Tumor Endothelial Cell Proliferation Research Articles

Abstract 3698: Epigenetic modification induced reprogramming of PUFAs-associated metabolism during trastuzumab resistance formation of HER2-positive breast cancer

Abstract Background: Secondary trastuzumab resistance seriously affects the treatment of HER2-positive breast cancer. Although studies have demonstrated several potential reasons that cause trastuzumab resistance, we still have poor knowledge about the changes in cell itself as well as its interaction with tumor microenvironment (TME) components during resistance formation. Here we suggested that altered histone modification reprograms polyunsaturated fatty acids (PUFAs) metabolism, which reduces cellular PUFAs and stimulates prostaglandin E2 (PGE2) production. Excessive PGE2 may further affect both cancer cells as well as immune cells in TME. Materials and Methods: Secondary trastuzumab-resistant cell line SKBR3_HR was generated from sensitive SKBR3 cells by raising trastuzumab concentration gently from 1ug/ml to 20ug/ml in 30 weeks. Cell viabilities were measured by CCK8 test. CUT&Tag with anti-H3K4me3 and anti-H3K27me3 antibodies were applied to make sequencing libraries. Total RNA and protein were collected for transcriptome and proteome analysis. Activity scores of metabolism processes were defined and calculated as the relative gene expression value averaged over all genes in this pathway in certain cell types. Oxidized PUFAs and PGE2 were assessed by flow cytometry with BODIPY-C11 and ELISA, respectively. Results: SKBR3_HR cells showed similar viability as primary resistant JIMT1 cells and were significantly higher than original SKBR3 cells. Pathway analysis indicated reduced PUFAs synthesis and activated arachidonic acid metabolism. As a consequence, oxidized PUFAs decreased while PGE2 accumulated heavily in SKBR3_HR cells, which may promote tumor and vascular endothelial cell proliferation but interrupt immune cell activity in TME. Transcriptional activity of two critical PUFAs synthesis-related genes, FASN and SCD, were reduced in resistant cells, while two key genes in arachidonic acid metabolism, PTGS1 and PTGES, were activated. H3K4me3 and H3K27me3 play opposite but critical rules in transcriptional regulation. During resistance formation, changes happened on 3256 H3K27me3 peaks as well as 316 H3K4me3 peaks. In detail, although little H3K27me3 changes were found at the promoter regions of four genes (FASN, SCD, PTGS1 and PTGES), reduced H3K4me3 levels may inhibit FASN and SCD expression while raised levels could stimulate PTGS1 and PTGES transcription. Conclusions: Combining the transcriptome and histone modification data, we have a deeper understanding of the cellular adaptions to trastuzumab. Altered trimethylation of H3K4 but not H3K27 remodels the expressions of FASN, SCD, PTGS1 and PTGES, drives the reprogramming of PUFAs-associated metabolism, and finally reduces cellular PUFAs while stimulates PGE2 production. High PGE2 level may affect the activities of tumor and TME-related cells. Citation Format: Yongmei Yin, Ningjun Duan, Yijia Hua, Shuang Hu. Epigenetic modification induced reprogramming of PUFAs-associated metabolism during trastuzumab resistance formation of HER2-positive breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3698.

Thyroid Hormone Enhances Angiogenesis and the Warburg Effect in Squamous Cell Carcinomas

