Nicht-professionelle Phagozytose von Tumorzellen

Background and aims The phenomenon of one cell being internalized into another has been described in the literature for over a century, and has amongst others been observed in human tumour tissue sections. The term cell-in-cell (CiC) structure refers to this characteristic phenotype and has been used in the in vitro studies described below. The aim of this study is to investigate the molecular background of non-professional phagocytic activity of human tumour cells. Besides the possible engagement of external stimuli and specific molecules in the uptake process, we also wanted to study the fate of the internalized cell. Methods An assay including the human pancreatic cancer cell line BxPC-3 was established to investigate the molecular mechanism of CiC genesis. Cell samples were labelled green or red with a fluorescent membrane-permeable life cell stain. One of the so dyed samples was heat-treated in a water bath at various temperatures while the other sample remained untreated. Subsequently, the two samples were co-incubated and the adhesion tendency was analyzed by flow cytometry. The incorporation process was verified by confocal and fluorescence microscopy. Image analysis software was used for quantification of CiC events. The expression of various proteins was assessed using indirect immunofluorescence. Western blot was used to detect the expression of specific proteins after heat treatment. In order to study the fate of the internalized cell over 3 days, the nucleus of the necrotic cell was labelled with the thymidine analogue 5-ethynyl-2´-deoxyuridine (EdU) and imaged by fluorescence microscopy. Results Heat treatment in a water bath at various temperatures and subsequent incubation of heat-treated with untreated pancreatic tumour cells showed differences in adhesion and consequently varying CiC rates depending on temperature and time. An increase in temperature from 37 to 56°C as well as in co-incubation time (up to 4 - 5 h) enhanced the incorporation process. A higher expression of β-catenin, P-cadherin and E-cadherin – proteins with prominent roles in cell adhesion and motility – was investigated at contact sites between heat-treated and untreated cells. Hyperthermic exposure of cells, however, caused a loss of these proteins dependent on the temperature. Functional blocking of E-cadherin affected neither adhesion tendency nor CiC rates. The arrangement of the microtubule-organizing center (MTOC) within the cell did not seem to follow a distinct orientation pattern during the engulfment process. Furthermore, heat-treated cells, in contra-distinction to untreated cells, lost their ability to phosphorylate ezrin – a mechanism required for cytoskeletal reorganization. Within one day after internalization, the incorporated cells lost the integrity of their nuclear envelopes and died. Conclusion Although the molecular background of CiC formation is still not fully understood, our results suggest that numerous factors can influence non-professional phagocytosis by tumour cells. Necrosis that follows heat treatment provokes cell adhesion, and is a necessary preliminary stage in the formation of CiC structures. Moreover, the host-cell must be able to rearrange its membrane cytoskeleton by accumulating cell adhesion molecules at the contact site with the heat-treated cell. It has also been shown that higher levels of heat exposure lead to increased CiC rates. Although there is evidence that CiC formation is a targeted process, orientation of the MTOC as a possible mechanism for cell polarization seems rather unlikely. Whereas phosphorylation of ezrin – a capability limited only to viable cells – is considered to play a crucial role in CiC genesis, E-cadherin is probably not responsible for the essential cell-cell interaction. As the incorporated cells die within 24 h, the whole process might be a physiologically relevant elimination mechanism in tumour tissues that often include necrotic areas. Necrotic cells in cancer tissue usually evoke a host immune response against tumor growth by releasing pro-inflammatory damage-associated molecular pattern molecules, and CiC formation might be a strategy of preventing this. Previous studies have highlighted the prognostic relevance of CiC structures in numerous tumour tissues. However, the presence of CiC structures might have varying effects on patient survival depending on the tumour entity. Revealing the triggering mechanisms for cell uptake might help to evolve patient-tailored therapies for the improvement of survival rates.Hintergrund und Ziele Phänomene, bei denen Zellen vollständig in das Zytoplasma anderer Zellen aufgenommen werden, lassen sich seit über einem Jahrhundert in der Literatur zurückfinden und wurden z.B. in Tumorgewebsschnitten beschrieben. Der Begriff Zell-in-Zell (CIC) Struktur bezieht sich auf dieses Erscheinungsbild und wurde für die nachfolgend aufgeführten in vitro Studien nicht-professioneller Phagozytose von humanen Tumorzellen verwendet. Es wurde untersucht ob verschiedene Behandlungsweisen von Zellen den