal role in orchestrating innate and adaptive antitumor immunity. Activated DCs can produce large amounts of various proinflammatory cytokines, initiate T-cell responses, and exhibit direct cytotoxicity against tumor cells. They also efficiently enhance the antitumoral properties of NK cells and T lymphocytes. Based on these capabilities, immunogenic DCs promote tumor elimination and are associated with improved survival of patients. Furthermore, they can essentially contribute to the clinical efficacy of immunotherapeutic strategies for cancer patients. However, depending on their intrinsic properties and the tumor microenvironment, DCs can be rendered dysfunctional and mediate tolerance by producing immunosuppressive cytokines and activating Treg cells. Such tolerogenic DCs can foster tumor progression and are linked to poor prognosis of patients. Here, we focus on recent studies exploring the phenotype, functional orientation, and clinical relevance of tumor-infiltrating conventional DC1, conventional DC2, plasmacytoid DCs, and monocyte-derived DCs in translational and clinical settings. In addition, recent findings demonstrating the influence of DCs on the efficacy of immunotherapeutic strategies are summarized.

<p>UVOD: Karcinom kolona (KK) je velik javnozdravstveni problem usled visoke incidence i stope mortaliteta. Kod KK je stadijum bolesti najvažniji pojedinačni nezavisni faktor prognoze. U prisustvu nepovoljnih prognostičkih parametara, u koje spadaju visok histolo&scaron;ki gradus, ileus, limfo-vaskularna i perineuralna invazija, nakon potencijalno kurativne operacije se kod pacijenata u stadijumu II indikuje primena adjuvantne hemioterapije koja ima pozitivan uticaj na ukupno preživljavanje i na produženje perioda bez bolesti. Međutim, relapsi bolesti nastaju kod nekih bolesnika bez negativnih prognostičkih faktora, &scaron;to ukazuje na moguće postojanje drugih tkivnih faktora lo&scaron;e prognoze. U novije vreme se sve veća pažnja posvećuje fenomenu tumorskog pupljenja koje predstavlja pojavu tumorskih pupoljaka (TP), odnosno oligocelularnih grupa tumorskih ćelija koje se na invazivnom frontu tumora odvajaju od glavne tumorske mase. Ove tumorske ćelije poprimaju fenotip mezenhimnih ćelija i stiču sposobnost ameboidnog kretanja kroz ekstracelularni matriks, uz pomoć citoplazmatskih podija koje se na dvodimenzionalnim histolo&scaron;kim rezovima vizualizuju kao citoplazmatski pseudofragmenti (CPF). Značaj gustine TP i CPF je jo&scaron; uvek nedovoljno ispitan, ali postoje indicije da se radi o moćnom prediktoru biolo&scaron;kog pona&scaron;anja tumora. CILJ: Cilj je bio da se ispita zavisnost dužine perioda bez relapsa, veličine primarnog tumora, gustine peritumorske limfocitne infiltracije i konfiguracije tumorske margine od gustine TP i CPF kod bolesnika sa KK u stadijumu II. METODOLOGIJA: Istraživanjem je obuhvaćeno 114 bolesnika operisanih od KK u stadijumu II na Institutu za onkologiju Vojvodine, bez nepovoljnih prognostičkih faktora i bez indikacija za primenu adjuvantne hemioterapije. Mikroskopskom analizom rutinskih histolo&scaron;kih i imunohistohemijskih preparata utvrđivana je gustina TP i CPF, koja je zatim korelirana sa vremenom pojave relapsa, veličinom primarnog tumora, gustinom peritumorske limfocitne infiltracije i konfiguracijom tumorske margine. REZULTATI: Velika gustina TP i/ili CPF nađena je kod 45 tumora (39,5%). U ovoj grupi se relaps dogodio kod 26 bolesnika (57,8%). U grupi bolesnika sa malom gustinom TP/CPF relaps je registrovan u 4 slučaja (5,8%). Poređenje krivih preživljavanja pokazalo je da je verovatnoća relapsa značajno veća ako se u tumoru nalazi velika gustina TP/CPF (p&lt;0,0001). Tumori sa velikom gustinom TP/CPF su imali najveći prečnik koji je varirao u rasponu od 25 do 100 mm, dok su tumori sa malom gustinom TP/CPF bili najvećeg prečnika od 20 do 110 mm (p=0,6744). Intenzitet peritumorskog limfoidnog odgovora je bio velik kod 13 tumora sa velikom gustinom TP/CPF (28,9%) i kod 17 tumora sa malom gustinom TP/CPF (24,6%), p=0,7747. Konfiguracija tumorske margine je bila infiltrativna u svim tumorima sa velikom gustinom TP/CPF, kao i kod 42 tumora sa malom gustinom TP/CPF (60,9%). ZAKLJUČAK: Velika gustina TP/CPF je nezavisan tkivni indikator lo&scaron;e prognoze kod bolesnika sa KK u stadijumu II, koji je ne korelira ni sa veličinom primarnog tumora ni sa intenzitetom peritumorskog limfoidnog odgovora. Velika gustina TP/CPF nije kompatibilna sa ekspanzivnom konfiguracijom tumorske margine, ali infiltrativna konfiguracija tumorske margine nije prediktor velike gustine TP/CPF.</p> / <p>INTRODUCTION: Colonic carcinoma (CC) is a serious public health problem due to its high incidence and mortality rate. Stage is the single most important independent prognosticator in patients with CC. In the presence of indicators of poor prognosis, including high histologic grade, ileus, lympho-vascular invasion and perineural invasion, there is a need for adjuvant chemotherapy after a potentially curative operation in patients with stage II CC, because the therapy improves both overall survival and disease-free survival. However, some patients with no documented poor prognostic factors suffer recurrences, which indicates that there may be some other tissue features that confer poor prognosis. In the recent publications there is an increasing interest in the phenomenon of tumor budding, a term assigned to the presence of small groups of discohesive tumor cells at the invasive front of the tumor &ndash; tumor buds (TB&#39;s). These cells acquire mesenchymal phenotype and gain the ability to migrate through the extracellular matrix by means of cytoplasmic extrusions which are visible on the two-dimensional immunohistologic sections and are called cytoplasmic pseudofragments (CPF&#39;s). Significance of density of TB&#39;s and CPF&#39;s is still to be evaluated, but the pool of evidence suggests that this is a powerful predictor of biologic behaviour of CC. AIM: The aim of this study was to determine the influence of density of TB&#39;s and CPF&#39;s on the risk of recurrence in patients with stage II CC. This research also attempted to establish whether there is a correlation between the density of TB&#39;s and CPF&#39;s and several other morphologic features such as tumor diameter, peritumoral lymphocytic response and the configuration of the tumor margin. METHODS: 114 patients with stage II CC were enrolled in the study. All the patients received surgery at the Institute of Oncology in Sremska Kamenica and no patient had indication for adjuvant chemotherapy. Microscopic analysis of routine histologic and immunohistochemical slides was performed to establish the density of TB&#39;s and CPF&#39;s, to estimate the intensity of the peritumoral lymphocytic response and to determine the configuration of the tumor margin. RESULTS: High density of TB&#39;s and/or CPF&#39;s was found in 45 tumors (39.5%). In this group recurrence occured in 26 patients (57.8%). In the group of patients with low density of TB/CPF in the tumor tissue 4 patients relapsed (5.8%). Comparison of survival curves showed that the probability of recurrence was significantly greater if the density of TB/CPF&#39;s was high (p&lt;0.0001). Tumors with high density of TB/CPF&#39;s ranged from 25 to 100 mm in greatest diameter, while those with low density measured from 20 to 110 mm (p=0.6744). Intensity of peritumoral lymphocytic response was high in 13 tumors with high density of TB/CPF&#39;s (28.9%) and in 17 tumors with low density of TB/CPF&#39;s (24.6%), p=0.7747. All tumors with high density of TB/CPF&#39;s and 42 tumors with low density of TB/CPF&#39;s (60.9%) had infiltrative configuration of tumor margin. CONCLUSION: High density of TB/CPF&#39;s is an independent indicator of poor prognosis in patients with stage II CC and it correlates neither with tumor diameter nor with intensity of peritumoral lymphocytic response. High density of TB/CPF&#39;s is not compatible with the expansive configuration of tumor margin, but the infiltrative configuration of tumor margin is not a predictor of high density of TB/CPF&#39;s.</p>

