Matricellular protein SPARCL1 regulates tumor microenvironment–dependent endothelial cell heterogeneity in colorectal carcinoma
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
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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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 8

SPARCL1 is an antiangiogenic protein.

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SPARCL1 is an antiangiogenic protein. (A) HUVECs were stably transduced ...
(A) HUVECs were stably transduced by a retroviral SPARCL1-encoding vector (pBABE-SPARCL1) and the corresponding control vector (pBABE). Increased SPARCL1 expression in these cells was confirmed by Western blotting and immunocytochemistry (Supplemental Figure 6A). In agreement with earlier results, the cells showed significant inhibition of angiogenic growth factor–induced (AGF-induced) proliferation by SPARCL1 (AGF = combined bFGF/VEGF, 10 ng/ml each; positive control) (Supplemental Figure 6B). SPARCL1 overexpression significantly reduced bFGF-induced 3D sprouts from spheroids embedded in collagen/methocel matrices. (B) MVECs were transiently transfected with an siRNA (50 nM) specifically targeting SPARCL1 and with a control siRNA. Reduction of SPARCL1 RNA expression in cells transfected with the SPARCL1 siRNA was confirmed by RT-qPCR. In parallel, the same cells were used for spheroid formation, and 3D sprouting was assessed after 24 hours. (C) HUVECs were either untreated (mock) or treated with AGF (positive control), AGF and IFN-γ (100 U/ml, negative control), or AGF with increasing concentrations of recombinant SPARCL1. Cell numbers after 6 days are shown. (D) HUVECs were plated on Transwell inserts (8 μm) and treated as in C for 6 hours. Migrated cells at the lower membrane side were determined by counterstaining with DAPI (Supplemental Figure 7B), and the mean values of cells counted per optical field are shown. (E) Spheroids from HUVECs were embedded in a collagen/methocel gel and stimulated as described in C, except that bFGF was used alone instead of AGF for 24 hours in duplicate experiments. The sprout lengths of 20 spheroids per stimulation were quantitatively determined and are indicated as the mean cumulative sprout length per spheroid. All experiments were performed 3 times in triplicate, except the spheroid assays, which were performed in duplicate, with 10 spheroids quantified per group. *P < 0.05 and ***P < 0.001, by Student’s t test (A and B) or ANOVA, with Levene and Bonferroni’s (equal variance) or Games-Howell (unequal variance) test (C–E). (A, B, and E) Scale bars: 250 μm. (A–E) Error bars indicate SD.