L1CAM protein expression is associated with poor prognosis in non-small cell lung cancer

Background: The L1 cell adhesion molecule (L1CAM) is potentially involved in epithelial-mesenchymal transition (EMT). EMT marker expression is of prognostic significance in non-small cell lung cancer (NSCLC). The relevance of L1CAM for NSCLC is unclear. We investigated the protein expression of L1CAM in a cohort of NSCLC patients. L1CAM protein expression was correlated with clinico-pathological parameters including survival and markers of epithelial-mesenchymal transition. Results: L1CAM protein expression was found in 25% of squamous cell carcinomas and 24% of adenocarcinomas and correlated with blood vessel invasion and metastasis (p < 0.05). L1CAM was an independent predictor of survival in a multivariate analysis including pT, pN, and pM category, and tumor differentiation grade. L1CAM expression positively correlated with vimentin, beta-catenin, and slug, but inversely with E-cadherin (all p-values < 0.05). E-cadherin expression was higher in the tumor center than in the tumor periphery, whereas L1CAM and vimentin were expressed at the tumor-stroma interface. In L1CAM-negative A549 cells the L1CAM expression was upregulated and matrigel invasion was increased after stimulation with TGF-beta1. In L1CAM-positive SK-LU-1 and SK-LC-LL cells matrigel invasion was decreased after L1CAM siRNA knockdown. Conclusions: A subset of NSCLCs with vessel tropism and increased metastasis aberrantly expresses L1CAM. L1CAM is a novel prognostic marker for NSCLCs that is upregulated by EMT induction and appears to be instrumental for enhanced cell invasion

Intratumoral macrophages contribute to epithelial-mesenchymal transition in solid tumors

<p>Abstract</p> <p>Background</p> <p>Several stromal cell subtypes including macrophages contribute to tumor progression by inducing epithelial-mesenchymal transition (EMT) at the invasive front, a mechanism also linked to metastasis. Tumor associated macrophages (TAM) reside mainly at the invasive front but they also infiltrate tumors and in this process they mainly assume a tumor promoting phenotype. In this study, we asked if TAMs also regulate EMT intratumorally. We found that TAMs through TGF-β signaling and activation of the β-catenin pathway can induce EMT in intratumoral cancer cells.</p> <p>Methods</p> <p>We depleted macrophages in F9-teratocarcinoma bearing mice using clodronate-liposomes and analyzed the tumors for correlations between gene and protein expression of EMT-associated and macrophage markers. The functional relationship between TAMs and EMT was characterized <it>in vitro </it>in the murine F9 and mammary gland NMuMG cells, using a conditioned medium culture approach. The clinical relevance of our findings was evaluated on a tissue microarray cohort representing 491 patients with non-small cell lung cancer (NSCLC).</p> <p>Results</p> <p>Gene expression analysis of F9-teratocarcinomas revealed a positive correlation between TAM-densities and mesenchymal marker expression. Moreover, immunohistochemistry showed that TAMs cluster with EMT phenotype cells in the tumors. <it>In vitro</it>, long term exposure of F9-and NMuMG-cells to macrophage-conditioned medium led to decreased expression of the epithelial adhesion protein E-cadherin, activation of the EMT-mediating β-catenin pathway, increased expression of mesenchymal markers and an invasive phenotype. In a candidate based screen, macrophage-derived TGF-β was identified as the main inducer of this EMT-associated phenotype. Lastly, immunohistochemical analysis of NSCLC patient samples identified a positive correlation between intratumoral macrophage densities, EMT markers, intraepithelial TGF-β levels and tumor grade.</p> <p>Conclusions</p> <p>Data presented here identify a novel role for macrophages in EMT-promoted tumor progression. The observation that TAMs cluster with intra-epithelial fibroblastoid cells suggests that the role of macrophages in tumor-EMT extends beyond the invasive front. As macrophage infiltration and pronounced EMT tumor phenotype correlate with increased grade in NSCLC patients, we propose that TAMs also promote tumor progression by inducing EMT locally in tumors.</p

Tumor associated macrophages regulate epithelial to mesenchymal transition in tumor cells in a TGF-β dependent manner