Aufnahmeprozess beeinflussen und in welcher Weise bestimmte Membranproteine an der Ausbildung von CiC Phänomenen beteiligt sind. Außerdem wurde das Schicksal der aufgenommenen Zelle studiert und mögliche Gründe für die Entstehung von CiC Strukturen im Tumorgewebe diskutiert. Methoden Um den Entstehungsmechanismus von CiC zu untersuchen wurde die humane Pankreaskarzinomzelllinie BxPC-3 verwendet. CiC Phänomene wurden in vitro generiert, indem eine hitzebehandelte Zellpopulation für einige Stunden mit einer unbehandelten Zellpopulation inkubiert wurde. Zellen wurden entweder mit einem rot oder einem grün fluoreszierenden, membranpermeablen Lebendzell-Farbstoff angefärbt. Eine der so gefärbten Proben wurde im Wasserbad bei verschiedenen Temperaturen hitzebehandelt, während die andere Probe unbehandelt blieb. Anschließend wurden die beiden Proben koinkubiert und die Adhäsionsfähigkeit zwischen behandelten und unbehandelten Zellen mit dem Durchflusszytometer gemessen. Der Prozess der Zell-Aufnahme wurde mithilfe von Konfokal- und Fluoreszenzmikroskopie analysiert. Eine Bild-Auswertungssoftware diente zur Quantifizierung von CiC Ereignissen. Die Expression verschiedener Proteine wurde mittels indirekter Immunfluoreszenz beurteilt. Western Blot wurde zur Detektion bestimmter Proteine nach Hitzebehandlung angewandt. Um das Schicksal der aufgenommenen Zelle über einen Zeitraum von drei Tagen mittels Fluoreszenzmikroskopie zu untersuchen, wurde der Zellkern der nekrotischen Zelle mit dem Thymidinanalogon 5-Ethynyl-2´-Deoxyuridine (EdU) markiert. Ergebnisse Hitzebehandlung im Wasserbad bei verschiedenen Temperaturen und anschließende Inkubation von hitzebehandelten mit unbehandelten Pankreastumorzellen, zeigte Unterschiede in der Adhäsion und als Folge unterschiedliche CiC Raten, abhängig von Temperatur und Zeit. Eine Steigerung der Temperatur von 37 auf 56°C, sowie eine Verlängerung der Koinkubationszeit (auf bis zu 4 - 5 h) förderten den Aufnahmeprozess. An den Kontaktstellen zwischen hitzebehandelten und unbehandelten Zellen wurde eine verstärkte Akkumulation von β-Catenin, P-Cadherin und E-Cadherin beobachtet – Proteine mit bedeutenden Rollen in Zelladhäsion und -beweglichkeit. Hyperthermiebehandlung hatte in Abhängigkeit von der Temperatur einen Verlust dieser Proteine zur Folge, was sowohl mit Immunfluoreszenzfärbung als auch im Western Blot gezeigt wurde. Eine funktionelle Blockade von E-Cadherin wirkte sich weder auf die Adhäsionsneigung noch auf die CiC Rate aus. Zu keinem Zeitpunkt des Aufnahmeprozesses schien die Orientierung des Mikrotubulus-Organisationszentrums (MTOC) in der vitalen Zelle bezüglich der hitzebehandelten Zelle gerichtet zu sein. Außerdem verloren hitzebehandelte im Gegensatz zu unbehandelten Zellen die Fähigkeit zur Phosphorylierung von Ezrin – ein notwendiger Mechanismus für die Umgestaltung des Zytoskeletts. Nach erfolgter Aufnahme durch eine vitale Zelle und innerhalb eines Tages starb die phagozytierte Zelle im Zytoplasma der lebenden Zelle, was durch den Integritätsverlust ihrer Kernmembran gezeigt werden konnte. Schlussfolgerungen Obwohl der Entstehungsmechanismus von CiC Ereignissen letztlich nicht vollständig geklärt ist, wurde beobachtet, dass zahlreiche Faktoren den Aufnahmeprozess beeinflussen können. Hitzebehandlung bei höheren Temperaturen führt zu gesteigerten CiC Raten. Durch Hyperthermiebehandlung hervorgerufene Nekrose fördert Adhäsion, eine wichtige Vorstufe der CiC Entstehung. Außerdem wird die Zell-Aufnahme durch die Fähigkeit der Wirtszelle zum aktiven Umbau ihres Membranzytoskeletts geregelt. Die vitale Zelle reichert Zelladhäsionsmoleküle an der Kontaktseite mit der hitzebehandelten Zelle an und nimmt sie anschließend vollständig in ihr Zytoplasma auf. Vieles deutet darauf hin, dass die CiC Entstehung ein gerichteter Prozess ist. Eine gezielte Ausrichtung des MTOC als möglicher Mechanismus der Zellorientierung ist jedoch unwahrscheinlich. Während der Phosphorylierung von Ezrin – eine Fähigkeit, die allein der vitalen Zelle vorbehalten ist – in der CiC Genese eine bedeutende Rolle zugeschrieben wird, ist E-Cadherin wahrscheinlich zumindest nicht für den ersten Zell-Zell Kontakt verantwortlich. Da die aufgenommene Zelle innerhalb von 24 h stirbt, kann der ganze Prozess als Eliminationsmechanismus für nekrotische Zellen angesehen werden und könnte deshalb eine wichtige physiologische Rolle in Tumorgeweben spielen, die häufig Areale nekrotischer Zellen beherbergen. Nekrotische Zellen rufen in Tumorgeweben normalerweise eine Immunantwort hervor, die sich gegen das Tumorwachstum richtet. Demzufolge könnte die Generierung von CiC Ereignissen ein Mechanismus sein, der die anti-Tumor Immunantwort unterdrückt. In früheren Studien wurde gezeigt, dass das Vorhandensein von CiC Strukturen in Tumorgeweben eine hohe prognostische Relevanz hat, sich jedoch abhängig von der Entität unterschiedlich auf das Überleben von Patienten auswirkt. Die Entdeckung der auslösenden Faktoren für die Zell-Aufnahme könnte es ermöglichen maßgeschneiderte Therapiestrategien zur Verbesserung des Patientenüberlebens zu entwickeln

