95. Jahresversammlung der Deutschen Gesellschaft für Hals-Nasen-Ohren-Heilkunde, Kopf- und Hals-Chirurgie

Prof.

NUR Wissenschaftliches Programm/ ONLY Scientific Program

Dr. med.

PD Dr.

Univ.-Prof. Dr. med.

Prof. Dr.

Targeted therapy with the antibody-drug conjugate sacituzumab-govitecan as a promising treatment strategy for head and neck squamous cell carcinoma

Zielgerichtete Therapie mit dem Antikörper-Wirkstoff-Konjugat Sacituzumab-Govitecan als vielversprechende Behandlungsstrategie für Plattenepithelkarzinome des Kopf- und Hals-Bereiches

Introduction: Systemic treatment of head and neck squamous cell carcinoma (HNSCC) is based on chemotherapy, anti-PD-1 immunotherapy and EGFR inhibition. New targeted therapies with antibody-drug conjugates (ADC) have improved progression-free and overall survival in other oncological entities, such as the ADC sacituzumab govitecan (SG) directed against the cell surface antigen TROP2 in metastatic triple-negative breast cancer. There is evidence that HNSCC tumors express TROP2 and other ADC targets. Methods: Tissue samples of HNSCC tumors (n = 26) and normal adjacent tissue (NAT) (n = 11) were analyzed by immunhistochemistry and 8 HNSCC cell lines were examined by flow cytometry for TROP2 expression. The response of these 8 HNSCC cell lines to treatment with SG was examined in vitro using the calorimetric XTT assay. Employing the chicken embryo chorioallantoic membrane assay intravenous treatment and topical treatment with SG was applied to HNSCC xenografts in vivo. Results: The HNSCC tumors showed heterogeneous expression of TROP2, with most having moderate (12 of 26) or strong staining intensity (10 of 26). None of the NAT showed a strong immunohistochemical detection response (0 of 11). In vitro, all HNSCC cell lines responded to treatment with SG. In vivo, SG significantly reduces tumor cell burden and cell proliferation and significantly induces apoptosis in HNSCC xenografts. Conclusion: Since TROP2 is expressed in all examined HNSCC tumors and a clear response of HNSCC cell lines and xenografts to SG treatment was shown in vitro and in vivo, SG appears to be a promising treatment strategy for HNSCC.Einleitung: Die systemische Behandlung von Plattenepithelkarzinomen des Kopf- und Halsbereiches (HNSCC) basiert auf Chemotherapie, Anti-PD-1-Immuntherapie und EGFR-Inhibition. Zielgerichtete Therapien mit Antikörper-Wirkstoff-Konjugaten (ADC) haben das progressionsfreie und das Gesamtüberleben in anderen onkologischen Entitäten verbessert, wie beispielsweise das gegen TROP2 gerichtete ADC Sacituzumab Govitecan (SG) bei metastasiertem dreifach-negativem Brustkrebs. Es gibt Hinweise darauf, dass HNSCC-Tumoren TROP2 und andere ADC-Zielstrukturen exprimieren. Methoden: HNSCC-Tumoren (n = 26) und an Tumoren grenzendes Normalgewebe (NAT) (n = 11) wurden immunhistochemisch analysiert und in 8 HNSCC-Zelllinien wurde mittels Durchflusszytometrie die TROP2-Expression bestimmt. Die Reaktion dieser 8 Zelllinien auf die Behandlung mit SG wurde mit dem XTT Assay in vitro untersucht. Mit dem Chorion-Allantois-Membran Assay wurden Effekte der Behandlung von HNSCC-Xenografts mit SG in vivo beleuchtet. Ergebnisse: Die HNSCC-Tumoren zeigten eine heterogene Expression von TROP2, wobei die meisten eine mäßige (12 von 26) oder starke Färbeintensität (10 von 26) aufwiesen. Keine der NAT wies eine starke Expression auf (0 von 11). In vitro sprachen alle HNSCC-Zelllinien auf die Behandlung mit SG an. Die Behandlung mit SG bewirkt eine signifikante Reduktion der Tumorzelllast, eine signifikante Verringerung der Proliferationsrate und eine signifikante Apoptose-Induktion in den HNSCC-Xenografts in vivo. Schlussfolgerung: Da TROP2 in allen untersuchten HNSCC-Tumoren vorkommt und sich in vitro und in vivo ein klares Ansprechen von HNSCC-Zelllinien und -Xenografts auf die Behandlung mit SG zeigte, erscheint SG als vielversprechende Strategie zur systemischen Behandlung von HNSCC.Die Autorinnen/Autoren geben an, dass kein Interessenskonflikt besteht.

