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 6

SPARCL1 is a marker of EC quiescence.

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SPARCL1 is a marker of EC quiescence. (A) MVECs were plated on chamber s...
(A) MVECs were plated on chamber slides and grown to subconfluent and confluent states. Cells were costained by IHC for SPARCL1/Ki-67 or SPARCL1/IL-33 (SPARCL1, green; Ki-67/nuclear IL-33, pink, arrowheads; DAPI, blue). Scale bar: 75 μm. Graph depicts the relative numbers (percentages) of Ki-67– and nuclear IL-33–positive cells per optical field. Error bars indicate SD. (B) HUVECs were transiently transfected with a human SPARCL1 expression plasmid. Cells were incubated 24 hours after transfection for 2 hours with 10 μM EdU. Afterward, the Click-iT reaction was performed according to the manufacturer’s protocol, and SPARCL1 was stained (EdU, pink; SPARCL1, green; DAPI, blue). Scale bar: 50 μm. The relative amount of EdU-positive cells was determined in both SPARCL1-positive and SPARCL1-negative cells (n = 1,883). Error bars indicate SD. (C) HUVECs were plated on chamber slides and grown until confluence was reached in some areas. The cells were immunocytochemically stained for SPARCL1/Ki-67 or SPARCL1/IL-33, and confocal images of areas with inhomogenous confluence were acquired (SPARCL1, green; Ki-67/nuclear IL-33, pink, arrowheads; DAPI, blue). Scale bar: 100 μm. (D) Confluent cell layers of MVECs were scratched and immunocytochemically stained for SPARCL1 after 0, 14, and 48 hours (SPARCL1, green, DAPI, blue). The migration front is marked by a dashed white line, and cells migrating into the scratch that had lost SPARCL1 expression are labeled with arrowheads. Scale bar: 250 μm. (E) HUVECs were grown in parallel dishes for 1, 15, or 22 days. On day 15, cells from 1 dish were reseeded at a low density (subcultivated - at day 15) and grown for an additional 8 days (subcultivated - at day 22). The slides were immunofluorescently costained for SPARCL1/Ki-67 and SPARCL1/IL-33 expression (SPARCL1, green; Ki-67/nuclear IL-33, pink, arrowheads; DAPI, blue). Scale bar: 100 μm. (A and B) ***P < 0.001, by Student’s t test . (A, B, and D) One representative experiment of two independent experiments is depicted.