Characterisation and prognostic value of tertiary lymphoid structures in oral squamous cell carcinoma

BACKGROUND: Oral squamous cell carcinomas are often heavily infiltrated by immune cells. The organization of B-cells, follicular dendritic cells, T-cells and high-endothelial venules into structures termed tertiary lymphoid structures have been detected in various types of cancer, where their presence is found to predict favourable outcome. The purpose of the present study was to evaluate the incidence of tertiary lymphoid structures in oral squamous cell carcinomas, and if present, analyse whether they were associated with clinical outcome. METHODS: Tumour samples from 80 patients with oral squamous cell carcinoma were immunohistochemically stained for B-cells, follicular dendritic cells, T-cells, germinal centre B-cells and high-endothelial venules. Some samples were sectioned at multiple levels to assess whether the presence of tertiary lymphoid structures varied within the tumour. RESULTS: Tumour-associated tertiary lymphoid structures were detected in 21 % of the tumours and were associated with lower disease-specific death. The presence of tertiary lymphoid structures varied within different levels of a tissue block. CONCLUSIONS: Tertiary lymphoid structure formation was found to be a positive prognostic factor for patients with oral squamous cell carcinoma. Increased knowledge about tertiary lymphoid structure formation in oral squamous cell carcinoma might help to develop and guide immune-modulatory cancer treatments

Presence of tumour high-endothelial venules is an independent positive prognostic factor and stratifies patients with advanced-stage oral squamous cell carcinoma