Zielgerichtete Therapie mit dem Antikörper-Wirkstoff-Konjugat Sacituzumab-Govitecan als vielversprechende Behandlungsstrategie für Plattenepithelkarzinome des Kopf- und Hals-Bereiches

Experimentelle Onkologie

Kopf-Hals-Onkologie

Visual Abstract

Cal27-derived Exosomes for site-specific Immunomodulation of natural killer cells in head and neck Squamous Cell Carcinoma

Von Cal27 abgeleitete Exosomen zur ortsspezifischen Immunmodulation natürlicher Killerzellen bei Plattenepithelkarzinomen im Kopf- und Halsbereich

Abstract HNSCC, being a global health concern, is the seventh most common cancer worldwide. The exosomes released by these tumor cells play a significant role in immunoregulation and HNSCC progression. However, the specific role of exosomes released by Cal27-HNSCC cells is yet to be studied Methods Cal27 cell lines are cultured in DMEM with 10% exosome-depleted FBS, for exosome isolation. Cell surface markers were determined on Cal27 tumor cells by flow cytometry. The exosomes are isolated from cell culture supernatants using size exclusion chromatography and the physical characteristics like size and morphology were measured using dynamic light scattering and transmission electron microscopy. The surface of these isolated exosomes was characterized for well-known tumor markers like Hsp70, CD9, CD63, and CD81 using flow cytometry. The binding affinity of surface Hsp70 positive exosomes to cmHsp70.1 monoclonal antibody (mAb) was determined by microscale thermophoresis and the influence of Cal27 derived exosomes on cytotoxic capacity of NK cells against Cal27 cells was investigated in cell death assays Results Cal27-derived exosomes, with an average size distribution of ~100 nm, exhibit various cell-surface markers and possess significant immunomodulatory effects on NK cells. Our study reveals that Cal27 cells, besides tetraspanins (CD9, CD63, and CD81), express different immunoregulatory ligands (eg, PD-L1, HLA-E, Hsp70) for NK cells. Upon co-incubation of monocytes with Cal27-derived exosomes, we observed an impaired immune response and cytotoxicity of treated NK cells against tumor cells Discussion Our findings highlight key implications for understanding HNSCC progression and developing a site-specific therapeutic strategy to modulate anti-cancer immune responseThe authors declare that they have no conflict of interest.

Von Cal27 abgeleitete Exosomen zur ortsspezifischen Immunmodulation natürlicher Killerzellen bei Plattenepithelkarzinomen im Kopf- und Halsbereich

Bioenergetic depression in platelets of Head and Neck cancer patients

Bioenergetische Defizite von Thrombozyten bei Patienten mit Kopf-Hals-Tumoren

Introduction Metabolic reprogramming is a hallmark of cancer cells that promotes tumor initiation and progression, with mitochondrial function as a key determinant. Profound evidence is pointing to a detrimental influence of tumors on the surrounding tissue, but also tumor-induced systemic metabolic alterations are increasingly unraveled. In this way, blood cells can be affected. We thus, asked whether mitochondrial alterations can be assessed in cancer patient platelets as they serve as major mitochondria reservoir in blood. Material and methods We established a protocol to measure mitochondrial respiration of isolated permeabilized platelets with high-resolution respirometry (Oroboros Instruments). The study enrolled 15 head and neck squamous cell carcinoma (HNSCC) patients and 15 healthy donors. Further electron microscopy imaging was used to analyze platelet morphology. RNA-sequencing data of platelet isolates completed the analysis of mitochondrial deficits. Results RNA-sequencing of platelets from HNSCC patients indicated mitochondrial functional alterations. While platelet mitochondrial structures appeared similar, high-resolution respirometry revealed a reduced electron transfer capacity in platelet mitochondria from HNSCC patients compared to healthy donors. Conclusion We find reduced respiratory chain function and impaired oxidative phosphorylation of platelet mitochondria in HNSCC patients. Clearly, metabolic alterations in the blood residing platelets add to implement a systemic ketogenic phenotype in such cancers. This could therefore not only become an important diagnostic tool to detect mitochondria-linked diseases but also plausibly provide a basis for new therapeutic approaches concerning tumor initiation and progression.Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht.

Bioenergetische Defizite von Thrombozyten bei Patienten mit Kopf-Hals-Tumoren

Tumormarker

Unveiling the prognostic role of IFNAR signaling in neutrophils in HNC

Der Einfluss des IFNAR Signalings auf Neutrophile Granulozyten, und dessen prognostischer Wert für das Oropharynx Karzinom

Type I interferons (IFNs) are known for their strong anti-tumoral properties, but tumors established ways to evade interferon control, by cleaving receptors for type I IFNs (IFNAR) in tumor microenvironment, which has been demonstrated for colon cancer. Little is known about the regulation of IFNAR expression in head and neck cancer (HNC) and its effect on the immune regulation. We aimed to investigate the dynamics and effects of IFNAR loss in tumor tissue on the functionality of tumor-associated neutrophils (TANs) and its prognostic value for HNC. We evaluated IFNAR expression in tissues (tumor, normal mucosa and invasive front) of 589 patients with HNC via multicolor immunohistochemistry and its impact on the outcome. To evaluate how IFNAR degradation influences TANs activity, we used the murine model of oropharyngeal carcinoma that was injected into WT and IFNAR-deficient animals. Tumor tissue associated with poor 5 years prognosis for patients with HNC, showed significant decrease of IFNAR. Similarly in murine model, IFNAR expression was also lost on neutrophils during tumor progression. IFNAR-deficient neutrophils showed impaired anti-tumoral properties, such as cytotoxicity or T cell stimulation, while maintaining pro-tumoral functions. Preliminary data demonstrated heterogeneous expression of the IFN-regulated molecules in subpopulations of TANs in mice and humans. Unbiased computed analysis will further unveil the spatial interactions of TAN subpopulations with tumor cells and their impact on HNC prognosis.All in all, we demonstrated the relevance of interferon signaling for anti-cancer immunity and correlation of IFNAR loss with immune suppression in HNC. IFNAR expression and its downstream molecules in TANs can serve as a biomarker for HNC progression.Elahi Pouya, Rücker Robin, Pylaeva Ekaterina, Anthony Squire, Siakaeva Elena, Thiel Ilona, Antczak Joanna, Kürten Cornelius, Lang Stephan, Jablonska Jadwiga

