in (partial) fulfillment of the requirements for obtaining the degree Dr. rer. nat. by Victoria Langer. Supporting grants were from the German Research Foundation (DFG) (KFO 257 [subproject 4 to M Stürzl and MJW], FOR 2438 [subproject 2 to EN and M Stürzl], SFB/TRR241 [subproject A06 to M Stürzl and NBL], and BR 5196/2-1 [to NBL]); the Interdisciplinary Center for Clinical Research (IZKF) of the Clinical Center Erlangen (D28 to EN and M Stürzl); the W. Lutz Stiftung (to M Stürzl); and the Forschungsstiftung Medizin am Universitätsklinikum Erlangen (to M Stürzl).
Magnetic resonance imaging (MRI) allows non-invasive evaluation of inflammatory bowel disease (IBD) by assessing pathologically altered gut. Besides morphological changes, relaxation times and diffusion capacity of involved bowel segments can be obtained by MRI. The aim of this study was to assess the use of multiparametric MRI in the diagnosis of experimentally induced colitis in mice, and evaluate the diagnostic benefit of parameter combinations using machine learning. This study relied on colitis induction by Dextran Sodium Sulfate (DSS) and investigated the colon of mice in vivo as well as ex vivo. Receiver Operating Characteristics were used to calculate sensitivity, specificity, positive- and negative-predictive values (PPV and NPV) of these single values in detecting DSS-treatment as a reference condition. A Model Averaged Neural Network (avNNet) was trained on the multiparametric combination of the measured values, and its predictive capacity was compared to those of the single parameters using exact binomial tests. Within the in vivo subgroup (n = 19), the avNNet featured a sensitivity of 91.3% (95% CI: 86.6–96.0%), specificity of 92.3% (95% CI: 85.1–99.6%), PPV of 96.9% (94.0–99.9%) and NPV of 80.0% (95% CI: 69.9–90.1%), significantly outperforming all single parameters in at least 2 accuracy measures (p < 0.003) and performing significantly worse compared to none of the single values. Within the ex vivo subgroup (n = 30), the avNNet featured a sensitivity of 87.4% (95% CI: 82.6–92.2%), specificity of 82.9% (95% CI: 76.1–89.7%), PPV of 88.9% (84.3–93.5%) and NPV of 80.8% (95% CI: 73.8–87.9%), significantly outperforming all single parameters in at least 2 accuracy measures (p < 0.015), exceeded by none of the single parameters. In experimental mouse colitis, multiparametric MRI and the combination of several single measured values to an avNNet can significantly increase diagnostic accuracy compared to the single parameters alone. This pilot study will provide new avenues for the development of an MR-derived colitis score for optimized diagnosis and surveillance of inflammatory bowel disease.
Lärm, Abgase und Unfallgefahr sind neben der Flächendominanz parkender Pkw zentrale Probleme für die derzeitige Stadt- und Verkehrsentwicklung. Das Projekt „City2Share“ gibt Anhaltspunkte für ökologische Alternativen.