Simple SummaryCancer cells rewire their metabolism to promote growth, survival, proliferation, and long-term maintenance. Aerobic glycolysis is a prominent trait of many cancers; contextually, glutamine addiction, enhanced glucose uptake and aerobic glycolysis sustain the metabolic needs of rapidly proliferating cancer cells. Thyroid hormone (TH) is a positive regulator of tumor progression and metastatic conversion of squamous cell carcinoma (SCC). Accordingly, overexpression of the TH activating enzyme, D2, is associated with metastatic SCC. The aim of our study was to assess the ability of TH and its activating enzyme in promoting key tracts of cancer progression such as angiogenesis, response to hypoxia and metabolic adaptation. By performing in vivo and in vitro studies, we demonstrate that TH induces VEGF-A in cancer cells and fosters aerobic glycolysis inducing pro-glycolytic mediators, thus implying that TH signal attenuation represents a therapeutic tool to contrast tumor angiogenesis and tumor progression.Cancer angiogenesis is required to support energetic demand and metabolic stress, particularly during conditions of hypoxia. Coupled to neo-vasculogenesis, cancer cells rewire metabolic programs to sustain growth, survival and long-term maintenance. Thyroid hormone (TH) signaling regulates growth and differentiation in a variety of cell types and tissues, thus modulating hyper proliferative processes such as cancer. Herein, we report that TH coordinates a global program of metabolic reprogramming and induces angiogenesis through up-regulation of the VEGF-A gene, which results in the enhanced proliferation of tumor endothelial cells. In vivo conditional depletion of the TH activating enzyme in a mouse model of cutaneous squamous cell carcinoma (SCC) reduces the concentration of TH in the tumoral cells and results in impaired VEGF-A production and attenuated angiogenesis. In addition, we found that TH induces the expression of the glycolytic genes and fosters lactate production, which are key traits of the Warburg effect. Taken together, our results reveal a TH–VEGF-A–HIF1α regulatory axis leading to enhanced angiogenesis and glycolytic flux, which may represent a target for SCC therapy.

Inhibition of glycolytic activator PFKFB3 suppresses tumor growth and induces tumor vessel normalization in hepatocellular carcinoma.

Glycolysis emerges as a new therapeutic target for malignancies. The inhibition of glycolytic activator, PFKFB3, repairs tumor endothelial cell function, and normalizing the tumor microenvironment. We aimed to investigate the significance of PFKFB3 in HCC, and the effects of the PFKFB3 inhibitor, PFK15, in HCC tumor cells and tumor endothelial cells. Double immunofluorescent staining of PFKFB3 and CD31 in HCC tissues revealed that high PFKFB3 expression in both tumor cells and tumor endothelial cells was significantly correlated with poor prognosis. Multivariate analysis identified PFKFB3 expression as an independent prognostic factor. PFK15 suppressed proliferation of HCC cell line and tumor endothelial cells in vitro. In a subcutaneous tumor model of the HCC cell line with tumor endothelial cells, PFK15 suppressed tumor growth and induced apoptosis. Moreover, PFK15 treatment induced tumor vessel normalization, decreasing vessel diameter with pericyte attachment and improving vessel perfusion. High PFKFB3 expression in both tumor cells and tumor endothelial cells was identified as a novel prognostic marker in HCC. Targeting PFKFB3 via PFK15 might be a promising strategy for suppressing tumor growth and inducing tumor vessel normalization.

Jet‐Lagged Nanoparticles Enhanced Immunotherapy Efficiency through Synergistic Reconstruction of Tumor Microenvironment and Normalized Tumor Vasculature

Lactic acid (LA), an anaerobic glycolysis metabolite normally oversecreted by tumor cells, can inhibit the activity of Tcells and stimulate the rapid proliferation and migration of tumor endothelial cells (TECs), thereby limiting the synergistic treatment efficiency of tumor immunotherapy and vascular normalization. Herein, Jet-lagged nanoparticles, apatinib (APA)-loaded TEC-targeting nanodrug (APA/MCP) and lonidamine (LND)-loaded tumor cell-targeting nanodrug (LND/MCA), are constructed to combine vascular normalization therapy and tumor cell metabolic treatment. APA/MCP can block VEGF/VEGFR2 to inhibit TEC proliferation and LND/MCA can inhibit LA efflux to remodel tumor acid metabolism. After treatment, Jet-lagged nanoparticles remarkably reduce the level of LA in tumor microenvironment (TME) through limiting LA efflux. Besides, the pericyte cell coverage ratio of tumor vasculature increased to 69%, which is significantly improved compared to the APA/MCP group (47%). Moreover, the results of in vivo pharmacodynamic studies show that after the above synergistic reconstruction of TME and normalized tumor vasculature, the therapeutic effect of programmed death 1 (PD-1) drug is improved 3-folds to that of the PD-1 group. Above all, the strategy in this paper may propose an innovative vision to facilitate the tumor immunotherapy through high-precision spatiotemporal delivery strategy of nanodrugs.

