Effekte von 2-Hydroxyglutarat auf humane Immunzellen

Somatische Mutationen der Isozitratdehydrogenase (IDH) 1 und 2 wurden in verschiedenen Tumorentitäten nachgewiesen. Das mutierte Enzym IDH1/2 erlangte die Fähigkeit, α-Ketoglutarat in den Onkometaboliten 2-Hydroxyglutarat (2-HG) zu konvertieren. Bei der akuten myeloischen Leukämie (AML) ist bekannt, dass die Mutation der IDH1/2 positiv mit der Tumorprogression und einem schlechteren Outcome korreliert. Im Gegensatz dazu ist für Glioma WHO II und III und sekundäre Glioblastoma beschrieben, dass die Mutation mit einer guten Prognose korreliert. Auch beim Mamma Karzinom konnten erhöhte Level von 2-HG nachgewiesen werden, auch wenn keine Mutation der IDH vorlag und hier beeinflusste die Höhe des 2-HG-Levels die Prognose ebenfalls negativ. Bisher wird angenommen, dass 2-HG vor allem zu epigenetischen Veränderungen führt und somit die Prognose der Patienten beeinflusst. Aufgrund der wichtigen Rolle von Immunzellen für die Tumorentstehung und -entwicklung, sollte im Rahmen der Arbeit der Effekt von 2-HG auf Immunzellen untersucht werden. Hierbei sollten insbesondere dendritische Zellen (DC) untersucht werden, da diese Antigen präsentierenden Zellen zum einen die Verbindung zwischen angeborener und adaptiver Immunität bilden und zum anderen essentiell sind für die Initiierung einer effektiven Anti-Tumor-Immunantwort. Vordaten der Arbeitsgruppe zeigten, dass 2-HG die Interleukin (IL)-12p70 Sekretion von DC signifikant hemmt. In der Arbeit sollte diese verminderte Sekretion bestätigt und der Mechanismus der Hemmung anhand verschiedener Parameter näher untersucht werden. Zudem sollte die Expression der Interleukin Untereinheiten p35 und p40 auf mRNA Ebene, sowie die Biosynthese der Untereinheiten des Interleukins unter Einfluss von 2-HG analysiert werden. Da eine effektive Differenzierung der DC die Basis der DC Aktivität darstellt, sollten weitere mögliche Effekte auf die Differenzierung und Aktivierung der DC untersucht werden. In der Arbeit konnte gezeigt werden, dass das Dinatriumsalz von 2-HG spezifisch die IL-12p70 Produktion von LPS-aktivierten DC beeinflusst, jedoch keine Veränderung der Produktion anderer Zytokine bewirkt. Es zeigte sich zwar eine leichte Erhöhung der IL-10 Konzentration sowie eine leichte Reduktion der TNF Konzentration im Überstand der Zellen, diese Veränderungen waren jedoch nicht signifikant. Auch die Genexpression der IL-12p70 Untereinheiten p35 und p40 zeigte sich durch Na2-2-HG reduziert, jedoch ohne Signifikanz. Auf Proteinebene, mittels Durchflusszytometrie untersucht, führte Na2-2-HG zu einer Reduktion des IL-12p70-Dimers sowie zur Verminderung der p40 Untereinheit. Zur Verifizierung dieser Beobachtung sollten noch weitere Untersuchungen folgen. In der MLR schien sich ein leichter Effekt vor allem durch das L-Enantiomer von Na2-2-HG im Vergleich zur LPS-Kontrolle vorzuliegen, v.a. bei einer DC-Zellzahl von 5000 pro Well, da im Rahmen meiner Arbeit jedoch nur ein Versuch durchgeführt wurde, müssen weitere Versuche folgen. Bei der Untersuchung des Stoffwechsels der DC, wurden das Enzym Indolamin 2,3-Dioxigenase (IDO), die Atmung und die Laktatproduktion der Zellen unter Einfluss von Na2-2-HG analysiert. Die Untersuchung von IDO sowie der Laktatproduktion zeigten keinen Effekt von Na2-2-HG auf diese Stoffwechselwege. Die Untersuchung der Atmung der Zellen mittels PreSens ergab eine leichte Verstärkung der Atmung nach ca. drei Stunden, dieser Effekt war aber nur vorübergehend und sollte in weiteren Versuchen genauer untersucht werden. Um den Mechanismus der Beeinflussung des Zellmetabolismus durch Na2-2-HG genauer zu untersuchen, wurden erste Analysen von LPS-Signaltranduktionswegen mittels WesternBlot durchgeführt. Weder die Degradation von IκBα, noch dessen Phosphorylierung wurden hierbei durch Na2-2-HG beeinflusst. Auch bei der Untersuchung der MAPK Akt und p38 und deren Phosphorylierung konnte kein Effekt von Na2-2-HG festgestellt werden. Da in dieser Arbeit der spezifische Effekt von Na2-2-HG auf die IL-12p70 Produktion in DC bestätigt werden konnte, bei den weiteren Untersuchungen sich jedoch nur bei der Zellatmung ein signifikanter Effekt durch den Metaboliten ergab, sollte in weiteren Experimenten der mögliche Zusammenhang zwischen beiden Effekten analysiert werden