In colorectal carcinoma (CRC), a Th1-tumor microenvironment (TME) is associated with improved prognosis of the patients. The lead cytokine of the Th1-response is interferon (IFN)-γ. IFN-γ is predominantly regarded as an immunomodulatory cytokine. At present, its putative tumor blood vessel-directed anti-tumorigenic effects have not been investigated comprehensively. We demonstrate that the vascular effects of IFN-γ in CRC trigger a two-step anti-tumorigenic chain reaction. First, IFN-γ exerts direct angiostatic activity on endothelial cells. This was detected in vitro using primary endothelial cell cultures and in vivo in Th1-dominated inflammatory reactions of the colon using mouse models with specific deletion of the IFN-γ receptor-2 in endothelial cells and treatment with neutralizing anti-IFN-γ antibodies. Angiostatic IFN-γ effects could be validated in human CRC tissues using the cellular IFN-γ activation marker guanylate binding protein-1 which confirmed reduced angiogenic activity of vessels exposed to IFN-γ at that single cell level. IFN-γ-induced angiostasis was associated with a highly significantly improved cancer-related 5-year survival of the CRC patients (n=388). In a second step, angiostatic activity of IFN-γ resulted in the maintenance of mature tumor vessels in CRC tissues which expressed and secreted the anti-tumorigenic protein SPARCL1 in human CRC tissues (n = 42). This was significantly repressed in CRC tissues lacking a Th1 response. Functional analyses showed that SPARCL1 inhibited angiogenic activity of cultivated endothelial cells in different in vitro tests of angiogenesis (endothelial cell proliferation, migration, spreading, 3D-sprouting and capillary formation in matrigel), both after retrovirally triggered recombinant expression in endothelial cells or after adding the recombinantly purified human SPARCL1 protein. Interestingly, secreted SPARCL1 has also been shown to inhibit proliferation and migration of human CRC tumor cell lines. We could confirm the anti-tumor cell activity of soluble purified SPARCL1 using different tumor cell lines derived from mouse colon tumors (MC38) or mouse melanomas (B16F10), respectively. SPARCL1 inhibited proliferation and migration of both cell lines significantly. Using a mouse model system with a general knock out of the SPARCL1 gene, we obtained first evidence that the growth of metastatically spread MC38 cells in the lungs is repressed by SPARCL1 in vivo. Altogether our study demonstrates that the tumor vessel-directed effects of an IFN-γ-dominated tumor microenvironment in CRC trigger a two-step anti-tumorigenic chain reaction. This provides further insight into the potent anti-tumorigenic activity of a Th1-TME in CRC that may be of relevance for selection of patients' for anti-angiogenic therapy regiments. Moreover, our findings indicate novel pathways which may be prone to tumor immune evasion. Citation Format: Michael Stürzl, Victoria Langer, Daniela Regensburger, Clara Tenkerian, Annika Klingler, Maximilian Waldner, Christoph Becker, Valerie Meniel, Robert Grützmann, Carol-Immanuel Geppert, Nathalie Britzen-Laurent, Elisabeth Naschberger. Interferon-γ triggers an anti-tumorigenic chain reaction in the tumor vessels of colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2048.
Colorectal carcinoma (CRC) is the second leading cause of cancer-related mortality worldwide. The contribution of the tumor microenvironment (TME) to CRC pathogenesis is well established, whereby the dominance of one immune response (Th1) over another (Th17) can yield opposite effects on patient prognosis. We recently reported on TME-dependent vascular plasticity in CRC and identified the secreted protein, acidic and rich in cysteine-like 1 (SPARCL1) as a marker thereof. SPARCL1 is a matricellular protein expressed and secreted exclusively by the vascular system (endothelial and mural cells). In previous studies, gene expression analyses in CRC tumor tissues of different patient cohorts congruently showed a significant loss of SPARCL1 in colon and rectum adenocarcinomas as compared to uninvolved colon tissues and indicated that this loss is TME-dependent. Specifically, high SPARCL1 expression was associated with a Th1-TME, reduced incidence of distant metastasis and increased cancer-related survival. In contrast, SPARCL1 expression was lost in aggressive CRC with a non-Th1-TME. Here we show in functional analyses that SPARCL1 inhibits angiogenesis in different in vitro (endothelial cell proliferation, capillary formation on matrigel, 3D spheroid sprouting), ex vivo (fetal mouse metatarsal explant) and in vivo (FITC-dextran vascular permeability) assays. Moreover, increased tumor cell colonization is detected in the lungs of SPARCL1-knockout mice, further consolidating the anti-tumorigenic function of SPARCL1. To characterize the molecular mechanism of SPARCL1 anti-angiogenic function, we identified the TGF-β co-receptor Endoglin as a cellular receptor for SPARCL1 and found that SPARCL1 regulates ERK1/2 activity. The RAS-ERK pathway is amplified in more than half of all CRC tumors and as such is considered an important therapeutic target. Interestingly, SPARCL1 regulates ERK1/2 not only through phosphorylation but also through its subcellular localization. We show that SPARCL1 induces ERK1/2 phosphorylation preferentially in the cytoplasm and activates specifically cytoplasmic substrates downstream of it. Finally, we show that the regulation of ERK1/2 by SPARCL1 is not limited to endothelial cells but extends to epithelial CRC cells, indicative of autocrine and paracrine functions of SPARCL1. In summary, our study indicates that SPARCL1 is a TME-dependent angiocrine tumor suppressor in CRC acting through the regulation of ERK1/2 activity. This finding opens novel perspectives elucidating the crosstalk between the TME and the ERK signaling pathway and accordingly may enable new strategies to overcoming drug resistance and/or therapy-oriented patient stratification. Citation Format: Clara Tenkerian, Daniela Regensburger, Victoria Langer, Anika Klingler, Anne Borau, Heinrich Sticht, Andreas Ramming, Thomas Wohlfahrt, Benjamin Schmid, Valérie Méniel, Robert Grützmann, Nathalie Britzen-Laurent, Vera Schellerer, Michael Stürzl, Elisabeth Naschberger. SPARCL1 is an angiocrine inhibitor of tumorigenesis in colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 195.