Der Einfluss des IFNAR Signalings auf Neutrophile Granulozyten, und dessen prognostischer Wert für das Oropharynx Karzinom

Temporary tumor cell reverse migration through EGFR stimulation: potential implications for HNSCC therapy

Temporäre Tumorzell-Rückmigration durch EGFR-Stimulation: potenzielle Implikationen für die HNSCC-Therapie

In squamous cell carcinomas of the upper aerodigestive tract (HNSCCs), metastasis plays a crucial role in determining prognosis. Current therapies primarily focus on slowing down tumor cell migration, while the direction of migration is scarcely investigated and insufficiently understood. The aim of this study was to investigate the influence of the epidermal growth factor (EGF) on the migration behavior of HNSCC cells. Three HNSCC cell lines (CAL-27, CLS-354, PE/CA-PJ15) were stimulated in vitro with EGF, and their movement into a gap was analyzed over 27 hours (migration). The influence on cell number (proliferation) and size over 7 days was separately examined, and EGFR expression was detected immunohistochemically. In two out of three cell lines, EGF treatment resulted in gap widening during the first 6 hours, although this gap later closed simultaneously with the negative control despite the increased distance. Initially, EGFR was expressed more strongly in the membrane between the cells than at the migration front toward the free gap. Addition of EGF led to EGFR internalization, increased tumor cell size, and reduced cell count. The growth factor accelerated migration and temporarily induced the reverse migration of tumor cells. The reduction in proliferation and cell enlargement further supported the increased motility caused by EGF. The direction of migration appears to depend on EGFR distribution in the membrane. Cellular receptor localization and the mechanisms of reverse migration should be further investigated. A deeper understanding of these processes could serve as the basis for new therapeutic approaches, particularly in controlling tumor growth and metastasis.Bei Plattenepithelkarzinomen des oberen aerodigestiven Traktes (HNSCCs) ist die Metastasierung für die Prognose mitentscheidend. Aktuelle Therapien fokussieren primär auf die Verlangsamung der Tumorzellmigration, wohingegen die Migrationsrichtung kaum untersucht und nur unzureichend verstanden ist. Ziel dieser Arbeit war die Untersuchung des Einflusses des epithelialen Wachstumsfaktors (EGF) auf das Migrationsverhalten von HNSCC Zellen. Drei HNSCC-Zelllinien (CAL-27, CLS-354, PE/CA-PJ15) wurden in-vitro mit EGF stimuliert und die Bewegung in einen freien Spalt über 27 h analysiert (Migration). Der Einfluss auf Zellzahl (Proliferation) und -größe über 7 Tage wurde separat untersucht und die EGFR-Expression immunhistochemisch detektiert. Bei zwei von drei Zelllinien zeigte sich nach EGF-Behandlung in den ersten 6 h eine Spalt-erweiterung, wobei sich dieser später trotz größerer Distanz zeitgleich mit der Negativkontrolle schloss. EGFR wurde zunächst stärker in der Membran zwischen den Zellen exprimiert als an der Migrationsfront zum freien Spalt hin. Eine Zugabe von EGF führte zur EGFR-Internalisierung, erhöhter Tumorzellgröße und verringerter Zellzahl. Der Wachstumsfaktor beschleunigte die Migration und führte zur vorübergehenden Rückmigration der Tumorzellen. Die Reduktion der Proliferation und Zellvergrößerung belegt die Steigerung der Motilität durch EGF zusätzlich. Die Migrationsrichtung scheint von der EGFR-Verteilung in der Membran abzuhängen. Die zelluläre Lokalisation von Rezeptoren und die Mechanismen der Rückmigration sollten zukünftig näher untersucht werden. Ein tieferes Verständnis dieser Prozesse könnte die Grundlage neuer therapeutischer Ansätze sein, insbesondere im Hinblick auf die Kontrolle von Tumorwachstum und Metastasenbildung.keine

Temporäre Tumorzell-Rückmigration durch EGFR-Stimulation: potenzielle Implikationen für die HNSCC-Therapie

Development of in vitro and ex vivo models for testing of new therapeutic options in HNSCC

Entwicklung von in vitro und ex vivo Modellen für die Testung neuer Therapieoptionen im HNSCC

