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).
SPARCL1 is a matricellular protein with anti-adhesive, anti-proliferative and anti-tumorigenic functions and is frequently downregulated in tumors such as colorectal carcinoma or non-small cell lung cancer. Studies have identified SPARCL1 as an angiocrine tumor suppressor secreted by tumor vessel endothelial cells, thereby exerting inhibitory activity on angiogenesis and tumor growth, in colorectal carcinoma. It is unknown whether SPARCL1 may exert these homeostatic functions in all organs and in other species. Therefore, SPARCL1 expression was comparatively analysed between humans and mice in a systematic manner. Murine Sparcl1 (mSparcl1) is most strongly expressed in the lung; expressed at an intermediate level in most organs, including the large intestine; and absent in the liver. In human tissues, SPARCL1 (hSPARCL1) was detected in all organs, with the strongest expression in the stomach, large intestine and lung, mostly consistent with the murine expression pattern. A striking difference between human and murine tissues was the absence of mSparcl1 expression in murine livers, while human livers showed moderate expression. Furthermore, mSparcl1 was predominantly associated with mural cells, whereas hSPARCL1 was detected in both mural and endothelial cells. Human SPARCL1 expression was downregulated in different carcinomas, including lung and colon cancers. In conclusion, this study revealed species-, organ-and cell-type-dependent expression of SPARCL1, suggesting that its function may not be similar between humans and mice.
Background The understanding of vascular plasticity is key to defining the role of blood vessels in physiologic and pathogenic processes. In the present study, the impact of the vascular quiescence marker SPARCL1 on angiogenesis, capillary morphogenesis, and vessel integrity was evaluated. Methods Angiogenesis was studied using the metatarsal test, an ex vivo model of sprouting angiogenesis. In addition, acute and chronic dextran sodium sulfate colitis models with SPARCL1 knockout mice were applied. Results This approach indicated that SPARCL1 inhibits angiogenesis and supports vessel morphogenesis and integrity. Evidence was provided that SPARCL1-mediated stabilization of vessel integrity counteracts vessel permeability and inflammation in acute and chronic dextran sodium sulfate colitis models. Structure-function analyses of purified SPARCL1 identified the acidic domain of the protein necessary for its anti-angiogenic activity. Conclusions Our findings inaugurate SPARCL1 as a blood vessel–derived anti-angiogenic molecule required for vessel morphogenesis and integrity. SPARCL1 opens new perspectives as a vascular marker of susceptibility to colitis and as a therapeutic molecule to support blood vessel stability in this disease.
Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.
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.
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