Das Wachstum eines Tumors hängt von einer Reihe von limitierenden Schritten ab, die in höchstem Masse von der Mikroumgebung des Tumors beeinflusst werden. In diesem Zusammenhang gelten Tumor-assoziierte Makrophagen (TAM) als hauptsächliche Regulatoren des Tumorwachstums; dies einerseits wegen deren Einfluss auf die Angiogenese, auf den sogenannten „angiogenic switch“, und andererseits wegen deren vielfältigen Funktionen im Modellieren der Tumormikroumgebung. Diese Ereignisse erleichtern und orchestrieren die Invasion und Verbreitung von Tumorzellen ins benachbarte Gewebe, weshalb man auch begünstigende Effekte der TAM auf die Bildung von Metastasen vermutet. Ihr genauer Einfluss in diesen komplexen Prozessen ist allerdings noch nicht vollständig aufgeklärt. Schon früher haben wir und andere die vielseitige Rolle von TAM im Tumorwachstum studiert, indem wir TAM in Tumor tragenden Mäusen mit einer pharmakologischen Methode depletierten. Diese Studien haben erfolgreich eine kausale Verbindung zwischen TAM Depletion und reduziertem Tumorwachstum hergestellt, wobei die TAM-abhängige Tumorangiogenese als hauptsächlicher Mechanismus identifiziert wurde. In der präsentierten Arbeit habe ich diese auf Bisphosphonat-Liposomen basierte Technik der TAM Depletion im F9-Teratokarzinom Mausmodell angewendet, und mit Hilfe einer Genexpressionsanalyse dieser Tumore Genprofile identifiziert, welche zusätzliche und neuartige Rollen für TAM in Tumorwachstum und -entwicklung aufdeckten. Resultate dieser Analyse haben eine Korrelation zwischen TAM-Dichte und einem Genprofil ergeben, das der sogenannten epithelial-mesenchymalen-Transition (EMT) entspricht. EMT ist ein gut charakterisierter Mechanismus, durch welchen epithelartige Tumorzellen einen invasiven mesenchymalen Phänotyp erlangen können, der als limitierender Schritt in der Tumorinvasion und Metastasierung betrachtet wird. Die gefundene Korrelation zwischen TAM-Dichte und EMT-assoziiertem Genprofil suggeriert demzufolge eine Rolle der TAM in der Regulation der EMT-assoziierten Tumorinvasion. Dieser Befund wurde weiter in Zellkulturen von F9- und NMuMG-Zellen und mit molekularen und zellbiologischen Techniken untersucht. Dabei wurde der TAM-abhängige Faktor TGF-β1, und die durch diesen Faktor induzierte Aktivierung der β-Catenin Signalkaskade, als Hauptmechanismus der Tumorzell-EMT identifiziert. Die TAM-induzierte EMT führte zu erhöhter Tumorzellinvasion als Antwort auf Makrophagen abhängige Chemokine. Die Invasion wurde jedoch nach Neutralisierung von TGF-β1 wieder aufgehoben. TGF-β1 alleine erwies sich nicht als genügend starkes Chemoattraktans für die untersuchten Zelllinien. Demzufolge zeigen die Daten einen direkten Einfluss der TAM auf die intrinsische Regulierung invasiver Eigenschaften von Tumorzellen. TAM sind eine wichtige Quelle verschiedenster Faktoren, wie z.B. SDF-1, VEGF und EGF und die Ergebnisse suggerieren demnach ein Modell in welchem TAM durch eine TGF-β abhängige Signalübertragung einen invasiven Tumorzellphänotyp via EMT ausbilden und gleichzeitig durch Signalübertragung von Chemokinen den Tumorzellen wachstumsgerichtete Stimuli bieten. Ein wichtiger Aspekt dieser Studie war die Evaluation der klinischen Relevanz der TAM-induzierten Tumor-EMT im Verlauf einer Tumorerkrankung. Diese Relevanz wurde durch eine histopathologische Analyse von 491 Tumorproben von nicht kleinzelligen Lungenkarzinomen („non-small cell lung cancer“, NSCLC) bestätigt, wobei eine signifikante Korrelation zwischen intra-tumoraler Makrophagendichte, mesenchymalem Tumorzell-Phänotyp und dem Entwicklungsgrad der Tumore gezeigt werden konnte. Diese klinische Relevanz der grundlegenden in vivo und in vitro Ergebnisse weist darauf hin, dass therapeutische Ansätze gegen TAM den Krankheitsverlauf von EMT-assoziierten Tumorformen möglicherweise verbessern könnten. Insgesamt kann durch die Resultate aus meiner Doktorarbeit eine neuartige Dimension im komplexen Zusammenspiel von Tumorzellen und der Tumormikroumgebung erkannt werden. Summary Tumor progression depends on a number of rate-limiting steps that are highly influenced by the tumor microenvironment. To this end, tumor-associated macrophages (TAMs) are regarded as key regulators of tumor progression. This is in part due to their involvement in the “angiogenic switch” and their functions in modulating tumor growth. Moreover, TAMs secrete a vast amount of chemokines through which they attract motile cancer cells. These events facilitate and orchestrate tumor cell invasion and dissemination, thus TAMs are generally believed to promote tumorigenesis and metastatic disease. However, their exact involvement in these processes and the mechanisms of action remain elusive. Our laboratory and others have previously studied the versatile roles of TAMs in tumor progression by performing pharmacologic depletion of TAMs in tumor-bearing mice. These studies successfully linked TAM-depletion to reduced tumor growth and identified TAM-dependent tumor-angiogenesis as a major regulator hereof. In the presented work, I have used this bisphosphonate-liposome based technique to deplete TAMs in F9-teratocarcinoma tumors. I have combined this technique with gene expression analysis performed on the isolated F9-tumors and identified gene expression profiles revealing additional and novel implications for TAMs in tumor growth and progression. Data obtained from this comparative gene expression analysis revealed a correlation between TAM density and a gene expression profile characteristic of epithelial to mesenchymal transition (EMT). EMT is a well characterized mechanism through which epithelial tumor cells can acquire an invasive, mesenchymal phenotype, and it is therefore regarded to be a rate-limiting step in tumor invasion and progression. Thus, the correlation between TAM density and EMT-associated gene expression suggested a role for TAMs in regulation of EMT-associated tumor invasion. This was confirmed in cell cultures of murine F9- and mammary gland NMuMG-cells by using molecular and cell biological techniques.Importantly, TAM-derived TGF-β and consecutive activation of the β-catenin pathway was identified as a main inducer of tumor cell EMT. TAM-induced EMT led to increased invasion in response to macrophage-derived chemokines, however invasion was abrogated upon neutralization of TGF-β1. TGF-β1 alone did not prove to be a potent chemoattractant for the tested cell lines, thus the data suggests that TGF-β regulates tumor cell invasion by induction of a chemotactic phenotype, rather than providing chemoattracting signaling. The data demonstrate a direct involvement of TAMs in the intrinsic regulation of invasive properties in tumor cells. An important aspect of this study was to evaluate the clinical relevance of TAM-induced tumor EMT in disease progression. This was done by the histopathologic analysis of 491 non-small cell lung cancer specimens, which revealed a significant correlation between intra-tumoral macrophage density, mesenchymal tumor cell phenotype and tumor grade. This underscores the clinical relevance of the murine data and suggests that therapeutic targeting of TAMs may improve management of cancers susceptible to stroma induced EMT-associated disease progression. Collectively, the data obtained during the course of my PhD unravel a novel dimension in the complex interplay between tumor cells and the tumor stroma and provide novel insight valuable for the development of therapies abrogating EMT-associated disease progression