Can Exercise Enhance the Efficacy of Checkpoint Inhibition by Modulating Anti-Tumor Immunity?

Immune checkpoint inhibition (ICI) has revolutionized cancer therapy. However, response to ICI is often limited to selected subsets of patients or not durable. Tumors that are non-responsive to checkpoint inhibition are characterized by low anti-tumoral immune cell infiltration and a highly immunosuppressive tumor microenvironment. Exercise is known to promote immune cell circulation and improve immunosurveillance. Results of recent studies indicate that physical activity can induce mobilization and redistribution of immune cells towards the tumor microenvironment (TME) and therefore enhance anti-tumor immunity. This suggests a favorable impact of exercise on the efficacy of ICI. Our review delivers insight into possible molecular mechanisms of the crosstalk between muscle, tumor, and immune cells. It summarizes current data on exercise-induced effects on anti-tumor immunity and ICI in mice and men. We consider preclinical and clinical study design challenges and discuss the role of cancer type, exercise frequency, intensity, time, and type (FITT) and immune sensitivity as critical factors for exercise-induced impact on cancer immunosurveillance

Expression of pH-Sensitive TRPC4 in Common Skin Tumors

TRPCs (transient receptor potential classical or cation channels) play a crucial role in tumor biology, especially in the Ca2+ homeostasis in cancer cells. TRPC4 is a pH-sensitive member of this family of proteins. As solid tumors exhibit an inversed pH-gradient with lowered extracellular and increased intracellular pH, both contributing to tumor progression, TRPC4 might be a signaling molecule in the altered tumor microenvironment. This is the first study to investigate the expression profiles of TRPC4 in common skin cancers such as basal cell carcinoma (BCC), squamous cell carcinoma (SCC), malignant melanoma (MM) and nevus cell nevi (NCN). We found that all SCCs, NCNs, and MMs show positive TRPC4-expression, while BCCs do only in about half of the analyzed samples. These data render TRPC4 an immunohistochemical marker to distinguish SCC and BCC, and this also gives rise to future studies investigating the role of TRPC4 in tumor progression, and especially metastasis as BCCs very rarely spread and are mostly negative for TRPC4

Combined Metabolic Targeting With Metformin and the NSAIDs Diflunisal and Diclofenac Induces Apoptosis in Acute Myeloid Leukemia Cells

