Nanoparticle-based Therapeutic Intervention of cAMP-mediated Immunosuppression in Malignant Melanoma

Malignant melanoma is a form of skin cancer which, even though it represents its least prevalent form, is accountable for 90% of skin cancer related deaths. While treatment of malignant melanoma up until Stage III promises great success, the same so far does not apply to Stage IV melanoma patients. In recent years, the introduction of novel treatment regiments has raised patients hopes for survival, but limited treatment success requires further development and combination of current and novel strategies. Modifications in metabolic pathways such as the accumulation of the intracellular second messenger cyclic adenosine monophosphate (cAMP) have been recognized as a hallmark of cancer. In melanoma, elevated intracellular cAMP levels correlate with a greater metastatic potential and are believed to be the cause of regulatory T cell (Treg)-mediated immunosuppression in the tumour. In the present study, it was investigated how a micellar formulation of the adenylyl cylase inhibitor MDL-12, 330A hydrochloride made from the amphiphilic polypeptoid-block-polypeptide copolymer polysacrcosine-block-polyglutamatic acid benzylester (PSar-b-PGlu(OBn)) can be used to intervene in the formation of cAMP. The in vivo testing of cAMP reduction using the adenylyl cyclase inhibitor MDL-12, 330A hydrochloride showcased its potential as a therapeutic agent by significantly reducing the tumour burden in the context of the murine B16F10-OVA melanoma model. A detailed examination of the tumour immune cell infiltrate following cAMP reduction revealed drastic quantitative and functional changes. Based on these results, a treatment strategy for complete tumour rejection was successfully established, combining cAMP reduction with punctual Treg removal. In addition, the potential of azide functionalized polysarcosine brushes with polylysine backbone and polysarcosine side chains labelled with the fluorescent dye Alexa Fluor 647 and coupled with antigen and adjuvant could be demonstrated as a protective and therapeutic cancer vaccine. In order to completely eliminate tumour growth in a therapeutic approach, it would be interesting to try to combine it with cAMP reduction.Das maligne Melanom ist eine Form von Hautkrebs, die, obwohl sie die am wenigsten verbreitete Form ausmacht, für 90% der durch Hautkrebs verursachten Todesfälle ursächlich ist. Während die Behandlung des malignen Melanoms bis zum Stadium III große Erfolge verspricht, gilt dies bisher nicht für Pateinten im Stadium IV. In den letzten Jahren hat die Einführung neuer Behandlungsansätze die Hoffnung vieler Patienten auf ein Überleben geweckt. Begrenzte Behandlungserfolge machen eine Weiterentwicklung und Kombination von aktuell verwendeten und neuen Strategien notwendig. Veränderungen in Stoffwechselwegen wie zum Beispiel die Anreicherung des intrazellulären Signalbotenstoffes zyklisches Adenosinmonophosphat (cAMP) sind unter anderem ein Kennzeichen von Krebs. In Melanomen korrelieren erhöhte intrazelluläre cAMP Spiegel mit einem größeren metastatischen Potential und sind vermutlich für die durch regulatorische T Zellen (Treg)-vermittelte Immunsuppression im Tumor mit verantwortlich. In der vorliegenden Arbeit wurde untersucht, wie unter Verwendung mizellarer Formulierungen des Adenylylzyklaseinhibitors MDL-12, 330A Hydrochloride bestehend aus dem amphiphilen polypeptoid-block-polypeptide Copolymer Polysacrcosine-Block-Polyglutaminsäure-benzylester (PSar-b-PGlu(OBn)) in die cAMP-Bildung in Melanomen eingegriffen werden kann. Die in vivo Erprobung der cAMP-Absenkung durch die Verwendung des cAMP Inhibitors MDL-12, 330A Hydrochloride zeigte sein Potenzial als Therapeutikum, indem es im Kontext des murinen B16F10-OVA Melanommodells die Tumorlast signifikant reduzieren konnte. Eine detaillierte Untersuchung des Tumorimmunzellinfiltrates nach cAMP Absenkung zeigte drastische quantitative und funktionelle Veränderungen. Basieren auf diesen Ergebnissen wurde erfolgreich eine Behandlungsstrategie zur kompletten Abstoßung des Tumors etabliert, welche die cAMP Absenkung mit einer punktuellen Entfernung der Tregs verband. Darüber hinaus konnte das Potential von azid-funktionalisierten Polysarkosinbürsten mit Polylysinrückrat und Polysarkosinseitenketten, die mit dem Fluoreszenzfarbstoff Alexa Fluor 647 markiert und mit Antigen und Adjuvans gekoppelt wurden, als protektiver und therapeutischer Krebsimpfstoff aufgezeigt werden. Um das Tumorwachstum in einem therapeutischen Ansatz vollständig zu eliminieren, wäre es interessant zu versuchen, diesen mit der cAMP-Absenkung zu kombinieren