Introduction: Infiltration, differentiation and function of immune cells are crucial for an effective anti-tumour immune response. The metabolic tumour microenvironment decisively shapes the function and polarisation of infiltrating immune cells and thus represents a potential therapeutic target structure. Valid ex vivo and in vitro models reflecting this complex interplay are essential to identify possible therapeutic strategies. Material/Methods: We established a co-culture protocol of HNSCC spheroids with whole immune cell populations from peripheral blood for testing metabolic conditions or pharmacological modulators. For the transfer to the patient situation we developed an ex vivo model of freshly obtained tumour tissue cultivated in autologous serum to test various metabolic conditions and clinically applicable metabolic modulators. The tissue was processed to a single cell suspension and the immune infiltrate and function analysed. Results: As observed in primary tumours, glutamine restriction diminished T cell function; accordingly, treatment with the glutaminase inhibitor BPTES increased IFNγ production in a portion of patients. In addition, a partial reduction in the tumour-promoting cytokine IL-6 was observed. In contrast, treatment with the glutamine analogue DON, shown to positively impact anti-tumor response of T cells in mice, had a negative effect on human intra-tumoral T cell function. Conclusion: We were able to establish an in vitro tumour spheroid and ex vivo tumour fragment model for HNSCC in which effects of metabolic manipulation on tumour and immune cells can be tested. The ex vivo model is to be expanded by the addition of autologous immune cells in order to analyse effects on infiltration.Einleitung: Infiltration, Differenzierung und Funktion sind von entscheidender Bedeutung für eine effektive antitumorale Immunantwort. Das metabolische Tumormikromilieu prägt Funktion und Polarisierung infiltrierender Immunzellen entscheidend und stellt eine mögliche therapeutische Zielstruktur dar. Um Angriffspunkte zu identifizieren, sind valide in vitro und ex vivo Modelle, die das komplexe Zusammenspiel reflektieren, von großer Bedeutung. Material/Methoden: Für die Testung pharmakologischer Modulatoren bzw. metabolischer Bedingungen wurden Kokulturen aus HNSCC Sphäroiden mit allen Immunzellpopulationen aus peripherem Blut etabliert. Für die Übertragung in die Patientensituation wurde ein ex vivo Modell aus frisch gewonnenem Tumorgewebe in autologem Serum unter verschiedenen metabolischen Konditionen und klinisch anwendbarer metabolischer Modulatoren etabliert. Nach Aufarbeitung des Gewebes zur Einzelzellsuspension wurden Immuninfiltrat und ‑funktion analysiert. Ergebnisse: Wie in primären Tumoren beobachtet führte Glutaminrestriktion zur schlechteren T-Zellfunktion, dementsprechend erhöhte die Behandlung mit dem Glutaminaseinhibitor BPTES bei einem Teil der Patienten die IFNγ-Produktion und verminderte das tumorfördernde Zytokin IL-6. Dagegen wirkte sich die Behandlung mit dem Glutaminanalogon DON, welches im murinen System die T-Zell vermittelte Tumorimmunität positiv beeinflusste, negativ auf humane intratumorale T-Zellen aus. Fazit: Wir konnten ein ex vivo Tumorfragment- sowie ein in vitro Tumorsphäroid-Modell etablieren, in welchen die Auswirkungen einer metabolischen Manipulation auf Tumor- und Immunzellen getestet werden können. Das ex vivo Modell soll um die Zugabe autologer Immunzellen erweitert werden, um Effekte auf die Immuninfiltration zu analysieren.Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht.

Entwicklung von in vitro und ex vivo Modellen für die Testung neuer Therapieoptionen im HNSCC

Protein cargo and functional properties of salivary exosomes from head and neck cancer patients are stage dependent

Die Proteinladung und funktionellen Charakteristika von Speichelexosomen von Kopf-Hals-Tumorpatienten sind stadienabhängig

Introduction: Salivary exosomes have high potential as non-invasive diagnostic biomarkers for Head and Neck cancer (HNC). In this study, we investigated the protein cargo and functional impact of salivary exosomes from HNC patients and healthy donors (HD). Methods: Exosomes were isolated from the saliva of 19 HNC patients and 8 HDs by ultracentrifugation. The characterization of exosomes was conducted by Transmission electron microscopy (TEM), Nanoparticle tracking analysis (NTA), protein concentration, and Western blotting (WB). The cargo of immunomodulatory proteins (PD-L1, TGF-β, Fas-L) on exosomes from HNC patients and HDs was compared using spectral flow cytometry and Western blots. The functional assessment of exosomes included Annexin V apoptosis assays using CD8+ T cells and wound healing assays with human umbilical vein endothelial cells (HUVECs). Results: Salivary exosomes showed the typical shape and size in TEM and contained the typical markers for extracellular vesicles (e.g. TSG 101, CD 9). There was significantly higher particle and protein concentration in HNC patients than in HDs, particularly in progressed HNC. HNC patients carried more PD-L1, Fas-L and TGF-β than HDs, which was also associated with tumor stage and HPV status. There was a higher induction of apoptosis of CD8+ T cells after 24h co-incubation with salivary exosomes from HNC patients than HDs. Salivary exosomes of HNC stimulated HUVEC migration more than HDs. Discussion: These results indicate that the immunosuppressive and proangiogenic effects of salivary exosomes depend on the disease severity of HNC patients. To better assess the biomarker potential of salivary exosomes, further studies involving larger, serially-monitored patient cohorts are necessary.Einleitung: Speichelexosomen haben großes Potential nicht invasive diagnostische Biomarker für Kopf-Halstumore (HNC) zu werden. In dieser Studie wurde der Proteingehalt und die funktionellen Auswirkungen von Speichelexosomen von HNC-Patienten und gesunden Spendern (HDs) untersucht. Methoden: Exosomen wurden aus dem Speichel von 19 HNC-Patienten und 8 HDs durch Ultrazentrifugation isoliert. Die Charakterisierung der Exosomen erfolgte durch Transmissionselektronenmikroskopie (TEM), Nanopartikel-Tracking-Analyse (NTA), BCA und Western Blots. Immunmodulatorische Proteine (PD-L1, TGF-β, FAS-L) auf Speichelexosomen von HNC-Patienten und HDs wurden mittels Durchflusszytometrie und Western Blots verglichen. Die funktionelle Untersuchung von Exosomen umfasste Annexin-V-Apoptosetests mit CD8+ T-Zellen und Wundheilungsassays mit humanen Nabelvenenendothelzellen (HUVECs). Ergebnisse: Speichelexosomen zeigten im TEM die typische Form und Größe und enthielten die typischen Marker für extrazelluläre Vesikel. HNC-Exosomen enthalten signifikant höhere Partikel- und Proteinkonzentrationen, welche mit fortschreitendem Tumorstadium ansteigen. HNC-Patienten wiesen mehr PD-L1, Fas-L und TGF-β auf als HDs, was auch mit dem Tumorstadium und dem HPV-Status zusammenhing. Nach einer 24-stündigen Ko-Inkubation mit Speichelexosomen von HNC-Patienten wurde die Apoptose von CD8+ T-Zellen stärker induziert als bei HDs. Speichelexosomen von HNC stimulierten die HUVEC-Migration stärker als HDs. Diskussion: Diese Studie zeigt, dass die immunsuppressiven und proangiogenen Effekte der Speichelexosomen mit dem fortschreitenden Tumorwachstum korreliert. Um das Biomarker-Potenzial von Speichelexosomen besser beurteilen zu können, sind weitere longitudinale Studien mit größeren Patientenkohorten erforderlich.Die Autorinnen/Autoren geben an, dass kein Interessenskonflikt besteht.

