Angiogenesis involves dynamic interactions between specialized endothelial tip and stalk cells that are believed to be regulated in part by VEGF and Dll4-Notch signaling. However, our understanding of this process is hampered by limited knowledge of the heterogeneity of endothelial cells and the role of different signaling pathways in specifying endothelial phenotypes. Here, we characterized by single-cell transcriptomics the heterogeneity of mouse endothelial cells and other stromal cells during active angiogenesis in xenograft tumors as well as from adult normal heart, following pharmacologic inhibition of VEGF and Dll4-Notch signaling. We classified tumor endothelial cells into three subpopulations that appeared to correspond with tip-like, transition, and stalk-like cells. Previously identified markers for tip and stalk cells were confirmed and several novel ones discovered. Blockade of VEGF rapidly inhibited cell-cycle genes and strongly reduced the proportion of endothelial tip cells in tumors. In contrast, blockade of Dll4 promoted endothelial proliferation as well as tip cell markers; blockade of both pathways inhibited endothelial proliferation but preserved some tip cells. We also phenotypically classified other tumor stromal cells and found that tumor-associated fibroblasts responded to antiangiogenic drug treatments by upregulating hypoxia-associated genes and producing secreted factors involved in angiogenesis. Overall, our findings better define the heterogeneity of tumor endothelial and other stromal cells and reveal the roles of VEGF and Dll4-Notch in specifying tumor endothelial phenotype, highlighting the response of stromal cells to antiangiogenic therapies. These findings provide a framework for defining subpopulations of endothelial cells and tumor-associated fibroblasts and their rapid changes in gene expression following antiangiogenic treatment. .
Anti-VEGF therapies benefit several cancer types, but drug resistance that limits therapeutic response can emerge. We generated cell lines from anti-VEGF-resistant tumor xenografts to investigate the mechanisms by which resistance develops. Of all tumor cells tested, only A431 (A431-V) epidermoid carcinoma cells developed partial resistance to the VEGF inhibitor aflibercept. Compared with the parental tumors, A431-V tumors secreted greater amounts of IL6 and exhibited higher levels of phospho-STAT3. Notably, combined blockade of IL6 receptor (IL6R) and VEGF resulted in enhanced activity against A431-V tumors. Similarly, inhibition of IL6R enhanced the antitumor effects of aflibercept in DU145 prostate tumor cells that displays high endogenous IL6R activity. In addition, post hoc stratification of data obtained from a clinical trial investigating aflibercept efficacy in ovarian cancer showed poorer survival in patients with high levels of circulating IL6. These results suggest that the activation of the IL6/STAT3 pathway in tumor cells may provide a survival advantage during anti-VEGF treatment, suggesting its utility as a source of response biomarkers and as a therapeutic target to heighten efficacious results.
The liver is a common host organ for cancer, either through lesions that arise in liver epithelial cells [e.g., hepatocellular carcinoma (HCC)] or as a site of metastasis by tumors arising in other organs (e.g., colorectal cancer). However, the changes that occur in liver stromal cells in response to cancer have not been fully characterized, nor has it been determined whether the different sources of liver cancer induce distinct stromal changes. Here, we performed single-cell profiling of liver stromal cells from mouse models of induced spontaneous liver cancer or implanted colorectal liver metastases, with a focus on tumor endothelial cells (ECs). While ECs in liver tissue adjacent to cancerous lesions (so-called adjacent normal) corresponded to liver zonation phenotypes, their transcriptomes were also clearly altered by the presence of a tumor. In comparison, tumor EC transcriptomes show stronger similarities to venous than sinusoidal ECs. Further, tumor ECs, independent of tumor origin, formed distinct clusters displaying conserved “tip-like” or “stalk-like” characteristics, similar to ECs from subcutaneous tumors. However, they also carried liver-specific signatures found in normal liver ECs, suggesting an influence of the host organ on tumor ECs. Our results document gene expression signatures in ECs in liver cancer and show that the host organ, and not the site of tumor origin (liver versus colorectal), is a primary determinant of EC phenotype. In addition, primarily in tumors, we further defined a cluster of chimeric cells that expressed both myeloid and endothelial cell markers and might play a role in tumor angiogenesis.
