Search

Interferon (IFN)-α and IFN-γ are cytokines with potent immunomodulating and anti-tumor activities. It is unknown which of the two IFNs may be more potent in the regulation of an anti-tumorigenic response in colorectal carcinoma or whether both cytokines cooperate. We, therefore, established human myxovirus resistance protein A and human guanylate-binding protein-1 as markers for the differential detection of IFN-α– and IFN-γ–driven tumor micromilieus, respectively. In vitro studies with different cultures of tumor cells from colorectal carcinoma and stroma cells showed that the expression of myxovirus resistance protein A was exclusively induced by IFN-α, whereas guanylate-binding protein-1 was strongly induced by IFN-γ and only weakly by IFN-α. This expression pattern was used to distinguish cell activation caused by the two cytokines in a clinical cohort of patients with colon carcinoma ( n = 378). Patients with primary tumors expressing only guanylate-binding protein-1 exhibited the highest cancer-specific 5-year survival (94.0%, P = 0.006) compared with those expressing both factors (90.3%, P = 0.006), myxovirus resistance protein A alone (83.5%, P = 0.096), or none (72.8%). Our study describes a successful proof-of-principle approach that complex cytokine interaction networks can be dissected in human tissues and demonstrates that an IFN-γ–driven tumor microenvironment exhibits a superior prognostic effect compared with an IFN-α–driven tumor microenvironment in colon carcinoma.

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.

Different tumor microenvironments (TMEs) induce stromal cell plasticity that affects tumorigenesis. The impact of TME-dependent heterogeneity of tumor endothelial cells (TECs) on tumorigenesis is unclear. Here, we isolated pure TECs from human colorectal carcinomas (CRCs) that exhibited TMEs with either improved (Th1-TME CRCs) or worse clinical prognosis (control-TME CRCs). Transcriptome analyses identified markedly different gene clusters that reflected the tumorigenic and angiogenic activities of the respective TMEs. The gene encoding the matricellular protein SPARCL1 was most strongly upregulated in Th1-TME TECs. It was also highly expressed in ECs in healthy colon tissues and Th1-TME CRCs but low in control-TME CRCs. In vitro, SPARCL1 expression was induced in confluent, quiescent ECs and functionally contributed to EC quiescence by inhibiting proliferation, migration, and sprouting, whereas siRNA-mediated knockdown increased sprouting. In human CRC tissues and mouse models, vessels with SPARCL1 expression were larger and more densely covered by mural cells. SPARCL1 secretion from quiescent ECs inhibited mural cell migration, which likely led to stabilized mural cell coverage of mature vessels. Together, these findings demonstrate TME-dependent intertumoral TEC heterogeneity in CRC. They further indicate that TEC heterogeneity is regulated by SPARCL1, which promotes the cell quiescence and vessel homeostasis contributing to the favorable prognoses associated with Th1-TME CRCs.

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.

Complex regional pain syndrome (CRPS) is a condition that is characterized by severe pain and exaggerated neurogenic inflammation, which may develop after injury or surgery. Neurogenic inflammation is mediated by neuropeptides, such as calcitonin gene-related peptide (CGRP) and substance P (SP) that are released from nociceptors. Genetic factors may play a role in CRPS as was suggested by the occurrence of familial cases and several genetic association studies investigating mainly the human leukocyte antigen (HLA) system. Here we investigated the role of neutral endopeptidase (NEP), a key enzyme in neuropeptide catabolism. NEP dysfunction resulting in reduced inactivation of neuropeptides may be a possible pathomechanism in CRPS. To this end, we tested a GT-repeat polymorphism, in the NEP promoter region as well as 18 tag-SNPs in six linkage disequilibrium (LD) blocks in the NEP gene region in 320 CRPS patients and 376 controls. No significant genetic association was observed. Thus, we conclude that the NEP gene does not seem to be a major risk factor for CRPS. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

It is well known that tumor cell heterogeneity and the tumor microenvironment (TME) contribute to tumorigenesis. In contrast, stromal cell heterogeneity and plasticity in response to different TMEs and its impact on tumorigenesis has not been conclusively investigated in human tumors, as yet. Here we analyzed TME-induced heterogeneity of tumor endothelial cells (TECs) in colorectal carcinoma (CRC). To this goal vital and highly pure TEC cultures were isolated from CRC with a Th1-like TME (Th1-TME) and from CRC with a non-Th1-like TME (Ctrl-TME). Tumors with a Th1-TME exhibited a significantly reduced angiogenic activity and were associated with an improved prognosis of CRC patients. Comparative transcriptome analyses of the established TEC cultures identified a cluster of genes which was homogeneously expressed in TECs from Th1-TME but significantly different in TECs from Ctrl-TME. The gene encoding secreted protein, acidic and rich in cysteine-like 1 (SPARCL1) was identified to be most strongly up-regulated in TECs derived from a Th1-TME. We could confirm with in situ hybridization and immunohistochemistry that in normal colon and CRC tissues SPARCL1 expression is associated with endothelial cells (EC). Of note, SPARCL1 was consistently and highly expressed in normal colon, to a lower extent in CRC with a Th1-TME and low or absent in CRC tissues with a Ctrl.-TME. In cell culture SPARCL1 expression was absent in actively proliferating EC, but highly induced in EC reaching a confluent quiescent state. The SPARCL1 induction in confluent cells was reversible after the reduction of cell density. In functional studies SPARCL1 inhibited proliferation, migration and 3D sprouting of cultivated EC indicating potent anti-angiogenic activity. At the morphological level vessels in normal colon and in CRC with a Th1-TME were larger as compared to vessels in tumors with Ctrl-TME. Accordingly, SPARCL1 characterizes quiescent mature vessels that are larger as compared to rapidly sprouting microvessels. Altogether our results show that TECs isolated from CRC with different TMEs exhibit specific and differential long-term gene expression profiles. Characterization of the respective genes identified SPARCL1 as an anti-angiogenic gene associated with EC quiescence. These results demonstrate for the first time that human tumor endothelial cells retain differential TMEs by cellular memory allowing the identification of genes functionally imprinting the TME in these cellular sensors. Citation Format: Andrea Liebl, Elisabeth Naschberger, Vera S. Schellerer, Ute Schaal, Patrick Kölbel, Sandra Grenz, Nathalie Britzen-Laurent, Tilman T. Rau, Susanne Merkel, Barbara Dietel, Ludger Klein-Hitpass, Werner Hohenberger, Roland S. Croner, Michael Stürzl. Endothelial cells isolated from colorectal carcinoma exhibit tumor microenvironment-dependent plasticity allowing the identification of SPARCL1 as a novel endothelial cell quiescence factor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2375. doi:10.1158/1538-7445.AM2015-2375

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...