Die Proteinladung und funktionellen Charakteristika von Speichelexosomen von Kopf-Hals-Tumorpatienten sind stadienabhängig

Characterization and functional analysis of exosomes derived from the supernatant of head and neck cancer cells: Implications for Immunomodulation

Introduction Head and neck squamous cell carcinoma (HNSCC) ranks as the seventh most common cancer worldwide. Exosomes play a key role in the progression of tumors including HNSCC. This study investigated the role of exosomes derived from the HNSCC cell line (UD5), with a focus on characterizing their surface markers and assessing their immunomodulatory potential on natural killer (NK) cells Methods Cell surface markers were determined on UD5 tumor cells by flow cytometry and extracellular vesicles were isolated from cell culture supernatants. Size and morphology were measured by dynamic light scattering and transmission electron microscopy, and typical exosomal surface markers were determined by flow cytometry. The binding affinity of Hsp70-positive exosomes to cmHsp70.1 mAb was assessed by microscale thermophoresis. The impact of exosomes on the cytotoxic activity of NK cells against UD5 cells was investigated in cell death assays. Results UD5 cells express immunosuppressive ligands for NK cells such as PD-L1 and HLA-E. Extracellular vesicles of UD5 cells present typical exosomal markers (CD9, CD63, CD81, Hsp70), PD-L1 and HLA-E, show a uniform size distribution of around 100 nm and bind the cmHsp70.1 mAb with a high affinity. A co-incubation of the exosomes with NK cells results in a down-regulation of the activatory receptor NKG2D. Discussion We characterized extracellular vesicles derived from the supernatant of the HNSCC cell line UD5 as exosomes, showed a phenotypical characterization of their surface markers and provided evidence for an immunosuppressive activity of tumor cell-derived exosomes to NK cells. Our findings provide valuable insights into the interaction of exosomes with NK cells and their potential to modulate anti-cancer immune responses.The authors declare that there is no conflict of interest.

Characterization and functional analysis of exosomes derived from the supernatant of head and neck cancer cells: Implications for Immunomodulation﻿

Development of anti-mHsp70 CAR cells targeting HNSCC tumors

Entwicklung von anti-mHsp70 CAR cells bei HNSCC Tumoren

Introduction Chimeric antigen receptors (CAR) consisting of an extracellular anti-body based single chain variable fragment (scFv) linked to a transmembrane and intracellular signaling domain can boost anti-tumor activities of effector cells. In this study, we focused on membrane-bound heat shock protein 70 (mHsp70) as a tumor specific target for the recognition by an anti-mHsp70 CAR in NK cell lines. We focused on targeting HNSCC tumors, due to limited treatment options available for late-stage tumors. Material/Methods The NK cell line KHYG1 and primary NK cells of a healthy donor were selected as CAR cell platforms and three HNSCC served as tumor target cells. The expression of activatory and inhibitory receptors and mHsp70 on the different NK cell types was performed by flow cytometry. The CAR expression was confirmed by confocal microscopy. Results Receptors expression pattern differed significantly in KHYG1 and primary NK cells. A successful transfection of scFv anti-mHsp70 CAR in KHYG1 cells could be confirmed. Due to a mHsp70 positivity of KHYG1 cells the anti-mHsp70 CAR resulted in fratricide among the effector cells. Therefore, we aim to continue with NK92 cells and primary NK cells lacking in mHsp70. Validation of the efficacy of anti-mHsp70 CAR will be performed by cytotoxicity assays. Discussion In contrast to CAR T cells causing partially severe side effects such as cytokine release syndrome (CRS) and graft-versus-host disease, our focus is to introduce NK cells of different origins as a platform for the transfection with an anti-mHsp70 to produce an over-the-counter NK cell-based drug.Conflict of Interest GM. Is founder and Chief Scientific Officer of multimmune GmbH

Entwicklung von anti-mHsp70 CAR cells bei HNSCC Tumoren

Univ.-Prof. Dr.med.