Polymorphisms in the dopamine (DA) signaling to predict outcome in patients (pts) with metastatic colorectal cancer (mCRC): Data from TRIBE, MAVERICC, and FIRE-3 phase III trials.

3048 Background: Strong evidence supports the critical role of the gut-brain axis in modulating gastrointestinal function and homeostasis. Available data suggest an involvement of the dopaminergic pathway in CRC dynamics. DA could inhibit proliferation and migration of tumor endothelial cells and enhanced 5-fluorouracil efficacy in CRC preclinical models. Hence, we hypothesized that genetic variants in DA signaling may predict treatment outcomes in mCRC pts. Methods: The impact on outcome of 22 selected single nucleotide polymorphisms (SNPs) in 9 genes of the DA signaling pathway ( DRD1, DRD2, DRD3, DRD4, DRD5, TAAR1, SLC6A3, SLC18A2, PPP1R1B) was analyzed on a total of 884 pts enrolled in three independent randomized first-line trials: TRIBE (n = 324), MAVERICC (n = 324), and FIRE-3 (n = 236). Genomic DNA from blood samples of pts was genotyped through the OncoArray, a custom array manufactured by Illumina. A meta-analysis approach using the METASOFT software was used to quantify SNPs prognostic effects and heterogeneities across treatment arms. P values were adjusted for multiple testing using the false discovery rate (FDR) method. Results: Overall, DRD3 rs3732790, rs9817063 and rs2134655 showed a significant nominal p value ( P) in association with tumor response (TR) across trials ( P= 0.032, P= 0.021, P= 0.027, respectively). TAAR1 rs8192620 showed an association with both progression free survival (PFS) ( P= 0.01) and overall survival (OS) ( P= 0.033), similar to DA transporter SLC6A3 rs6347 ( P= 0.016 and P= 0.002, respectively). SLC6A3 rs6347 association with OS remained significant after FDR ( PFDR= 0.045). Subgroup analyses showed a significant association with PFS for DRD1 rs267410 and SLC6A3 rs2652510 in females ( PFDR= 0.056), and between SLC6A3 rs6347 and OS ( PFDR= 0.041) and SLC6A3 rs6876890 and TR ( PFDR= 0.05) in KRAS wild type. Conclusions: Our results suggest that SNPs in DA signaling may have a prognostic value in mCRC pts receiving first-line treatment. Upon validation, these findings may provide novel insight on the role of DA signaling in CRC and possibly contribute to open novel therapeutic perspectives.

The effects of microRNA-1224-5p on hepatocellular carcinoma tumor endothelial cells.

