<div>Abstract<p>Endothelial Notch signaling is critical for tumor angiogenesis. Notch1 blockade can interfere with tumor vessel function but causes tissue hypoxia and gastrointestinal toxicity. Notch4 is primarily expressed in endothelial cells, where it may promote angiogenesis; however, effective therapeutic targeting of Notch4 has not been successful. We developed highly specific Notch4-blocking antibodies, 6-3-A6 and humanized E7011, allowing therapeutic targeting of Notch4 to be assessed in tumor models. Notch4 was expressed in tumor endothelial cells in multiple cancer models, and endothelial expression was associated with response to E7011/6-3-A6. Anti-Notch4 treatment significantly delayed tumor growth in mouse models of breast, skin, and lung cancers. Enhanced tumor inhibition occurred when anti-Notch4 treatment was used in combination with chemotherapeutics. Endothelial transcriptomic analysis of murine breast tumors treated with 6-3-A6 identified significant changes in pathways of vascular function but caused only modest change in canonical Notch signaling. Analysis of early and late treatment timepoints revealed significant differences in vessel area and perfusion in response to anti-Notch4 treatment. We conclude that targeting Notch4 improves tumor growth control through endothelial intrinsic mechanisms.</p>Significance:<p>A first-in-class anti-Notch4 agent, E7011, demonstrates strong antitumor effects in murine tumor models including breast carcinoma. Endothelial Notch4 blockade reduces perfusion and vessel area.</p></div>
<p>Treatment of human tumor xenografts with 6-3-A6 delays growth of tumors which express endothelial Notch4. <b>A,</b> IF staining of Notch4 (red) and CD31<sup>+</sup> tumor vessels (green) in human tumor xenografts. Arrowheads indicate Notch4-expressing vessels (Scale bar = 200 μm). <b>B</b> and <b>C,</b> Tumor growth curves of subcutaneously implanted tumors from the indicated cell lines. <b>B,</b> MDA-MB-231 xenografts treated intravenously with 6-3-A6 (<i>n</i> = 5). <b>C,</b> Calu-6 human NSCLC xenograft tumors treated intravenously with 6-3-A6. All treatments started 12 hours after tumor implantation. Statistical analysis performed with two-way ANOVA for tumor growth curves, plotted as average ± SEM; *, <i>P</i> < 0.05.</p>
<p>Notch4 is expressed on tumor ECs, and treatment alters the tumor vessel transcriptional profile. <b>A,</b> Representative flow plots of gating for CD31<sup>+</sup> cells in Py8119 and B16F10 tumors. <b>B,</b> Histograms comparing E7011 and human IgG binding to CD31<sup>+</sup> and CD31<sup>−</sup> cell populations from dissociated Py8119 and B16F10 tumors (<i>n</i> = 3). Left, E7011 samples were concatenated and merged into a single representative histogram. Right, Graph of concatenated MFI from Py8119 and B16F10 samples bound with E7011 or human IgG. <b>C,</b> Heatmaps of significantly upregulated and downregulated differentially expressed genes isolated from IP polysomes from tumor ECs treated with 6-3-A6 or control IgG (<i>n</i> = 3). Boxes indicate genes implicated in GPCR control of angiogenesis (red), endothelial nitric oxide regulation (blue), and inflammatory/immune response (green). <b>D,</b> Reactome pathway analysis of upregulated and downregulated pathways was performed using g:Profiler, and significance threshold by Benjamini–Hochberg FDR was set at 0.05. <b>A,</b> Fold cutoff of −log<sub>10</sub>(<i>P</i><sub>adj</sub>) > 2 was used to select the displayed pathways. Statistical analysis performed with the <i>t</i> test for perfusion studies, plotted as average ± SEM; *, <i>P</i> < 0.05; **, <i>P</i> < 0.005. Heatmap data presented as <i>Z</i>-scores, which integrate both fold change and significance.</p>
<p>A, Schematic of experimental design of Py8119 orthotopically implanted tumors in RiboTag<sup>EC</sup> mice. Female RiboTag<sup>EC</sup> mice with 75mg/kg of tamoxifen (20mg/mL) for 5 days by IP injection. Py8119 tumors were implanted at 8 weeks of age and then treated once tumors reached 50mm3 with twice weekly IV control IgG or 6-3-A6 for a total of 10 days (n=3). B, Growth curve of RiboTagEC treated with 25mg/kg of IV control IgG or 6-3-A6 twice weekly for 10 days (n=3). C, Heatmap of endothelial expressed genes segregated by total mRNA input and ribosomal IP mRNA from control IgG treated and 6-3-A6 treated Py8119 tumors. D, PCA plots of the ribosomal IP fraction from control IgG and 6-3-A6 treated tumors. Two samples from the control IgG and 6-3-A6 groups were identified as outliers and removed from analysis. E, Heatmap of downstream Notch regulated genes from endothelial IP of the control IgG or 6-3-A6 treated tumors and quantitative rt-PCR of the canonical Notch target gene <i>Hes1</i> and <i>Rnd1</i> amplified from 6-3-A6 treated and control RiboTag IP fractions (n=4). F, Heatmaps of genes associated with significantly enriched or downregulated signaling pathways involved in inflammation and extracellular matrix interactions. All heatmap data presented as Z-scores. Statistics by t-test for qPCR studies</p>
Abstract E7130 is a novel anticancer agent created from total synthetic study of the natural compound norhalichondrin B. In addition to inhibiting microtubule dynamics, E7130 also ameliorates tumor-promoting aspects of the tumor microenvironment (TME) by suppressing cancer-associated fibroblasts (CAF) and promoting remodeling of tumor vasculature. Here, we demonstrate TME amelioration by E7130 using multi-imaging modalities, including multiplexed mass cytometry [cytometry by time-of-flight (CyTOF)] analysis, multiplex IHC analysis, and MRI. Experimental solid tumors characterized by large numbers of CAFs in TME were treated with E7130. E7130 suppressed LAP-TGFβ1 production, a precursor of TGFβ1, in CAFs but not in cancer cells; an effect that was accompanied by a reduction of circulating TGFβ1 in plasma. To our best knowledge, this is the first report to show a reduction of TGFβ1 production in TME. Furthermore, multiplex IHC analysis revealed reduced cellularity and increased TUNEL-positive apoptotic cells in E7130-treated xenografts. Increased microvessel density (MVD) and collagen IV (Col IV), an extracellular matrix (ECM) component associated with endothelial cells, were also observed in the TME, and plasma Col IV levels were also increased by E7130 treatment. MRI revealed increased accumulation of a contrast agent in xenografts. Moreover, diffusion-weighted MRI after E7130 treatment indicated reduction of tumor cellularity and interstitial fluid pressure. Overall, our findings strongly support the mechanism of action that E7130 alters the TME in therapeutically beneficial ways. Importantly, from a translational perspective, our data demonstrated MRI as a noninvasive biomarker to detect TME amelioration by E7130, supported by consistent changes in plasma biomarkers.
The complications of dural and direct cavernous sinus fistula (CCF) arise mainly from the specific venous route. However, embolization at an inappropriate site within the cavernous sinus (CS) is also a major factor. Therefore, we first diagrammed the surrounding structures of the CS to elucidate the specificity of the venous routes. Next, we divided the inside structure of the CS into four compartments, to examine orifices at which part we can start embolization with the least danger of causing complications when we have to embolize them within the CS. We obtained findings which will be useful to prevent complications such as subarachnoid haemorrhage, glaucoma, central retinal vein thrombosis and marked neurological impairment.