Accepted manuscript version. The final publication is available at Springer via http://dx.doi.org/10.1007/s13277-015-4036-4Background: Staging of oral squamous cell carcinoma is based on the TNM system, which has been deemed insufficient for prognostic purposes. Hence, better prognostic tools are needed to reflect the biological diversity of these cancers. Previously, high numbers of specialized blood vessels called high-endothelial venules have been reported to be associated with prolonged survival in patients with breast cancer. In this study, we analysed the prognostic value and morphological characteristics of tumour-associated high-endothelial venules in oral cancer. Methods: The presence of tumour-associated high-endothelial venules was evaluated by immunohistochemistry in 75 patients with oral squamous cell carcinoma and analysed with correlation to clinicopathological parameters, patients’ survival, and vessel morphology. Ten of the samples were analysed at multiple levels to evaluate intratumoural heterogeneity. Results: The presence of tumour-associated high-endothelial venules was found to be associated with lower disease-specific death in multivariate regression analyses (P=0.002). High-endothelial venules were present in all (n=53) T1-T2 tumours, but only in two-thirds (n=14) of the T3-T4 tumours. The morphology of high-endothelial venules was heterogeneous and correlated with lymphocyte density. High-endothelial venules were found to be distributed homogeneously within the tumours. Conclusion: We found the presence of tumour-associated high-endothelial venules to be an easy-to-use, robust, and independent positive prognostic factor for patients with oral cancer. Absence of these vessels in advancedstage tumours might identify patients with more aggressive disease. Evaluating the presence of tumour-associated high-endothelial venules might help to tailor the treatment of oral cancer patients to their individual need

Presence of high-endothelial venules correlates with a favorable immune microenvironment in oral squamous cell carsinoma

Oral squamous cell carcinomas (OSCC) are associated with a poor prognosis, which may be partly due to functional impairment of the immune response. Lymphocyte recruitment to the tumor site is facilitated by high-endothelial venules (HEV), whereas expression of programmed-death ligand 1 (PD-L1) can impair T cell function. Thus, we hypothesize that these factors are important in shaping the immune response in OSCC. In the present study, we characterized the immune infiltrate in formalin-fixed, paraffin-embedded tumor samples from 75 OSCC patients. We used immunohistochemistry to determine the distribution of immune cell subsets, HEV and PD-L1, as well as quantitative real-time polymerase chain reaction to assess the expression of inflammatory cytokines and chemokines associated with lymphocyte trafficking. Finally, we calculated correlations between the presence of immune cell subsets, the gene expression patterns, HEV, PD-L1 and the clinicopathological parameters including patient survival. The presence of HEV correlated with increased number of CD3+ T cells and CD20+ B cells, higher levels of the chemokines CXCL12 and CCL21, and lower levels of CCL20, irrespective of the tumors’ T-stage. In univariate analysis, high levels of CD20+ B cells and CD68+ macrophages, positive HEV-status, and low T- and N-stages predicted longer patient survival. However, only the presence of HEV and a low T-stage were independent positive prognosticators. This indicates that HEV are important mediators and a convenient marker of an antitumor immune response in OSCC. Our findings support HEV as a potential immunomodulatory target in OSCC. PD-L1 staining in tumor cells correlated with lower T-stage, increased infiltration of CD4+ cells and higher expression of several inflammation-related cytokines. Thus, OSCC tumors rich in CD4+ cells may preferentially respond to PD-1/PD-L1 blockade therapy

Spillbasert læring: motivasjon for å ta i bruk ny teknologi

Vitenskapelig foredrag på MNT-konferansen 16.03. - 17.03.23, Stavanger: https://www.uis.no/nb/det-teknisk-naturvitenskapelige-fakultet/mnt-konferansen-2023.Spillbasert læring bruker prinsipper fra spill for å engasjere og motivere studenter i sin læring. I kombinasjon med innsikter om effektive læringsstrategier kan spillbaserte læringsressurser sikre både motivasjon til å lære og en effektiv læringsprosess. En forutsetning for å lykkes med introduksjon av ny teknologi generelt og spillbaserte læringsressurser spesifikt er at teknologien aksepteres av studentene og at teknologien er vel egnet til hva den brukes for. CranialGame er en nyutviklet digital spillbasert læringsressurs som tester om studentene mestrer navn, utspring og funksjoner til de tolv hjernenervene hos mennesker. I dette bidraget undersøker vi studentenes motivasjon til å ta i bruk CranialGame spesifikt og spillbaserte læringsressurser mer generelt. Et kull medisinstudenter har blitt introdusert til læringsressursen både individuelt og gruppevis i en modul om nevroanatomi. Studentene har deretter blitt oppfordret til å bruke CranialGame i sin læring. Gjennom en spørreundersøkelse har vi undersøkt bruken av CranialGame spesifikt og holdninger om spillbasert læring generelt. Vi bruker en kombinasjon av de to modellene Technology Acceptance Model og Task-Technology Fit for å måle studentenes opplevelse av velegnethet, brukervennlighet og nytte av spillbaserte læringsressurser. Selv om de aller fleste studentene fikk lyst til å bruke CranialGame etter dets introduksjon og var motivert til å bruke spillbaserte læringsressurser generelt, rapporterte bare omtrent en tredjedel at de faktisk har brukt denne læringsressursen. Vi diskuterer her hvorfor ikke flere studenter har tatt i bruk CranialGame, og mer generelt hvordan vi kan lykkes med å motivere studenter til å ta i bruk ny teknologi eller nye læringsressurser. Innsikt i studentenes motivasjon i møte med ny teknologi er viktig for planlegging og utvikling av nye ressurser