The aim of this study was to investigate the effect of microRNA-1224-5p (miR-1224-5p) on tumor endothelial cells (TECs) of human hepatocellular carcinoma (HCC). Oligonucleotides were chemically synthesized and transfected into TECs using Lipofectamine 2000. TECs were divided into three groups, namely a control (CON) group without transfection, a negative control (NC) group transfected with negative control oligonucleotides and green fluorescent protein (GFP), and a micro-up (MU) group transfected with miR-1224-5p mimic and GFP. The expression of miR-1224-5p was quantified via quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The proliferation of TECs was detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the optical density value at 490 nm was measured after every 24 h. Apoptosis was detected via flow cytometry using a 7-aminoactinomycin/APC Annexin V kit. The migration and invasion of TECs were detected using transwell assay. The tube formation ability was evaluated using the tube formation assay. Oligonucleotides were successfully transduced into TECs, and the expression of miR-1224-5p was specifically upregulated. The results of qRT-PCR analysis showed that the expression of miR-1224-5p was significantly upregulated in the MU group (2-ΔΔCt = 3.27 ± 0.15) than in the CON group (2-ΔΔCt = 1) and NC group (2-ΔΔCt = 1.08 ± 0.11) (P < 0.01). The results of MTT assay showed that the cell proliferation was significantly inhibited in the MU group at four time points than in the CON and NC groups (P < 0.01). Flow cytometry analysis revealed the significant increase in apoptosis of cells from the MU group (19.29% ± 0.95%) than those from the CON (8.73% ± 0.64%) and NC (9.51% ± 0.56%) (P < 0.01) groups. The migration ability was significantly inhibited in MU group (51.0 ± 3.6) as compared with CON (77.7 ± 2.5) and NC (79.2 ± 3.5) groups (P < 0.01). The invasion ability of TECs was significantly inhibited in MU group (9.8 ± 1.3) than in CON (15.8 ± 0.8) and NC (15.4 ± 0.9) groups (P < 0.01). The ability of tube formation of TECs was completely inhibited in MU group but remained unaffected in CON and NC groups. miR-1224-5p may serve as a potential tumor suppressor in HCC. Upregulation in miR-1224-5p expression may decrease proliferation, induce apoptosis, inhibit migration and invasion, and suppress tube formation in TECs of human HCC.

Effect of the monoclonal antibody TRC105 in combination with Sunitinib on renal tumor derived endothelial cells.

Anti-angiogenic therapy is an important strategy to limit growth, development and expansion of solid tumors. However, resistance to VEGF-targeting agents may develop, due to activation of alternative pro-angiogenic pathways, indicating the need of multiple target strategy. Here we obtained tumor endothelial cells (TEC) either from total renal carcinomas or from renal cancer stem cells (CSC-TEC) and we tested the effect of a CD105 targeting monoclonal antibody, TRC105, alone or in association with anti-VEGF drugs. We demonstrated that TRC105 impaired the ability of TEC and CSC-TEC to organize in tubular structures, whereas it did not limit proliferation or survival. The combination of TRC105 with different anti-angiogenic drugs showed a synergistic effect of TRC105 only in combination with the tyrosine kinase inhibitor Sunitinib. In particular, TRC105 plus Sunitinib reduced tubulogenesis, proliferation and survival of CSC-TEC and tumor-derived TEC in a similar manner. At a molecular level, we showed that the combination of TRC105 and Sunitinib induced the phosphorylation of Smad 2/3 to promote endothelial cell death. Moreover, TRC105 enhanced the inhibitory effect of Sunitinib on VEGF signaling and reduced VEGFR2-Akt-Creb activation, suggesting a molecular cooperation between the two drugs. Our results highlight that the combined inhibition of VEGF and TGF-β pathway may have a potential use in renal cell carcinoma therapy.

Tissue-engineered 3D microvessel and capillary network models for the study of vascular phenomena.

Advances in tissue engineering, cell biology, microfabrication, and microfluidics have led to the development of a wide range of vascular models. Here, we review platforms based on templated microvessel fabrication to generate increasingly complex vascular models of (i) the tumor microenvironment, (ii) occluded microvessels, and (iii) perfused capillary networks. We outline fabrication guidelines and demonstrate a number of experimental methods for probing vascular function such as permeability measurements, tumor cell intravasation, flow characterization, and endothelial cell morphology and proliferation.