Salmonella typhi, a Gram negative bacterium, has become multidrug resistant (MDR) to wide classes of antibacterials which necessitate an alarming precaution. This study focuses on the binding potential and therapeutic insight of Nano-Fullerene C60 towards virulent targets of Salmonella typhi by computational prediction and preliminary in vitro assays. The clinical isolates of Salmonella typhi were collected and antibiotic susceptibility profiles were assessed. The drug targets of pathogen were selected by rigorous literature survey and gene network analysis by various metabolic network resources. Based on this study, 20 targets were screened and the 3D structures of few drug targets were retrieved from PDB and others were computationally predicted. The structures of nanoleads such as Fullerene C60, ZnO and CuO were retrieved from drug databases. The binding potential of these nanoleads towards all selected targets were predicted by molecular docking. The best docked conformations were screened and concept was investigated by preliminary bioassays. This study revealed that most of the isolates of Salmonella typhi were found to be MDR (p < .05). The theoretical models of selected drug targets showed high stereochemical validity. The molecular docking studies suggested that Fullerene C60 showed better binding affinity towards the drug targets when compared to ZnO and CuO. The preliminary in vitro assays suggested that 100 μg/L Fullerene C60 posses significant inhibitory activities and absence of drug resistance to this nanoparticle. This study suggests that Fullerene C60 can be scaled up as probable lead molecules against the major drug targets of MDR Salmonella typhi.
Angiopoietin-1 (Ang1) and -2 (Ang2) regulate angiogenesis via the endothelial cell-specific receptor tyrosine kinase Tie2. Blocking Ang2 binding to Tie2 decreases vessel density and inhibits tumor growth in various human xenograft models. However, not all tumors respond to Ang2 blockade, which could be due to high Ang1 levels acting as another ligand for Tie2, to Ang2 activities independent of Tie2, or to a weak role for Ang-2/Tie2 signaling in these tumors. To elucidate if Ang1 plays an integral role in tumor angiogenesis and growth, we compared the effects of blocking Ang2 or Tie2 in various tumor xenografts with differing levels of Ang1 expression. We found that in six out of seven tumor models, antibodies that specifically bind Ang2 (REGN910, 10 mg/kg 2x/wk) or that bind Tie2 and block binding of both Ang1 and Ang2 (REGN1376, 10 mg/kg 2x/wk) were equally efficacious at inhibiting tumor growth and decreasing vessel density. In only one tumor model (Calu-6), blockade of Tie2 with REGN1376 was more effective than blockade of Ang2 (REGN910) at reducing tumor growth and decreasing tumor vessel density. Although Calu-6 tumors express high Ang1 levels, other tumors tested (Lox, LS174T) have comparable or higher Ang1 levels and respond similarly to Ang2 and Tie2 blockade, thus the levels of Ang1 do not account for the differential response of Calu-6 tumors. Taken together, our data suggest that Ang2 is the dominant ligand for Tie2 in the microenvironment of most tumors, and further, that the effects of Ang2 are mediated primarily via Tie2. These findings support the approach to specifically block the binding of Ang2 to Tie2 as an anti-angiogenic therapy. Citation Format: Alexander P. Adler, Christopher Daly, Asma A. Parveen, Thomas Nevins, Jing Shan, Jeanette Fairhurst, Tammy Huang, Joel Martin, Nicholas Papadopoulos, George D. Yancopoulos, Gavin Thurston, Gavin Thurston, Alexandra Eichten. Blockade of angiopoietin-2 or Tie2 is equally effective at inhibiting tumor growth and reducing tumor vessel density in most human tumor xenograft models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4492. doi:10.1158/1538-7445.AM2014-4492
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