PD Dr. med

Tumour–associated inflammation in patients with advanced and relapsing hypopharyngeal carcinoma

Tumor-assoziierte Entzündung in Patienten mit fortgeschrittenem und rezidivierenden Hypopharynxkarzinomen

Therapy with immune checkpoint blockade became a first-line-therapy in relapsing head and neck squamous cell carcinoma (HNSCC) [1]. Neutrophils are a potential target in immunotherapy. For CD68 positive Macrophages expressing Factor 10 (FX) a prognostic role in tumorbiology is discussed [2]. For these reasons we aimed at better describing the tumour microenvironment (TME) with a focus on neutrophilic infiltration in the framework of either active acute or chronic inflammation and the prognostic role of macrophages in HPSCC (hypopharyngeal SCC) patients. In a retrospective analysis 32 HPSCC patients were included. Severity of chronic inflammation was scored depending on the accumulation of cells and lymph follicles. Acute inflammation was scored depending on the density of neutrophil granulocyte infiltration. We analysed the number of cells, which were double positive for CD68 and Factor 10 (FX). The primary tumour samples showed in 90% an active inflammation and 83% in relapsing tumours. Patients who developed a relapse or a distant metastasis had in 67% a score of ≥5 cells / mm² double positive for FX_CD68. We found evidence that HPSCC are immunogenic cancers with a strong accumulation of inflammatory cells at first diagnosis. Furthermore, an increased infiltration with CD68 positive macrophages is more often found in patients with relapse. These patterns deserve further study as prognostic markers to different treatment modalities. 1)Parmar K et al. Immunotherapy in head and neck squamous cell carcinoma: An updated review. Cancer Treat Res Commun. 2022;33:100649. 2)Zhang Y et al. Coagulation Factor X Regulated by CASC2c Recruited Macrophages and Induced M2 Polarization in Glioblastoma Multiforme. Front Immunol. 2018 Jul 6;9:1557. Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht.

Klinische Studien / Immunonkologie

Turning Neutrophils into guardians: Targeting neutrophil STAT3 signaling impairs tumor growth via the modulation of their anti-tumor bias

Turning Neutrophils into Guardians: Targeting neutrophil STAT3 signaling impairs tumor growth via the modulation of their anti-tumor bias

Stat3 signaling pathway play a crucial role in pro-tumoral polarization of neutrophils. In this study, we aim to investigate the role of neutrophil-specific STAT3 deficiency (NStat3-/-) on the tumor development in head and neck cancer (HNC).We found that, NStat3-/- mice show significantly impaired tumor growth as compared to WT counterparts. TANs of such mice show distinct anti-tumoral phenotype: with shortened survival, low CD62L expression and elevated expression of molecules involved in activation of adaptive immune. Neutrophils from tumor-draining lymph nodes (TDLNs) significantly downregulate PD-L1 expression and support proliferation of CD8+ T-cells. Stat3 signaling is an important driver of pro-tumoral activity of neutrophils. Here, we show that neutrophil-specific deficiency in Stat3 not only supports their anti-tumor activity, but also significantly impairs tumor development and growth, once again demonstrating the essential role of neutrophils in the regulation of tumorigenesis and suggesting them as powerful targets in cancer immunotherapy.The authors declare that there is no conflict of interest

Turning Neutrophils into Guardians: Targeting neutrophil STAT3 signaling impairs tumor growth via the modulation of their anti-tumor bias

Addressing the Three R principle in microvascular research – Measurements of Vascular development and proangiogenic factors with the HET cam assay

Das Drei-R-Prinzip in der mikrovaskulären Forschung - Messung der Gefäßentwicklung und der proangiogenen Faktoren mit dem HET Cam Assay

