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).

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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.

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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.