Low-Density LiDAR and Optical Imagery for Biomass Estimation over Boreal Forest in Sweden

Knowledge of the forest biomass and its change in time is crucial to understanding the carbon cycle and its interactions with climate change. LiDAR (Light Detection and Ranging) technology, in this respect, has proven to be a valuable tool, providing reliable estimates of aboveground biomass (AGB). The overall goal of this study was to develop a method for assessing AGB using a synergy of low point density LiDAR-derived point cloud data and multi-spectral imagery in conifer-dominated forest in the southwest of Sweden. Different treetop detection algorithms were applied for forest inventory parameter extraction from a LiDAR-derived canopy height model. Estimation of AGB was based on the power functions derived from tree parameters measured in the field, while vegetation classification of a multi-spectral image (SPOT-5) was performed in order to account for dependences of AGB estimates on vegetation types. Linear regression confirmed good performance of a newly developed grid-based approach for biomass estimation (R2 = 0.80). Results showed AGB to vary from below 1 kg/m2 in very young forests to 94 kg/m2 in mature spruce forests, with RMSE of 4.7 kg/m2. These AGB estimates build a basis for further studies on carbon stocks as well as for monitoring this forest ecosystem in respect of disturbance and change in time. The methodology developed in this study can be easily adopted for assessing biomass of other conifer-dominated forests on the basis of low-density LiDAR and multispectral imagery. This methodology is hence of much wider applicability than biomass derivation based on expensive and currently still scarce high-density LiDAR data

Innate effector-memory T-Cell activation regulates post-thrombotic vein wall inflammation and thrombus resolution

Rationale: Immune cells play an important role during the generation and resolution of thrombosis. T cells are powerful regulators of immune and nonimmune cell function, however, their role in sterile inflammation in venous thrombosis has not been systematically examined. Objective: This study investigated the recruitment, activation, and inflammatory activity of T cells in deep vein thrombosis and its consequences for venous thrombus resolution. Methods and Results: CD4+ and CD8+ T cells infiltrate the thrombus and vein wall rapidly on deep vein thrombosis induction and remain in the tissue throughout the thrombus resolution. In the vein wall, recruited T cells largely consist of effector-memory T (TEM) cells. Using T-cell receptor transgenic reporter mice, we demonstrate that deep vein thrombosis–recruited TEM receive an immediate antigen-independent activation and produce IFN-γ (interferon) in situ. Mapping inflammatory conditions in the thrombotic vein, we identify a set of deep vein thrombosis upregulated cytokines and chemokines that synergize to induce antigen-independent IFN-γ production in CD4+ and CD8+ TEM cells. Reducing the number of TEM cells through a depletion recovery procedure, we show that intravenous TEM activation determines neutrophil and monocyte recruitment and delays thrombus neovascularization and resolution. Examining T-cell recruitment in human venous stasis, we show that superficial varicose veins preferentially contain activated memory T cells. Conclusions: TEM orchestrate the inflammatory response in venous thrombosis affecting thrombus resolution

Tumor immunoevasion via acidosis-dependent induction of regulatory tumor-associated macrophages

Many tumors evolve sophisticated strategies to evade the immune system, and these represent major obstacles for efficient antitumor immune responses. Here we explored a molecular mechanism of metabolic communication deployed by highly glycolytic tumors for immunoevasion. In contrast to colon adenocarcinomas, melanomas showed comparatively high glycolytic activity, which resulted in high acidification of the tumor microenvironment. This tumor acidosis induced Gprotein–coupled receptor–dependent expression of the transcriptional repressor ICER in tumor-associated macrophages that led to their functional polarization toward a non-inflammatory phenotype and promoted tumor growth. Collectively, our findings identify a molecular mechanism of metabolic communication between non-lymphoid tissue and the immune system that was exploited by high-glycolytic-rate tumors for evasion of the immune system

Tumor immunoevasion via acidosis-dependent induction of regulatory tumor-associated macrophages

Many tumors evolve sophisticated strategies to evade the immune system, and these represent major obstacles for efficient antitumor immune responses. Here we explored a molecular mechanism of metabolic communication deployed by highly glycolytic tumors for immunoevasion. In contrast to colon adenocarcinomas, melanomas showed comparatively high glycolytic activity, which resulted in high acidification of the tumor microenvironment. This tumor acidosis induced Gprotein-coupled receptor-dependent expression of the transcriptional repressor ICER in tumor-associated macrophages that led to their functional polarization toward a non-inflammatory phenotype and promoted tumor growth. Collectively, our findings identify a molecular mechanism of metabolic communication between non-lymphoid tissue and the immune system that was exploited by high-glycolytic-rate tumors for evasion of the immune system