Hsin Chieh Lin

DDR2 receptor promotes MMP-2–mediated proliferation and invasion by hepatic stellate cells

Type I collagen provokes activation of hepatic stellate cells during liver injury through mechanisms that have been unclear. Here, we tested the role of the discoidin domain tyrosine kinase receptor 2 (DDR2), which signals in response to type I collagen, in this pathway. DDR2 mRNA and protein are induced in stellate cells activated by primary culture or in vivo during liver injury. The receptor becomes tyrosine phosphorylated in response to either endogenous or exogenous type I collagen, whereas its expression is downregulated during cellular quiescence induced by growth on Matrigel. We developed stellate cell lines stably overexpressing either wild-type DDR2, a constitutively active chimeric DDR2 receptor (Fc-DDR2), a truncated receptor expressing the extracellular domain, or a kinase-dead DDR2 Cells overexpressing DDR2 showed enhanced proliferation and invasion through Matrigel, activities that were directly related to increased expression of active matrix metalloproteinase 2 (MMP-2). These data show that DDR2 is induced during stellate cell activation and implicate the phosphorylated receptor as a mediator of MMP-2 release and growth stimulation in response to type I collagen. Moreover, type I collagen-dependent upregulation of DDR2 expression establishes a positive feedback loop in activated stellate cells, leading to further proliferation and enhanced invasive activity.

Angiogenic sprouting into neural tissue requires Gpr124, an orphan G protein-coupled receptor

The vasculature of the CNS is structurally and functionally distinct from that of other organ systems and is particularly prone to developmental abnormalities and hemorrhage. Although other embryonic tissues undergo primary vascularization, the developing nervous system is unique in that it is secondarily vascularized by sprouting angiogenesis from a surrounding perineural plexus. This sprouting angiogenesis requires the TGF-β and Wnt pathways because ablation of these pathways results in aberrant sprouting and hemorrhage. We have genetically deleted Gpr124, a member of the large family of long N-terminal group B G protein-coupled receptors, few members of which have identified ligands or well-defined biologic functions in mammals. We show that, in the developing CNS, Gpr124 is specifically expressed in the vasculature and is absolutely required for proper angiogenic sprouting into the developing neural tube. Embryos lacking Gpr124 exhibit vascular defects characterized by delayed vascular penetration, formation of pathological glomeruloid tufts within the CNS, and hemorrhage. In addition, they display defects in palate and lung development, two processes in which TGF-β and/or Wnt pathways also play important roles. We also show that TGF-β stimulates Gpr124 expression, and ablation of Gpr124 results in perturbed TGF-β pathway activation, suggesting roles for Gpr124 in modulating TGF-β signaling. These results represent a unique function attributed to a long N-terminal group B–type G protein-coupled receptor in a mammalian system.

Use of the Fluidigm C1 platform for RNA sequencing of single mouse pancreatic islet cells

Significance Pancreatic islets are complex structures composed of four cell types whose primary function is to maintain glucose homeostasis. Owing to the scarcity and heterogeneity of the islet cell types, little is known about their individual gene expression profiles. Here we used the Fluidigm C1 platform to obtain high-quality gene expression profiles of each islet cell type from mice. We identified cell-type–specific transcription factors and pathways providing previously unrecognized insights into genes characterizing islet cells. Unexpectedly, our data uncover technical limitations with the C1 Fluidigm cell capture process, which should be considered when analyzing single-cell transcriptomics data. This study provides an assessment of the Fluidigm C1 platform for RNA sequencing of single mouse pancreatic islet cells. The system combines microfluidic technology and nanoliter-scale reactions. We sequenced 622 cells, allowing identification of 341 islet cells with high-quality gene expression profiles. The cells clustered into populations of α-cells (5%), β-cells (92%), δ-cells (1%), and pancreatic polypeptide cells (2%). We identified cell-type–specific transcription factors and pathways primarily involved in nutrient sensing and oxidation and cell signaling. Unexpectedly, 281 cells had to be removed from the analysis due to low viability, low sequencing quality, or contamination resulting in the detection of more than one islet hormone. Collectively, we provide a resource for identification of high-quality gene expression datasets to help expand insights into genes and pathways characterizing islet cell types. We reveal limitations in the C1 Fluidigm cell capture process resulting in contaminated cells with altered gene expression patterns. This calls for caution when interpreting single-cell transcriptomics data using the C1 Fluidigm system.

