Sox17 promotes tumor angiogenesis and destabilizes tumor vessels in mice Hanseul Yang, … , Gou Young Koh, Injune Kim J Clin Invest. 2013;123(1):418-431. https://doi.org/10.1172/JCI64547. Research Article Oncology Little is known about the transcriptional regulation of tumor angiogenesis, and tumor ECs (tECs) remain poorly characterized. Here, we studied the expression pattern of the transcription factor Sox17 in the vasculature of murine and human tumors and investigated the function of Sox17 during tumor angiogenesis using Sox17 genetic mouse models. Sox17 was specifically expressed in tECs in a heterogeneous pattern; in particular, strongS ox17 expression distinguished tECs with high VEGFR2 expression. Whereas overexpression of Sox17 in tECs promoted tumor angiogenesis and vascular abnormalities, Sox17 deletion in tECs reduced tumor angiogenesis and normalized tumor vessels, inhibiting tumor growth. Tumor vessel normalization by Sox17 deletion was long lasting, improved anticancer drug delivery into tumors, and inhibited tumor metastasis. Sox17 promoted endothelial sprouting behavior and upregulated VEGFR2 expression in a cell-intrinsic manner. Moreover, Sox17 increased the percentage of tumor- associated CD11b+Gr-1+ myeloid cells within tumors. The vascular effects of Sox17 persisted throughout tumor growth. Interestingly, Sox17 expression specific to tECs was also observed in highly vascularized human glioblastoma samples. Our findings establish Sox17 as a key regulator of tumor angiogenesis and tumor progression. Find the latest version: https://jci.me/64547/pdf Research article Sox17 promotes tumor angiogenesis and destabilizes tumor vessels in mice Hanseul Yang,1 Sungsu Lee,1 Seungjoo Lee,1 Kangsan Kim,1 Yeseul Yang,1 Jeong Hoon Kim,2 Ralf H. Adams,3,4 James M. Wells,5 Sean J. Morrison,6 Gou Young Koh,1 and Injune Kim1 1Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea. 2Department of Neurological Surgery, Asan Medical Center, College of Medicine, University of Ulsan, Seoul, Republic of Korea. 3Department of Tissue Morphogenesis, Max Plank Institute for Molecular Biomedicine, Münster, Germany. 4Faculty of Medicine, University of Münster, Münster, Germany. 5Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA. 6Howard Hughes Medical Institute, University Texas Southwestern Medical Center, Dallas, Texas, USA. Little is known about the transcriptional regulation of tumor angiogenesis, and tumor ECs (tECs) remain poorly characterized. Here, we studied the expression pattern of the transcription factor Sox17 in the vascu- lature of murine and human tumors and investigated the function of Sox17 during tumor angiogenesis using Sox17 genetic mouse models. Sox17 was specifically expressed in tECs in a heterogeneous pattern; in particular, strong Sox17 expression distinguished tECs with high VEGFR2 expression. Whereas overexpression of Sox17 in tECs promoted tumor angiogenesis and vascular abnormalities, Sox17 deletion in tECs reduced tumor angio- genesis and normalized tumor vessels, inhibiting tumor growth. Tumor vessel normalization by Sox17 dele- tion was long lasting, improved anticancer drug delivery into tumors, and inhibited tumor metastasis. Sox17 promoted endothelial sprouting behavior and upregulated VEGFR2 expression in a cell-intrinsic manner. Moreover, Sox17 increased the percentage of tumor-associated CD11b+Gr-1+ myeloid cells within tumors. The vascular effects of Sox17 persisted throughout tumor growth. Interestingly, Sox17 expression specific to tECs was also observed in highly vascularized human glioblastoma samples. Our findings establish Sox17 as a key regulator of tumor angiogenesis and tumor progression. Introduction not been well studied in the context of tumor angiogenesis. More In the last few decades, regulation of tumor angiogenesis has been studies on the transcription factors are needed to clarify transcrip- central to the fields of vascular biology and oncology, as tumor tional regulation of key players in tumor angiogenesis. growth critically depends on new blood vessel formation. Tumor Although tumor vessel morphogenesis is influenced by various angiogenesis, characterized by excessive angiogenesis and abnor- cells within the tumor (4), tumor ECs (tECs) may represent one mal morphogenesis, is mediated by multiple angiogenic regula- of the most important cell types. Nonetheless, it is not completely tors (1, 2). Many of the angiogenic regulators implicated in tumor clear how the characteristics of tECs differ from those of normal angiogenesis are surface receptors on ECs and their cognate ligands ECs, and the properties of tECs are extrapolated from studies on being expressed by the tumor cells and