AB112. Expression of brain-specific angiogenesis inhibitor 1 and association with p53, microvessel density and vascular endothelial growth factor in the tissue of human bladder transitional cell carcinoma

ObjectiveBrain-specific angiogenesis inhibitor 1 (BAI1) was initially described in 1997, and there have since been a number of studies on its expression in different types of cancer. The aim of the present study was to investigate the expression levels of BAI1 in bladder transitional cell carcinoma (BTCC) at different stages and the mechanism by which it inhibits tumor endothelial cell proliferation.MethodsNormal bladder mucosa biopsy specimens were obtained as the control group, and human BTCC biopsy specimens were used as the study group. Immunohistochemical assays were used to detect the expression levels of BAI1, vascular endothelial growth factor (VEGF) and mutant p53, in addition to microvessel density (MVD) in the tissues. Western blotting was used to analyze the differential expression of BAI1 in the two samples.ResultsStatistical analysis was performed, which indicated that BAI1 expression levels in the normal bladder mucosa group were significantly higher than those in the BTCC group and were associated with clinical staging. BAI1 levels in the T1 stage BTCC tissues were higher than those in the T2–4 stage BTCC tissues (P<0.05). BAI1 expression levels were negatively correlated with those of VEGF (r=−0.661, P<0.001), mutant p53 (r=−0.406, P=0.002) and with the MVD (r=−0.675, P<0.001).ConclusionsBAI1 may be involved in the negative regulation of BTCC microvascular proliferation, and its expression may be associated with a reduction in p53 mutations.

Development and future directions of antiangiogenic therapy in hepatocellular carcinoma.

Although the proliferation of tumor cells has been studied extensively, little is known about the proliferation of tumor endothelial cells (TEC) and their contribution to tumor angiogenesis [4]. Identifying molecules that are specific for TEC is important for the development of antiangiogenic therapies and would improve their efficacy. In this issue of The International Journal of Clinical Oncology, the first article is a review of the field of TEC. The authors focused on the heterogeneity of TEC according to the tumor status in addition to the differences compared with normal endothelial cells. The second article is a review of the antiangiogenic therapies for HCC with a special reference to clinical trials. We hope that these special combined reviews will provide valuable information in the field of antiangiogenic treatment and will help readers to understand the current advances and future prospectives in the treatment of HCC.

Higher proliferation of peritumoral endothelial cells to IL-6/sIL-6R than tumoral endothelial cells in hepatocellular carcinoma.

BackgroundThis study aimed to explore the responses to the interleukin-6 (IL-6)/soluble interleukin-6 receptor (sIL-6R) complex in peritumoral endothelial cells (PECs) and tumor endothelial cells (TECs), as well as determine the signaling pathways in the angiogenesis of hepatocellular carcinoma (HCC).MethodsThe expression of IL-6, IL-6R, gp130, CD68, HIF-1α, and microvessel density (MVD) were assessed with an orthotopic xenograft model in nude mice. ECs were incubated under hypoxic conditions to detect IL-6 and gp130. The proliferation of PECs and TECs in the presence of IL-6 and sIL-6R, as well as the expression of gp130, JAK2/STAT3, PI3K/AKT in endothelial cells were measured.ResultsPeritumoral IL-6, IL-6R, gp130, CD68, and HIF-1α expression, as well as MVD, gradually increased during tumor growth. Hypoxia could directly induce IL-6 expression, but not gp130 in PECs. The co-culture of IL-6/sIL-6R induced much higher PEC proliferation and gp130 expression, as well as the elevated phosphorylation of JAK2 and STAT3, however not the phosphorylation of PI3K and AKT.ConclusionsPECs exhibited higher proliferation in response to IL-6/sIL-6R co-treatment compared with TECs in HCC via the up-regulation of gp130 /JAK2/STAT3. PEC and its associated peritumoral angiogenesis microenvironment may be a potential novel target for anti-angiogenic treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1763-2) contains supplementary material, which is available to authorized users.

TRPV4 channel activation selectively inhibits tumor endothelial cell proliferation.