Evaluation Challenges in the Validation of B7-H3 as Oral Tongue Cancer Prognosticator

B7-H3 was the only molecule identified with prognostic potential from a recent systematic review of the prognostic value of immune checkpoints in oral cancer. We aimed to validate this finding in a multicenter international cohort. We retrospectively retrieved 323 oral tongue squamous cell carcinoma (OTSCC) samples from three different countries (Brazil, Finland, and Norway) for immunostaining and scoring for B7-H3. We evaluated tumor immunogenicity by analyzing the amount of tumor-infiltrating lymphocytes and divided the tumors into immune hot and cold. To increase the reliability of the results, both digital and manual visual scoring were used. Survival curves were constructed based on the Kaplan-Meier method, and the Cox proportional hazard model was utilized for univariate and multivariate survival analysis. B7-H3 expression was not significantly associated with overall or disease-specific survival in the whole OTSCC cohort. When divided into immune hot and cold tumors, high B7-H3 expression was significantly associated with poor disease-specific and overall survival in the immune hot group, depending on the scoring method and the country of the cohort. This was achieved only in the univariate analysis. In conclusion, B7-H3 was a negative prognosticator for OTSCC patient survival in the subgroup of immune hot tumors, and was not validated as a prognosticator in the full cohort. Our findings suggest that the immune activity of the tumor should be considered when testing immune checkpoints as biomarkers.Peer reviewe

Evaluation Challenges in the validation of B7-H3 as oral tongue cancer prognosticator

B7-H3 was the only molecule identified with prognostic potential from a recent systematic review of the prognostic value of immune checkpoints in oral cancer. We aimed to validate this finding in a multicenter international cohort. We retrospectively retrieved 323 oral tongue squamous cell carcinoma (OTSCC) samples from three different countries (Brazil, Finland, and Norway) for immunostaining and scoring for B7-H3. We evaluated tumor immunogenicity by analyzing the amount of tumor-infiltrating lymphocytes and divided the tumors into immune hot and cold. To increase the reliability of the results, both digital and manual visual scoring were used. Survival curves were constructed based on the Kaplan-Meier method, and the Cox proportional hazard model was utilized for univariate and multivariate survival analysis. B7-H3 expression was not significantly associated with overall or disease-specific survival in the whole OTSCC cohort. When divided into immune hot and cold tumors, high B7-H3 expression was significantly associated with poor disease-specific and overall survival in the immune hot group, depending on the scoring method and the country of the cohort. This was achieved only in the univariate analysis. In conclusion, B7-H3 was a negative prognosticator for OTSCC patient survival in the subgroup of immune hot tumors, and was not validated as a prognosticator in the full cohort. Our findings suggest that the immune activity of the tumor should be considered when testing immune checkpoints as biomarkers

The immune microenvironment in oral squamous cell carcinoma – characterization and prognostic markers