Tumor and stromal cell plasticity induced by different tumor microenvironments (TMEs) has a significant impact on tumorigenesis of colorectal carcinoma (CRC). Specifically the role of the immunological TME has been investigated intensively. The prognosis of CRC is influenced by the density and localization of infiltrating T cells. Moreover, the presence of an mRNA expression profile indicative of a type 1 adaptive immune response (high IFN-γ and IFN-induced gene expression, cytotoxic T cell (CTL) signature) represents a positive prognostic factor for patients with CRC. IFN-γ is a major mediator of the Th1 immune response, which is produced by NK, NKT, Th1 cells and CTL. The role of IFN-γ in anti-tumor immune response has been mainly attributed to the immune-modulatory activity of the cytokine, such as the recruitment and activation of cytotoxic T-cells or monocytes. However, the expression of the IFN-γ receptor is ubiquitous and studies of IFN-γ-induced genes expression in CRC revealed that many different cell types are stimulated by the cytokine within a tumor, including tumor cells and stromal cells such as macrophages/monocytes and endothelial cells. In vitro, IFN-γ shows anti-tumorigenic activities in CRC cell lines, activates monocytes and exerts potent anti-angiogenic effects on endothelial cells. Here we investigated the cell-type-specific impact of the response to IFN-γ on CRC development using mouse strains with conditional knock-out of the IFN-γ receptor in intestinal epithelial cells (Villin-Cre), T-cells (CD4-Cre), myeloid cells (CD11b-Cre) and endothelial cells (Tie2-Cre + BMT). Tumor growth was chemically induced by injection of azoxymethane combined with three cycles of treatment with dextran-sodium sulfate. This model recapitulates inflammation-induced carcinogenesis. Our results revealed increased tumor numbers and load when the IFN-γ response was blocked in epithelial cells, despite an initial attenuation of inflammation. Tumors that developed in Ifngr2-Villin-Cre mice showed an attenuated IFN-γ response, and a decrease of CD8+ T cell infiltration, cell death and hypoxia. Abrogation of the IFN-γ-response in endothelial cells also fostered tumor growth, which could be attributed to an increase of angiogenesis. Surprisingly, only a modest effect was seen in the T-cell-specific knock-out, in comparison to the myeloid-specific knock-out, which was associated with a strong increase of tumor numbers and load. The latter indicated that the anti-tumorigenic activity mediated by IFN-γ relies to a larger extent on the innate than on the adaptive immune response. Our study documents that the anti-tumorigenic activity of IFN-γ is based on direct effects on epithelial tumor cells and on the vasculature, as well as on the involvement of the innate immune response. Citation Format: Nathalie Britzen-Laurent, Wei Guo, Victoria Langer, Svetlana Khoziainova, Thomas Weisenburger, Thomas H. Winkler, Julia Straube, Maximilian J. Waldner, Christoph Becker, Elisabeth Naschberger, Lisa Skottke, Tripal Philipp, Sergei Grivennikov, Michael Stürzl. Role of IFN-gamma-activation of distinct tumor and stromal cell populations in colorectal carcinoma pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5162.
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