The accelerated metabolism of tumor cells, inevitable for maintaining high proliferation rates, is an emerging target for tumor therapy. Increased glucose and lipid metabolism as well as mitochondrial activity have been shown in solid tumors but also in leukemic cells. As tumor cells are able to escape the blockade of one metabolic pathway by a compensatory increase in other pathways, treatment strategies simultaneously targeting metabolism at different sites are currently developed. However, the number of clinically applicable anti-metabolic drugs is still limited. Here, we analyzed the impact of the anti-diabetic drug metformin alone or in combination with two non-steroidal anti-inflammatory drugs (NSAIDs) diclofenac and diflunisal on acute myeloid leukemia (AML) cell lines and primary patient blasts. Diclofenac but not diflunisal reduced lactate secretion in different AML cell lines (THP-1, U937, and KG-1) and both drugs increased respiration at low concentrations. Despite these metabolic effects, both NSAIDs showed a limited effect on tumor cell proliferation and viability up to a concentration of 0.2 mM. In higher concentrations of 0.4–0.8 mM diflunisal alone exerted a clear effect on proliferation of AML cell lines and blocked respiration. Single treatment with the anti-diabetic drug metformin blocked mitochondrial respiration, but proliferation and viability were not affected. However, combining all three drugs exerted a strong cytostatic and cytotoxic effect on THP-1 cells. Comparable to the results obtained with THP-1 cells, the combination of all three drugs significantly reduced proliferation of primary leukemic blasts and induced apoptosis. Furthermore, NSAIDs supported the effect of low dose chemotherapy with cytarabine and reduced proliferation of primary AML blasts. Taken together we show that low concentrations of metformin and the two NSAIDs diclofenac and diflunisal exert a synergistic inhibitory effect on AML proliferation and induce apoptosis most likely by blocking tumor cell metabolism. Our results underline the feasibility of applying anti-metabolic drugs for AML therapy

MCT4 blockade increases the efficacy of immune checkpoint blockade

Background & Aims Intratumoral lactate accumulation and acidosis impair T-cell function and antitumor immunity. Interestingly, expression of the lactate transporter monocarboxylate transporter (MCT) 4, but not MCT1, turned out to be prognostic for the survival of patients with rectal cancer, indicating that single MCT4 blockade might be a promising strategy to overcome glycolysis-related therapy resistance. Methods To determine whether blockade of MCT4 alone is sufficient to improve the efficacy of immune checkpoint blockade (ICB) therapy, we examined the effects of the selective MCT1 inhibitor AZD3965 and a novel MCT4 inhibitor in a colorectal carcinoma (CRC) tumor spheroid model co-cultured with blood leukocytes in vitro and the MC38 murine CRC model in vivo in combination with an antibody against programmed cell death ligand-1(PD-L1). Results Inhibition of MCT4 was sufficient to reduce lactate efflux in three-dimensional (3D) CRC spheroids but not in two-dimensional cell-cultures. Co-administration of the MCT4 inhibitor and ICB augmented immune cell infiltration, T-cell function and decreased CRC spheroid viability in a 3D co-culture model of human CRC spheroids with blood leukocytes. Accordingly, combination of MCT4 and ICB increased intratumoral pH, improved leukocyte infiltration and T-cell activation, delayed tumor growth, and prolonged survival in vivo. MCT1 inhibition exerted no further beneficial impact. Conclusions These findings demonstrate that single MCT4 inhibition represents a novel therapeutic approach to reverse lactic-acid driven immunosuppression and might be suitable to improve ICB efficacy

Qualität in der Befundung von Kopf- und Halssonografien an Universitätskliniken - eine Stichprobe