Considering animal experimentation, 3R (Replace, Reduce, Refine) strategies in microvascular research are urgently needed. The Henn&quot;s Egg Chorioallantoic Membrane (CAM) assay offers immune deficiency and a highly perfused membrane where growth and vascularization of tumors can be observed. Yet, standards on vascular differentiation and levels of proangiogenic factors, especially concerning the influence of therapies like radiation, have yet to be established. After opening of the eggshell, the vascular network was daily documented using in vivo microscopy and quantified using the deep learning based IKOSA®️ CAM Assay Application. From day 6 till 14 of development CAM samples were analyzed via qPCR. To determine therapeutic effects on microvasculature, measurements from native CAM tissue were compared to tissue 6 h after irradiation with 8 Gy on day 10, 12, or 14, respectively by measuring the expression of 84 angiogenic factors with the RT² Profiler qPCR array from Qiagen®️. VEGF showed high expression from day 7-11 of embryonic development followed by a decrease towards day 14. The response to irradiation was characterized by a dynamic range of expression of proangiogenic factors. CDH5, KDR, TBX1, TEK, and TIE1 were significantly decreased by more than 1.5 in their fold change in comparison to the non-irradiated control group. The vascular analyses revealed a noticeable decrease in the total vessel area 6 h after irradiation. The CAM assay can be considered a legitimate alternative for rodent experimentation in microvascular research. Besides obvious ethical, pecuniary, and bureaucratic advantages, samples can be studied non-invasively in high numbers and in a reproducible setting.Steigendes Bewusstsein bezüglich Tierversuchen verlangt nach alternativen Strategien im Bereich 3R (Replace, Reduce, Refine). Das Henn&quot;s Egg Chorionallantoic Membrane (CAM) assay bietet Immundefizienz und eine vaskularisierte Membran, auf der Tumorwachstum und -vaskularisierung analysiert werden können. Jedoch fehlt es an quantitativen Standards zur Gefäßentwicklung auf makroskopischer und molekularer Ebene. Auch sind (radiotherapeutische) Effekte bislang nicht beschrieben. Nach Eröffnen der Eierschale wurde das Gefäßnetzwerk täglich mittels in-vivo-Mikroskopie abgelichtet und mittels IKOSA®️ CAM Assay-Applikation deep-learning basiert quantifiziert. Ferner wurde an Tag 6-14 die Expression von 84 proangiogenen Faktoren mittels qPCR (RT² Profiler qPCR array von Qiagen®️) analysiert. Effekte von Bestrahlung mit 8 gray auf die Mikrogefässe wurden in gleicher Weise 6 Stunden nach der Exposition an Tag 10, 12 und 14 bestimmt. Es zeigte sich eine hohe VEGF-Expression von Tag 7-11 der embryonalen Entwicklung gefolgt von einem Abfall bis Tag 14. Nach Bestrahlung kam es zu einer sehr variablen Expression proangiogener Faktoren. CDH5, KDR, TBX1, TEK, and TIE1 waren in Vergleich zu Nativgewebe signifikant um mehr als das 1,5-fach erniedrigt. Die Gefäßanalyse zeigte ferner eine Abnahme der Gesamtgefäßfläche 6 Stunden nach der Bestrahlung. Der CAM-Assay sollte als legitime Alternative für Tierversuche im Bereich mikrovaskulärer Forschung betrachtet werden. Neben ethischen, finanziellen und bürokratischen Vorteilen sind Proben leicht zugänglich und das Gefäßsystem kann in hoher Stückzahl und reproduzierbar untersucht werden.Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht.

Das Drei-R-Prinzip in der mikrovaskulären Forschung - Messung der Gefäßentwicklung und der proangiogenen Faktoren mit dem HET Cam Assay

Immunomodulating effects of tumor-educated platelet derived exosomes and plasma exosomes of HNSCC patients

Eine vergleichende Studie zur Optimierung der Exosomenisolation aus dem Blut von Patienten mit Plattenepithelkarzinom des Kopf-Hals-Bereiches

An optimized method for the isolation of extracellular vesicles (EVs) including exosomes is crucial to increase the knowledge on the immunomodulatory effects of tumor derived exosomes (TEX). Hence, this study compares Sepharose and ExoSpin midi columns (Cell Guidance Systems) with classical ultracentrifugation to obtain TEX from blood of HNSCC patients. The blood of healthy donors served as a control. Protein concentration of the isolated TEX was determined by using BCA Colorimetric and Bradford assays. The quality of the isolated TEX was validated by Dynamic Light Scattering (DLS) for measuring size, Transmission Electron Microscopy (TEM) for morphology, and flow cytometry for the expression level of typical exosomal markers. Our results indicate that all methods enrich particles with sizes in the standard range for exosomes (30-150 nm). The evaluation of the expression of tetraspanins CD9, CD63, CD81 and Hsp70 by flow cytometry showed variable expression patterns with the different isolation methods. The highest protein concentrations were obtained after ultracentrifugation, followed by ExoSpin midi and Sepharose-based isolation which resulted in the highest purity and most homogeneous TEX enrichment as demonstrated by TEM. Sepharose is a reliable method for small-scale applications along with pre-selecting small extracellular vesicle subpopulations. In ultracentrifugation, while EVs contain impurities, the isolated particles might more closely reflect the whole range of vesicles present in the blood. ExoSpin results in higher protein concentrations and achieves a more homogenous vesicle population than ultracentrifugation. Future functional studies with EVs will tell which isolation method most likely reflects the in vivo function of exosomes in HNSCC patients.Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht.

Eine vergleichende Studie zur Optimierung der Exosomenisolation aus dem Blut von Patienten mit Plattenepithelkarzinom des Kopf-Hals-Bereiches

PD Dr. med.

Interaction between Neutrophil Granulocytes and tumor cells based on head and neck Carcinoma

Interaktion zwischen Neutrophilen Granulozyten und Tumorzellen von Kopf-Hals-Tumoren