Oral squamous cell carcinomas (OSSCs) are aggressive tumors often associated with a low survival rate. The immune system influences the development of OSCCs, and the immune infiltrate surrounding the cancer cells is an indicator of the host’s anti-tumor response. The main aim of this thesis was to describe how the tumor microenvironment in OSCC is organized, and to determine whether different subsets of the immune infiltrate can be used to predict patient outcome. In tissue from 80 OSCC patients, we used immunohistochemistry to detect distinct cellular components of the immune infiltrate. In addition, we used qPCR to analyze the gene expression of a number of inflammation-related cytokines and chemokines. We correlated the presence of the various components to each other as well as to clinicopathological data and the patients’ survival. A systematic review of the literature was conducted to identify promising prognostic factors in OSCC and to evaluate the quality of the published studies. In our review, we found that CD163+ M2 macrophages and CD57+ mature natural killer cells were the most promising prognostic markers and that the data reported in the papers was often incomplete. In our Norwegian OSCC patient cohort, the presence of specialized blood vessels called high-endothelial venules (HEVs) was a reliable, easy-to-use, independent prognostic marker for improved survival and had the most promising potential of the factors analyzed. However, larger, standardized studies are needed to reliably conclude about the prognostic value of the markers identified in our Norwegian OSCC cohort and our literature review. HEVs are known to be gateways for lymphocyte entry to the tumor site. We found HEVs to be associated with a potentially tumor-suppressive immune microenvironment. Therefore, we hypothesize that HEVs may be master regulators of a favorable immune reaction and potential targets for immune-modulating therapies