Background Ultrasound diagnostics are widely used and are standard for radiologists, otolaryngologists, and oral and maxillofacial surgeons in the diagnostic work-up of various pathologies. There is agreement that digital documentation is urgently needed at present to improve and standardize the quality of sonographic documentation. There are more and more publications on the implementation of standardized documentation of findings in imaging diagnostics, including head and neck sonography. Objective The present work aims to determine the quality of routine head and neck sonography findings on a random basis, according to the criteria of the Bavarian Association of Statutory Health Insurance Physicians (KVB) at a selection of German university otolaryngology departments (ENT). Materials and methods A total of 70 randomly selected anonymized written findings including image documentation from seven ENT departments were retrospectively analyzed by an experienced KVB examiner concerning fulfilment of KVB criteria. The data were evaluated descriptively. Results Of the 70 reports, 69 were eligible for evaluation. The average documentation completeness was 80.6%. A total of 9 findings were correctly documented in full (13%). The documentation completeness of the individual departments was sorted in ascending order from 68.1% to 93%. With 88.5% vs. 75%, the hospitals with a structured report showed a higher level of completeness. In 75% of the cases the hospitals with structured reports also had digital solutions for reporting and image archiving. Conclusion In general, there is potential for optimization regarding the completeness and quality of routinely prepared head and neck sonography findings at the selected university ENT departments. The implementation of structured reporting masks and the conversion of analogue documentation into digital solutions as well as digital networking with the hospital information systems, picture archiving and communication systems should be promoted. Supervision by senior doctors is required to ensure the quality of findings of inexperienced colleagues and to help to achieve standards in reporting

Can Exercise Enhance the Efficacy of Checkpoint Inhibition by Modulating Anti-Tumor Immunity?

Immune checkpoint inhibition (ICI) has revolutionized cancer therapy. However, response to ICI is often limited to selected subsets of patients or not durable. Tumors that are non-responsive to checkpoint inhibition are characterized by low anti-tumoral immune cell infiltration and a highly immunosuppressive tumor microenvironment. Exercise is known to promote immune cell circulation and improve immunosurveillance. Results of recent studies indicate that physical activity can induce mobilization and redistribution of immune cells towards the tumor microenvironment (TME) and therefore enhance anti-tumor immunity. This suggests a favorable impact of exercise on the efficacy of ICI. Our review delivers insight into possible molecular mechanisms of the crosstalk between muscle, tumor, and immune cells. It summarizes current data on exercise-induced effects on anti-tumor immunity and ICI in mice and men. We consider preclinical and clinical study design challenges and discuss the role of cancer type, exercise frequency, intensity, time, and type (FITT) and immune sensitivity as critical factors for exercise-induced impact on cancer immunosurveillance

Zusammenspiel von Tumormetabolismus und Immuninfiltrat im Kopf-Hals-Plattenepithelkarzinom

Einleitung Im HNSCC sind niedrige T-Zelllevel bekannt und korrelieren mit schlechter Prognose. Zudem haben Checkpointinhibitoren eine limitierte Wirkung. Das Ansprechen auf Immuntherapie ist abhängig von der T-Zellinfiltration und -funktion, welche vom Tumormetabolom beeinflusst werden. Wir nehmen an, dass ein veränderter Tumormetabolismus, abhängig von Stadium und Grading, Immunzellinfiltration und -funktion beeinflusst. Ziel Analyse des metabolischen Profils im HNSCC und Korrelation mit Immunzellinfiltrat und –funktion erfolgten abhängig von Stadium und Grading. Da sich der Glutamin- und Glukosemetabolismus signifikant verändert zeigten, wurde der Einfluss von Glutamin- und Glukosekonzentrationen, wie im Tumor gemessen, in einer mixed leukocyte reaction (MLR) untersucht. Therapeutische Optionen wurden mit einem Kokulturmodell aus frischen HNSCC-Tumorproben und Leukozyten getestet. Material/ Methoden Immuninfiltrat und -funktion wurden durchflusszytometrisch, das metabolische Profil massenspektrometrisch analysiert. Ergebnisse Die veränderten Glutaminlevel korrelierten positiv mit der T-Zell- und negativ mit der myeloiden Zellzahl und die Glutaminlevel waren abhängig von Tumorstadium und -grading. Eine Hemmung des Glutaminmetabolismus in Tumorfragmenten verbesserte die T-Zellaktivität und verminderte die Bildung des protumorigenen IL-6. überraschenderweise verstärkten hohe Glutaminlevel den immunsuppressiven Effekt von Laktat in der MLR, hier müssen weitere Untersuchungen folgen. Fazit Wir konnten einen Einfluss des Tumormetabolismus auf Immuninfiltration und -funktion zeigen. Zudem fördert die Hemmung der Glutaminolyse oder Glykolyse die antitumorale Immunität. Die Ergebnisse unterstützen mögliche Kombinationen von Checkpoint- mit antimetabolischer Therapie im HNSCC

