Matricellular protein SPARCL1 regulates tumor microenvironment–dependent endothelial cell heterogeneity in colorectal carcinoma
Elisabeth Naschberger, … , Werner Hohenberger, Michael Stürzl
Elisabeth Naschberger, … , Werner Hohenberger, Michael Stürzl
Published October 10, 2016
Citation Information: J Clin Invest. 2016;126(11):4187-4204. https://doi.org/10.1172/JCI78260.
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Research Article Angiogenesis Oncology

Matricellular protein SPARCL1 regulates tumor microenvironment–dependent endothelial cell heterogeneity in colorectal carcinoma

Abstract

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.

Authors

Elisabeth Naschberger, Andrea Liebl, Vera S. Schellerer, Manuela Schütz, Nathalie Britzen-Laurent, Patrick Kölbel, Ute Schaal, Lisa Haep, Daniela Regensburger, Thomas Wittmann, Ludger Klein-Hitpass, Tilman T. Rau, Barbara Dietel, Valérie S. Méniel, Alan R. Clarke, Susanne Merkel, Roland S. Croner, Werner Hohenberger, Michael Stürzl

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Figure 4

SPARCL1 is an EC-associated protein that is highly expressed in normal colon and progressively lost in more aggressive CRCs.

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SPARCL1 is an EC-associated protein that is highly expressed in normal c...
(A) SPARCL1 expression (pink, arrows) was determined by IHC in normal colon and CRC tissues. Tumor cells are labeled with asterisks. Isotype antibody staining of consecutive sections was used as a negative control. Scale bar: 50 μm. (B) Colocalization of SPARCL1, α-SMA, and CD31 was determined by immunofluorescence triple staining (SPARCL1, green; α-SMA, blue; CD31, red). A triple staining with the respective isotype antibodies is depicted as a control. All tissues were counterstained using DAPI (white), but this is depicted only for the isotype staining. Inserts are representative higher-magnification images of the 296 vessels counted, showing colocalizations of SPARCL1 and CD31 (arrows) and SPARCL1 and α-SMA (arrowheads) (n = 10 normal colon, n = 20 CRCs). Scale bar: 25 μm; original magnification, ×4.3 (inserts). (C) SPARCL1 expression was quantified by RT-qPCR in normal colon and corresponding CRC tissues (n = 42). 40-ΔCt values are shown at the individual patient level and in a box and whisker plot (insert). ***P < 0.001, by Student’s t test. Line corresponds to mean value. (D) SPARCL1 and CD31 expression was detected after immunofluorescence staining in normal colon, GBP-1hi CRC, and GBP-1lo CRC (n = 9 each). The relative content of SPARCL1-positive ECs per vessel was categorized and determined for 10 vessels per patient (n = 270 total vessels). The relative number of vessels with different SPARCL1 expression (%) is indicated by different gray tones. ***P < 0.001, by χ2 test. (E) SPARCL1 and GBP1 expression was quantified in CRC tissues (n = 127) by RT-qPCR. Expression is indicated in 40-ΔCt values. Pearson’s correlation (r = Pearson’s correlation coefficient) was performed in order to determine statistical significance. Line corresponds to linear regression.