Introduction Neutrophil granulocytes are the most abundant immune cell type in the tumor microenvironment (TME). An increased infiltration of tumor tissue and a high neutrophil-to-lymphocyte-ratio are associated with bad prognosis for patients with HNSCC. The aim of this study is to investigate the role of neutrophils in the peripheral blood in the progression of HNSCC and gain information on functional aspects of activation and change in phenotype mediated by cytokines of the tumor. Material and methods Venous blood was drawn from 5 HNSCC patients and 5 healthy donors. The isolated neutrophils were polarized into pro-tumor and anti-tumor phenotypes by adding TGF-β/CCL20 as well as LPS/IFN-γ. Cell activation and polarization was analyzed using flow cytometry to measure the protein expression on the cell surface. Results Stimulating neutrophils with TGF-β and CCL20 didn&quot;t show any change in cell activity. Neutrophils in the blood stream of tumor patients didn&quot;t show any overexpression of the marker CD66b. The stimulation with LPS and IFN-γ did show a significant shift in the surface expression of the proteins CD66b, CCR6 and CD62L, indicating an activation of the neutrophils, degranulation and polarization towards an anti-tumor phenotype. Discussion Neutrophil granulocytes of the peripheral blood can be affected in their immunologic function through cytokines and seem to differ from tissue-resident neutrophils in HNSCC concerning surface protein expression. An investigation of further influential factors seems promising.Einleitung In der Tumormikroumgebung (TME) sind Neutrophile Granulozyten die am häufigsten vorkommenden Immunzellen. Eine erhöhte Gewebeinfiltration und ein hohes Neutrophilen-zu-Lymphozyten-Verhältnis sind mit einer schlechten Prognose bei Patienten mit HNSCC verbunden. Ziel dieser Studie ist es, die Rolle der neutrophilen Granulozyten im Blut bei der Progression von HNSCC zu untersuchen und Informationen über funktionelle Aspekte der Aktivierung und phänotypische Veränderungen zu gewinnen, die durch Zytokine des Tumors vermittelt werden. Materialien und Methoden Venöses Blut wurde von insgesamt 5 HNSCC-Patienten und 5 gesunden Probanden gewonnen. Die daraus isolierten Neutrophilen wurden durch Zugabe von TGF-β/CCL20 sowie LPS/IFN-γ in pro-Tumor- und anti-Tumor-Phänotypen polarisiert. Durch Messung der exprimierten Oberflächenproteine der Neutrophilen mittels Durchflusszytometrie erfolgte eine Analyse der Zellaktivität und der Polarisierung. Ergebnisse Durch Stimulation der Proben mit TGF-β und CCL20 ließ sich keine Veränderung der Aktivität der Neutrophilen feststellen. Neutrophile des Blutstroms zeigten keine Überexpression des Markers CD66b. Die Stimulation mittels LPS und IFN-γ konnte einen signifikanten Shift in der Oberflächenexpression der Proteine CD66b, CCR6 und CD62L bewirken, was auf eine Aktivierung und Degranulation der Zelle sowie eine Polarisierung in einen anti-tumorösen Phänotyp hinweist. Diskussion Die neutrophilen Granulozyten des peripheren Blutes bei HNSCC lassen sich durch Zytokine in ihrer immunologischen Funktion verändern und unterscheiden sich scheinbar in ihrer Proteinexpression gegenüber gewebsresidenten Neutrophilen bei HNSCC. Eine Untersuchung weiterer Einflussfaktoren scheint vielversprechend.Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht.

Interaktion zwischen Neutrophilen Granulozyten und Tumorzellen von Kopf-Hals-Tumoren

Prof. Dr. med.

Comparing nanocellulose (NC)-based vs gelatin methacrylate (GelMA) bioink for fabricating a 3D bioprinted head and neck squamous cell carcinoma (HNSCC) model

Comparing nanocellulose (NC)-based vs gelatin methacrylate (GelMA) bioink for fabricating a 3D bioprinted head and neck squamous cell carcinoma (HNSCC) model

Introduction3D-bioprinted preclinical tumour model enables the fabrication of spatially defined tumour microenvironment (TME) mimicking the in-vivo situation. It is challenging to opt a bioink that is biocompatible, with minimum to no cytotoxicity &amp; inflammatory-response. Here, we compare tunicate-derived nanocellulose (NC) against widely used semi-synthetic, gelatin methacrylate (GelMA) biomaterial for best mirroring HNSCC TME via bioprinting technology. MethodsUsing HNSCC-lines (UM-SCC-14C, 11B &amp; 22B), we bioprinted HNSCC cell-laden bioinks in NC-based hydrogels (TEMPO-oxidized NC (TEMPO-NC) &amp; carboxymethylated NC (Carboxy-NC)) comparing to GelMA in alginate (GelMAA). Culturing bioconstructs for 21days in tumour media, we investigated bioink compatibility subjecting to a luminescence-based viability assay &amp; immunofluorescence imaging for E-cadherin (n-3) within the 3D architecture. ResultsOver 21 days, UM-SCC-22B (metastatic) cell-laden constructs showed significantly higher viability than 11B &amp; 14C constructs. HNSCC cells in Carboxy-NC displayed significantly high viability than cells in TEMPO-NC. Over this period, viability in Carboxy-NC were comparable to GelMAA. HNSCC cells in Carboxy-NC constructs formed cell clusters indicative of cell proliferation. Overall, all HNSCC constructs expressed E-cadherin, an epithelial marker. DiscussionDifferent cell lines showed differing behaviour reflecting their origin. The differences in bio-chemical &amp; mechanical properties resulted in differing viability &amp; proliferation, demonstrating carboxymethyl backbone within Carboxy-NC supports viability to a similar level as GelMAA. Given, NC bioinks endure minimum to no inflammatory-response, Carboxy-NC is preferred over GelMAA, while establishing a multicellular HNSCC TME model.The authors declare that there is no conflict of interest

Comparing nanocellulose (NC)-based vs gelatin methacrylate (GelMA) bioink for fabricating a 3D bioprinted head and neck squamous cell carcinoma (HNSCC) model

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Experimentelle Kopf-Hals-Onkologie 2