Routine restaging after primary non-surgical treatment of laryngeal squamous cell carcinoma—a review

Purpose Treatment of patients with laryngeal squamous cell carcinoma with radiotherapy or chemoradiation is an established alternative to laryngeal surgery in many cases, but particularly for advanced tumors without cartilage invasion. Imaging modalities face the challenge of distinguishing between posttherapeutic changes and residual disease in the complex anatomic subsite of the larynx. Guidelines concerning restaging of head and neck squamous cell carcinomas (HNSCC) are presented by the National Comprehensive Cancer Network (NCCN) and other national guidelines, but clearly defined recommendations for routine restaging particularly for laryngeal cancer are lacking. Methods A systematic search was carried out in PubMed to identify studies evaluating routine restaging methods after primary non-surgical treatment of laryngeal squamous cell carcinoma from 2009 to 2020. Results Only three studies were deemed eligible, as they included at least ≥50% patients with laryngeal squamous cell carcinoma and evaluated imaging modalities to detect residual cancer. The small number of studies in our review suggest restaging with fluoro-deoxy-glucose positron-emission tomography/computed tomography (FDG PET/CT) 3 months after initial treatment, followed by direct laryngoscopy with biopsy of the lesions identified by FDG PET/CT. Conclusion Studies evaluating restaging methods after organ-preserving non-surgical treatment of laryngeal carcinoma are limited. As radiotherapy (RT), chemoradiotherapy (CRT), systemic therapy followed by RT and radioimmunotherapy are established alternatives to surgical treatment, particularly in advanced laryngeal cancers, further studies are needed to assess and compare different imaging modalities (e.g. PET/CT, MRI, CT, ultrasound) and clinical diagnostic tools (e.g., video laryngoscopy, direct laryngoscopy) to offer patients safe and efficient restaging strategies. PET or PET/CT 3 months after initial treatment followed by direct laryngoscopy with biopsy of the identified lesions has the potential to reduce the number of unnecessary laryngoscopies

D-2-Hydroxyglutarate and L-2-Hydroxyglutarate Inhibit IL-12 Secretion by Human Monocyte-Derived Dendritic Cells

Mutations in isocitrate dehydrogenase (IDH) or a reduced expression of L-2-hydroxyglutarate (HG)-dehydrogenase result in accumulation of D-2-HG or L-2-HG, respectively, in tumor tissues. D-2-HG and L-2-HG have been shown to affect T-cell differentiation and activation; however, effects on human myeloid cells have not been investigated so far. In this study we analyzed the impact of D-2-HG and L-2-HG on activation and maturation of human monocyte-derived dendritic cells (DCs). 2-HG was taken up by DCs and had no impact on cell viability but diminished CD83 expression after Lipopolysaccharides (LPS) stimulation. Furthermore, D-2-HG and L-2-HG significantly reduced IL-12 secretion but had no impact on other cytokines such as IL-6, IL-10 or TNF. Gene expression analyses of the IL-12 subunits p35/IL-12A and p40/IL-12B in DCs revealed decreased expression of both subunits. Signaling pathways involved in LPS-induced cytokine expression (NFkB, Akt, p38) were not altered by D-2-HG. However, 2-HG reprogrammed LPS-induced metabolic changes in DCs and increased oxygen consumption. Addition of the ATP synthase inhibitor oligomycin to DC cultures increased IL-12 secretion and was able to partially revert the effect of 2-HG. Our data show that both enantiomers of 2-HG can limit activation of DCs in the tumor environment