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Aberrant activation of the Wnt/β-catenin pathway plays a major role in the development of colorectal carcinoma (CRC). Axin 2 is a key protein of this pathway and is upregulated in CRC. Here, we investigated RNA- and protein expression of axin 2 in CRC tissues at the single cell level. Moreover, the association of axin 2 with prognosis and survival was investigated in a large cohort of CRC patients (n = 280). Localization and expression of axin 2 and β-catenin was investigated using in situ hybridization and immunohistochemical staining. The quantitative expression levels of axin 2 were determined using RT-qPCR. The association of axin 2 expression with prognosis and survival of the patients was determined by statistical analysis (logrank test, Kaplan–Meier). Our results confirmed the upregulation of axin 2 in CRC and showed that it is broadly expressed in the cytoplasm of the tumor epithelial cells both, in the tumor center and at the invasion front. Axin 2 was rarely expressed by tumor stromal cells and only weakly by normal colonic epithelial cells. Staining of β-catenin and axin 2 in consecutive CRC tissue sections revealed that nuclear translocation of β-catenin in the tumor front was not associated with changes in the cytoplasmic localization of axin 2. Axin 2 did not show any association with proven prognostic factors or survival of the CRC patients. The generally increased expression of axin 2 in all tumor stages as compared to normal tissue suggests an initiating pathogenic function in the development of CRC.

The tumor microenvironment (TME) and tumor cell heterogeneity contribute significantly to tumor pathogenesis. In contrast, potential stromal cell heterogeneity induced by the TME and its impact on human tumorigenesis has not been conclusively investigated. Therefore, pure tumor endothelial cells (TECs) from human colorectal carcinomas (CRCs) with differential TMEs (favorable Th1-TME and worse control-TME) were isolated and significantly different gene clusters reflecting the tumorigenic and angiogenic impact attributed to the respective TMEs were identified by transcriptome analysis. SPARCL1 - the most strongly upregulated gene in Th1-TME-derived TECs - was analyzed to prove this concept. SPARCL1 was expressed by endothelial cells (EC) in colon tissues, most prominently in mature normal vessels, similarly in CRC with Th1-TME and lost towards more aggressive and angiogenic control tumors. SPARCL1 was induced in confluent, quiescent EC in culture but absent or resolved in angiogenically active EC. SPARCL1 inhibited proliferation, migration and sprouting of cultivated EC in vitro. In human CRC tissues SPARCL1 expressing vessels were highly covered by mural cells and of larger vessel size (perimeter and area), indicting more progressed maturation. Similarly the vessels in the colon of wild type mice as compared to SPARCL1/Sc1 knockout mice exhibited a more mature vessel structure. In conclusion, exploiting cellular memory processes we demonstrated TME-dependent intratumoral TEC heterogeneity in CRC and identified SPARCL1 as novel EC-derived protein regulating vessel maturation and homeostasis. Citation Format: Michael Stürzl, Andrea Liebl, Vera S. Schellerer, Manuela Schütz, Patrick Kölbel, Ute Schaal, Lisa Haep, Ludger Klein-Hitpass, Timan T. Rau, Barbara Dietel, Valerie Meniel, Alan R. Clarke, Susanne Merkel, Roland S. Croner, Werner Hohenberger, Elisabeth Naschberger. Tumor-microenvironment-dependent imprinting of endothelial cells in human colorectal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3369.

e22012 Background: Tumor endothelial cells (TECs) were considered to be genetically stable and despite being different from normal endothelial cells (NECs) to exhibit a relatively homogeneous pheno...