Embryonic stem cell tumor model reveals role of vascular endothelial receptor tyrosine phosphatase in regulating Tie2 pathway in tumor angiogenesis

Inhibiting angiogenesis has become an effective approach for treating cancer and other diseases. However, our understanding of signaling pathways in tumor angiogenesis has been limited by the embryonic lethality of many gene knockouts. To overcome this limitation, we used the plasticity of embryonic stem (ES) cells to develop a unique approach to study tumor angiogenesis. Murine ES cells can be readily manipulated genetically; in addition, ES cells implanted subcutaneously in mice develop into tumors that contain a variety of cell types (teratomas). We show that ES cells differentiate into bona fide endothelial cells within the teratoma, and that these ES-derived endothelial cells form part of the functional tumor vasculature. Using this powerful and flexible system, the Angiopoietin/Tie2 system is shown to have a key role in the regulation of tumor vessel size. Endothelial differentiation in the ES teratoma model allows gene-targeting methods to be used in the study of tumor angiogenesis.

Abstract B4: Evaluation of VEGFxxxb isoforms as a predictive biomarker for anti-VEGF cancer therapy.

Biomarkers are critical tools for measuring efficacy of treatments or predicting which patients are mostly likely to respond to a particular therapy. Recently, the VEGF isoform, VEGF165b, was proposed to be a predictive marker for bevacizumab in metastatic colorectal cancer patients. VEGF165b is an anti-angiogenic splice variant that differs from its pro-angiogenic counterpart, VEGF165, by only six amino acids at the C-terminal end of the protein. Members of the VEGFxxxb isoform family are produced by alternative splicing of the 3’ untranslated region (UTR) to generate exon 8b, instead of the conventional exon 8 which produces exon 8a. The study of VEGF165b in colorectal cancer (Bates et al, Clinical Cancer Research, 2012) reported that patients with low VEGF165b:VEGFtotal ratio had improved PFS following bevacizumab + FOLFOX4 treatment, while individuals with higher VEGF165b:VEGFtotal ratios did not benefit. On the other hand, another recent study (Harris et al, PLoS One, 2012) reported that VEGFxxxb isoforms may be a RT-PCR artifact. In this study, we sought to test the role of VEGF165b in the response to anti-VEGF therapy using preclinical human tumor models, and to evaluate the prevalence of VEGFxxxb isoform in human cancer cells and normal tissues using RNA-Seq, a next generation sequencing (NGS) platform that is independent of RT-PCR. Our results show that xenograft tumors engineered to overexpress VEGF165b had similar growth kinetics, tumor vascular density and were equally sensitive to aflibercept or bevacizumab treatment in humanized VEGF SCID mice compared to their parental counterparts. These results indicate that VEGF165b does not have anti-angiogenic properties, and VEGF165b levels did not correlate with the efficacy of anti-VEGF therapy. Furthermore, although we were able to detect VEGF165b splice variants by NGS profiling in cells engineered to overexpress VEGF165b, no NGS evidence supporting the VEGFxxxb isoforms was found in a panel of 24 human cancer cell lines and 12 different types of human normal tissues. To our knowledge, this is the first report to survey VEGFxxxb isoforms using NGS RNA-seq technology. Overall, these studies suggest that closer inspection of VEGFxxxb isoforms may be required before employing VEGF165b as a predictive biomarker for anti-VEGF therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B4. Citation Format: Wen Zhang, Cristina Abrahams, Ying Huang, Baosheng Li, Asma Parveen, Min Ni, Yi Wei, Yueming Ding, Eric Zhang, Hsin Chieh Lin, Gavin Thurston, Qing Zhang. Evaluation of VEGFxxxb isoforms as a predictive biomarker for anti-VEGF cancer therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B4.