Infiltration of myeloid cells in the tumor microenvironment is often associated with enhanced angiogenesis and tumor progression, resulting in poor prognosis in many types of cancer. The polypeptide chemokine PK2 (Bv8, PROK2) has been shown to regulate myeloid cell mobilization from the bone marrow, leading to activation of the angiogenic process, as well as accumulation of macrophages and neutrophils in the tumor site. Neutralizing antibodies against PK2 were shown to display potent anti-tumor efficacy, illustrating the potential of PK2-antagonists as therapeutic agents for the treatment of cancer. In this study we demonstrate the anti-tumor activity of a small molecule PK2 antagonist, PKRA7, in the context of glioblastoma and pancreatic cancer xenograft tumor models. For the highly vascularized glioblastoma, PKRA7 was associated with decreased blood vessel density and increased necrotic areas in the tumor mass. Consistent with the anti-angiogenic activity of PKRA7 in vivo, this compound effectively reduced PK2-induced microvascular endothelial cell branching in vitro. For the poorly vascularized pancreatic cancer, the primary anti-tumor effect of PKRA7 appears to be mediated by the blockage of myeloid cell migration/infiltration. At the molecular level, PKRA7 inhibits PK2-induced expression of certain pro-migratory chemokines and chemokine receptors in macrophages. Combining PKRA7 treatment with standard chemotherapeutic agents resulted in enhanced effects in xenograft models for both types of tumor. Taken together, our results indicate that the anti-tumor activity of PKRA7 can be mediated by two distinct mechanisms that are relevant to the pathological features of the specific type of cancer. This small molecule PK2 antagonist holds the promise to be further developed as an effective agent for combinational cancer therapy. / Dissertation

Metastatic breast cancer treatment has seen few advances in recent years, yet treatment resistance continues to rise, causing disease recurrence. A pilot study was performed to determine the efficacy of ex vivo expansion and reprogramming of tumor-reactive immune cells from experimental metastatic tumor-sensitized mice. Also, phenotypic changes in tumors due to metastasis or tumor microenvironment influences were characterized. Metastatic neu+ mouse mammary carcinoma (mMMC) and its distant relapsing neu-antigen-negative variant (mANV) were investigated in FVBN202 mice. Tumor-reactive central memory CD8+ T cells and activated NK/NKT cells were successfully reprogrammed and expanded during 6-day expansion from mMMC- and/or mANV-sensitized mice, resulting in tumor-specific cytotoxicity. mMMC exhibited a flexible neu-expression pattern and acquired stem-like, tumorigenic phenotype following metastasis while mANV remained stable except decreased tumorigenicity. Myeloid-derived suppressor cell (MDSC) levels were not increased. Adoptive cellular therapy (ACT) with reprogrammed tumor-reactive immune cells may prove effective prophylaxis against metastatic or recurrent breast cancer.