Non-professional phagocytosis of tumour cells

Hintergrund und Ziele Phänomene, bei denen Zellen vollständig in das Zytoplasma anderer Zellen aufgenommen werden, lassen sich seit über einem Jahrhundert in der Literatur zurückfinden und wurden z.B. in Tumorgewebsschnitten beschrieben. Der Begriff Zell-in-Zell (CIC) Struktur bezieht sich auf dieses Erscheinungsbild und wurde für die nachfolgend aufgeführten in vitro Studien nicht-professioneller Phagozytose von humanen Tumorzellen verwendet. Es wurde untersucht ob verschiedene Behandlungsweisen von Zellen den Aufnahmeprozess beeinflussen und in welcher Weise bestimmte Membranproteine an der Ausbildung von CiC Phänomenen beteiligt sind. Außerdem wurde das Schicksal der aufgenommenen Zelle studiert und mögliche Gründe für die Entstehung von CiC Strukturen im Tumorgewebe diskutiert. Methoden Um den Entstehungsmechanismus von CiC zu untersuchen wurde die humane Pankreaskarzinomzelllinie BxPC-3 verwendet. CiC Phänomene wurden in vitro generiert, indem eine hitzebehandelte Zellpopulation für einige Stunden mit einer unbehandelten Zellpopulation inkubiert wurde. Zellen wurden entweder mit einem rot oder einem grün fluoreszierenden, membranpermeablen Lebendzell-Farbstoff angefärbt. Eine der so gefärbten Proben wurde im Wasserbad bei verschiedenen Temperaturen hitzebehandelt, während die andere Probe unbehandelt blieb. Anschließend wurden die beiden Proben koinkubiert und die Adhäsionsfähigkeit zwischen behandelten und unbehandelten Zellen mit dem Durchflusszytometer gemessen. Der Prozess der Zell-Aufnahme wurde mithilfe von Konfokal- und Fluoreszenzmikroskopie analysiert. Eine Bild-Auswertungssoftware diente zur Quantifizierung von CiC Ereignissen. Die Expression verschiedener Proteine wurde mittels indirekter Immunfluoreszenz beurteilt. Western Blot wurde zur Detektion bestimmter Proteine nach Hitzebehandlung angewandt. Um das Schicksal der aufgenommenen Zelle über einen Zeitraum von drei Tagen mittels Fluoreszenzmikroskopie zu untersuchen, wurde der Zellkern der nekrotischen Zelle mit dem Thymidinanalogon 5-Ethynyl-2´-Deoxyuridine (EdU) markiert. Ergebnisse Hitzebehandlung im Wasserbad bei verschiedenen Temperaturen und anschließende Inkubation von hitzebehandelten mit unbehandelten Pankreastumorzellen, zeigte Unterschiede in der Adhäsion und als Folge unterschiedliche CiC Raten, abhängig von Temperatur und Zeit. Eine Steigerung der Temperatur von 37 auf 56°C, sowie eine Verlängerung der Koinkubationszeit (auf bis zu 4 - 5 h) förderten den Aufnahmeprozess. An den Kontaktstellen zwischen hitzebehandelten und unbehandelten Zellen wurde eine verstärkte Akkumulation von β-Catenin, P-Cadherin und E-Cadherin beobachtet – Proteine mit bedeutenden Rollen in Zelladhäsion und -beweglichkeit. Hyperthermiebehandlung hatte in Abhängigkeit von der Temperatur einen Verlust dieser Proteine zur Folge, was sowohl mit Immunfluoreszenzfärbung als auch im Western Blot gezeigt wurde. Eine funktionelle Blockade von E-Cadherin wirkte sich weder auf die Adhäsionsneigung noch auf die CiC Rate aus. Zu keinem Zeitpunkt des Aufnahmeprozesses schien die Orientierung des Mikrotubulus-Organisationszentrums (MTOC) in der vitalen Zelle bezüglich der hitzebehandelten Zelle gerichtet zu sein. Außerdem verloren hitzebehandelte im Gegensatz zu unbehandelten Zellen die Fähigkeit zur Phosphorylierung von Ezrin – ein notwendiger Mechanismus für die Umgestaltung des Zytoskeletts. Nach erfolgter Aufnahme durch eine vitale Zelle und innerhalb eines Tages starb die phagozytierte Zelle im Zytoplasma der lebenden Zelle, was durch den Integritätsverlust ihrer Kernmembran gezeigt werden konnte. Schlussfolgerungen Obwohl der Entstehungsmechanismus von CiC Ereignissen letztlich nicht vollständig geklärt ist, wurde beobachtet, dass zahlreiche Faktoren den Aufnahmeprozess beeinflussen können. Hitzebehandlung bei höheren Temperaturen führt zu gesteigerten CiC Raten. Durch Hyperthermiebehandlung hervorgerufene Nekrose fördert Adhäsion, eine wichtige Vorstufe der CiC Entstehung. Außerdem wird die Zell-Aufnahme durch die Fähigkeit der Wirtszelle zum aktiven Umbau ihres Membranzytoskeletts geregelt. Die vitale Zelle reichert Zelladhäsionsmoleküle an der Kontaktseite mit der hitzebehandelten Zelle an und nimmt sie anschließend vollständig in ihr Zytoplasma auf. Vieles deutet darauf hin, dass die CiC Entstehung ein gerichteter Prozess ist. Eine gezielte Ausrichtung des MTOC als möglicher Mechanismus der Zellorientierung ist jedoch unwahrscheinlich. Während der Phosphorylierung von Ezrin – eine Fähigkeit, die allein der vitalen Zelle vorbehalten ist – in der CiC Genese eine bedeutende Rolle zugeschrieben wird, ist E-Cadherin wahrscheinlich zumindest nicht für den ersten Zell-Zell Kontakt verantwortlich. Da die aufgenommene Zelle innerhalb von 24 h stirbt, kann der ganze Prozess als Eliminationsmechanismus für nekrotische Zellen angesehen werden und könnte deshalb eine wichtige physiologische Rolle in Tumorgeweben spielen, die häufig Areale nekrotischer Zellen beherbergen. Nekrotische Zellen rufen in Tumorgeweben normalerweise eine Immunantwort hervor, die sich gegen das Tumorwachstum richtet. Demzufolge könnte die Generierung von CiC Ereignissen ein Mechanismus sein, der die anti-Tumor Immunantwort unterdrückt. In früheren Studien wurde gezeigt, dass das Vorhandensein von CiC Strukturen in Tumorgeweben eine hohe prognostische Relevanz hat, sich jedoch abhängig von der Entität unterschiedlich auf das Überleben von Patienten auswirkt. Die Entdeckung der auslösenden Faktoren für die Zell-Aufnahme könnte es ermöglichen maßgeschneiderte Therapiestrategien zur Verbesserung des Patientenüberlebens zu entwickeln.Background and aims The phenomenon of one cell being internalized into another has been described in the literature for over a century, and has amongst others been observed in human tumour