Loneliness and the risk of type 2 diabetes

Introduction The incidence of type 2 diabetes is increasing globally. Recent research suggests that loneliness could be a potential risk factor for the development of type 2 diabetes. We aimed to investigate the association between loneliness and type 2 diabetes and the modifying effect of mental disorders.Research design and methods We conducted a prospective study including 465 290 adults (aged ≥16 years) who participated in either the Danish Health and Morbidity Survey or the Danish National Health Survey between 2000 and 2017. Loneliness was based on self-report, while type 2 diabetes was measured using an algorithm combining several health registers including type 2 diabetes patients treated both within the hospital sector and general practice. Cox proportional hazards regressions were used to estimate hazard ratios (HRs) and 95% confidence intervals (95% CIs).Results During a mean follow-up time of 6.3 years, 13 771 individuals (3%) developed type 2 diabetes. Feeling lonely once in a while was associated with a 14% increased risk of type 2 diabetes (95% CI 1.09 to 1.20), while feeling lonely often was associated with a 24% increased risk (95% CI 1.14 to 1.34), independent of sociodemographic factors and body mass index. The association was stronger among individuals without a mental disorder (HR 1.21, 95% CI 1.10 to 1.34 among those feeling lonely often) compared with those with a mental disorder (HR 1.07, 95% CI 0.93 to 1.23).Conclusions Loneliness independently increased the risk of type 2 diabetes. The effect was more pronounced in individuals without a mental disorder, as having a mental disorder itself likely increases the risk of type 2 diabetes. These findings emphasize the importance of addressing loneliness as a modifiable risk factor in preventing type 2 diabetes