tumor stromal cells. The key ECs in vascular development. Although different ECs coexist in ligands and receptors, including VEGF/VEGFRs and the angiopoi- vascular development, as shown in tip, stalk, and phalanx cells that etin and Tie2 pathways, have been the main targets of therapies are distinct from each other in morphology and signaling charac- seeking to control tumor angiogenesis (3–5). Despite therapeutic teristics (16–19), the heterogeneity of tECs has never been studied success for some cancers, blockade of these pathways occasion- at the individual cell level. Analysis of tEC heterogeneity is impor- ally results in incomplete inhibition or evasive activation of tumor tant to better understand the mechanisms of tumor angiogenesis. angiogenesis (6–9), emphasizing the need for a more complete Sox17 is a transcription factor belonging to the Sox family, which understanding of angiogenic regulation. contains HMG box (20). Although Sox17 has been considered an Compared with information from the many studies on growth important regulator in biological processes, including endoderm factors, their receptors, and the subsequent signaling pathways that formation during gastrulation (21), segregation of ventral foregut govern tumor angiogenesis, little is known about transcriptional lineages (22), and maintenance of fetal identity in HSCs (23), its sig- regulation of tumor angiogenesis. For example, the transcriptional nificance in vascular biology is only beginning to be recognized. In regulation of VEGFR2, which encodes the most important receptor the context of the vascular system, Sox17 expression is observed spe- for angiogenesis, remains elusive in tumor angiogenesis, although cifically in ECs during vascular development (24). Although global many studies have investigated VEGFR2 expression in vascular deletion of Sox17 results in abnormal vascular development, the con- development (10–12). Tumor angiogenesis is a continuous process tribution of Sox17 to endothelial function remains unclear (25). In (2, 4); thus, transcription factors that elicit a delayed response are the human genome, the Sox17 locus is one of the most susceptible expected to function as long-term regulators of tumor angiogenesis. loci associated with intracranial aneurysms, suggesting its impor- However, the roles of transcription factors such as Etv2, COUF-TFII, tance for blood vessel maintenance (26). However, the expression and and Prox-1 (13–15), which are crucial for vascular development, have function of Sox17 in tumor angiogenesis have not been investigated. In this study, we found profoundly increased Sox17 expression in Authorship note: Hanseul Yang and Sungsu Lee contributed equally to this work. tECs during tumor progression. Using Sox17 genetic mouse mod- Conflict of interest: The authors have declared that no conflict of interest exists. els, we provide compelling evidence that endothelial Sox17 has Citation for this article: J Clin Invest. 2013;123(1):418–431. doi:10.1172/JCI64547. both an intrinsic effect on tECs and an extrinsic effect on the peri- 418 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013 research article Figure 1 Sox17 expression specific to tECs is coincident with increased angiogenesis. LLC and B16F10 tumors grown in Sox17GFP/+ mice (A–C and F) or wild-type mice (D, E, and G) were analyzed. (A–C) Images showing Sox17 expression (GFP) in tumor vessels (PECAM), but not in tumor- associated macrophages (CD11b+) or pericytes (α-SMA+), 2 weeks after implantation. (D) Sox17 transcripts specific to tECs purified from tumors 2 weeks after implantation. FACS plot shows cell populations purified from tumors. tHCs, tumor hematopoietic cells; OCs, other cells. E( ) Sox17 expression in tECs at 1, 2, and 3 weeks after implantation. (F) Sox17 expression in individual tECs, as determined by FACS. Percentages of Sox17high tECs are shown. Cells from Sox17+/+ mice were used to establish background expression. (G) Images of tumor vessels showing differ- ent patterns of angiogenesis in LLC and B16F10 tumors at 1, 2, and 3 weeks after implantation. (A–C and G) Scale bars: 100 μm. (D–F) n = 3 (D and E) or 3–5 (F) per group. *P < 0.05, †P < 0.01 versus LLC tECs; #P < 0.001 versus tECs. endo thelial compartment of tumor vessels. By analyzing the expres- sels. 2 weeks after Lewis lung carcinoma (LLC) implantation, GFP sion of Sox17 in tECs at the individual cell level, we showed that tracing in Sox17GFP/+ mice revealed that most of the tumor vessels tECs were heterogeneous. Taken together, our findings demon- expressed Sox17 at high levels in primary tumors (Figure 1A) and strated that transcriptional regulation by Sox17 plays a critical role in