tissue sections. The term cell-in-cell (CiC) structure refers to this characteristic phenotype and has been used in the in vitro studies described below. The aim of this study is to investigate the molecular background of non-professional phagocytic activity of human tumour cells. Besides the possible engagement of external stimuli and specific molecules in the uptake process, we also wanted to study the fate of the internalized cell. Methods An assay including the human pancreatic cancer cell line BxPC-3 was established to investigate the molecular mechanism of CiC genesis. Cell samples were labelled green or red with a fluorescent membrane-permeable life cell stain. One of the so dyed samples was heat-treated in a water bath at various temperatures while the other sample remained untreated. Subsequently, the two samples were co-incubated and the adhesion tendency was analyzed by flow cytometry. The incorporation process was verified by confocal and fluorescence microscopy. Image analysis software was used for quantification of CiC events. The expression of various proteins was assessed using indirect immunofluorescence. Western blot was used to detect the expression of specific proteins after heat treatment. In order to study the fate of the internalized cell over 3 days, the nucleus of the necrotic cell was labelled with the thymidine analogue 5-ethynyl-2´-deoxyuridine (EdU) and imaged by fluorescence microscopy. Results Heat treatment in a water bath at various temperatures and subsequent incubation of heat-treated with untreated pancreatic tumour cells showed differences in adhesion and consequently varying CiC rates depending on temperature and time. An increase in temperature from 37 to 56°C as well as in co-incubation time (up to 4 - 5 h) enhanced the incorporation process. A higher expression of β-catenin, P-cadherin and E-cadherin – proteins with prominent roles in cell adhesion and motility – was investigated at contact sites between heat-treated and untreated cells. Hyperthermic exposure of cells, however, caused a loss of these proteins dependent on the temperature. Functional blocking of E-cadherin affected neither adhesion tendency nor CiC rates. The arrangement of the microtubule-organizing center (MTOC) within the cell did not seem to follow a distinct orientation pattern during the engulfment process. Furthermore, heat-treated cells, in contra-distinction to untreated cells, lost their ability to phosphorylate ezrin – a mechanism required for cytoskeletal reorganization. Within one day after internalization, the incorporated cells lost the integrity of their nuclear envelopes and died. Conclusion Although the molecular background of CiC formation is still not fully understood, our results suggest that numerous factors can influence non-professional phagocytosis by tumour cells. Necrosis that follows heat treatment provokes cell adhesion, and is a necessary preliminary stage in the formation of CiC structures. Moreover, the host-cell must be able to rearrange its membrane cytoskeleton by accumulating cell adhesion molecules at the contact site with the heat-treated cell. It has also been shown that higher levels of heat exposure lead to increased CiC rates. Although there is evidence that CiC formation is a targeted process, orientation of the MTOC as a possible mechanism for cell polarization seems rather unlikely. Whereas phosphorylation of ezrin – a capability limited only to viable cells – is considered to play a crucial role in CiC genesis, E-cadherin is probably not responsible for the essential cell-cell interaction. As the incorporated cells die within 24 h, the whole process might be a physiologically relevant elimination mechanism in tumour tissues that often include necrotic areas. Necrotic cells in cancer tissue usually evoke a host immune response against tumor growth by releasing pro-inflammatory damage-associated molecular pattern molecules, and CiC formation might be a strategy of preventing this. Previous studies have highlighted the prognostic relevance of CiC structures in numerous tumour tissues. However, the presence of CiC structures might have varying effects on patient survival depending on the tumour entity. Revealing the triggering mechanisms for cell uptake might help to evolve patient-tailored therapies for the improvement of survival rates

Tissue-infiltrating immune cells as prognostic markers in oral squamous cell carcinoma: a systematic review and meta-analysis

Background - Various immune cells have been suggested as prognostic markers for cancer patients. In this article, we present a systematic review and meta-analysis of studies assessing the prognostic value of tissue-infiltrating immune cells in oral cancer and discuss the reporting quality of these studies. Methods - We performed a systematic literature search and included studies using immunohistochemistry and survival analysis to assess the prognostic value of tumour-infiltrating T cells, B cells, macrophages, dendritic cells, mast cells and natural killer cells in oral cancer. We performed meta-analysis of studies providing necessary statistical data and investigated the studies’ adherence to the REporting recommendations for tumour MARKer prognostic studies (REMARK) guidelines. Results - Of the 1960 articles identified, 33 were eligible for this systematic review and 8 were included in the meta-analysis. CD163+ M2 macrophages and CD57+ natural killer cells were the most promising predictors of survival in oral cancer patients. Many studies lacked important information on their design and conduct. Conclusion - Deficiencies in the reporting of study design and conduct make it difficult to draw reliable conclusions about the suggested markers. The prognostic value of CD163+ M2 macrophages and CD57+ natural killer cells should be validated in large, standardised studies