Endothelial cell proliferation is a critical event during angiogenesis, regulated by both soluble factors and mechanical forces. Although the proliferation of tumor cells is studied extensively, little is known about the proliferation of tumor endothelial cells (TEC) and its contribution to tumor angiogenesis. We have recently shown that reduced expression of the mechanosensitive ion channel TRPV4 in TEC causes aberrant mechanosensitivity that result in abnormal angiogenesis. Here, we show that TEC display increased proliferation compared to normal endothelial cells (NEC). Further, we found that TEC exhibit high basal ERK1/2 phosphorylation and increased expression of proliferative genes important in the G1/S phase of the cell cycle. Importantly, pharmacological activation of TRPV4, with a small molecular activator GSK1016790A (GSK), significantly inhibited TEC proliferation, but had no effect on the proliferation of NEC or the tumor cells (epithelial) themselves. This reduction in TEC proliferation by TRPV4 activation was correlated with a decrease in high basal ERK1/2 phosphorylation. Finally, using a syngeneic tumor model revealed that TRPV4 activation, with GSK, significantly reduced endothelial cell proliferation in vivo. Our findings suggest that TRPV4 channels regulate tumor angiogenesis by selectively inhibiting tumor endothelial cell proliferation.

Regulation of tumorigenesis and metastasis of hepatocellular carcinoma tumor endothelial cells by microRNA-3178 and underlying mechanism

This study explored the effects of microRNA-3178 (miR-3178) on hepatocellular carcinoma (HCC) tumor endothelial cells (TECs) and on the target mRNA. Real-time polymerase chain reaction (PCR) was performed to detect the differential expression of miR-3178 in hepatic sinusoidal endothelial cells (HSECs) and HCC TECs. Furthermore, HCC TECs were transfected with miR-3178 mimic/inhibitor or their respective negative controls. The expression of miR-3178 before and after transfection was confirmed through RT-PCR. The effects of miR-3178 on the proliferation, apoptosis, cell cycle, invasion, migration, and angiogenesis of HCC TECs were also investigated through methyl thiazol tetrazolium assay, flow cytometry, matrigel invasion assay, transwell migration assay, and tube formation assay. Early growth responsive gene 3 (EGR3), as the putative target of miR-3178, was detected through RT-PCR and Western blot. Compared with HSECs, HCC TECs had lower miR-3178 expression levels (P < 0.001). MiR-3178 mimic inhibited proliferation, arrested cell cycle in G1 phase, and increased apoptosis. The numbers of migrated and invaded cells and capillary-like structures were significantly less in the mimic group than in the other groups. MiR-3178 mimic significantly decreased the mRNA and protein expression levels of EGR3. By contrast, miR-3178 inhibitor induced opposite effects. We conclude that miR-3178 was lowly expressed in HCC TECs, and miR-3178 mimic specifically inhibited the proliferation, migration, invasion, and angiogenesis and promoted the apoptosis and G1 phase arrest of HCC TECs in vitro through the inhibition of EGR3 expression. Thus, miR-3178 might be a critical target in HCC therapy.

Expression of brain‑specific angiogenesis inhibitor‑1 and association with p53, microvessel density and vascular endothelial growth factor in the tissue of human bladder transitional cell carcinoma.

The aim of the present study was to investigate the expression levels of brain‑specific angiogenesis inhibitor‑1 (BAI‑1) in bladder transitional cell carcinoma (BTCC) at different stages and the mechanism by which it inhibits tumor endothelial cell proliferation. Normal bladder mucosa biopsy specimens were obtained as the control group, and human BTCC biopsy specimens were used as the study group. Immunohistochemical assays were used to detect the expression levels of BAI‑1, vascular endothelial growth factor (VEGF) and mutant p53, in addition to microvessel density (MVD) in the tissues. Western blotting was used to analyze the differential expression of BAI‑1 in the two samples. Statistical analysis was performed, which indicated that BAI‑1 expression levels in the normal bladder mucosa group were significantly higher than those in the BTCC group and were associated with clinical staging. BAI‑1 levels in the T1 stage BTCC tissues were higher than those in the T2‑4 stage BTCC tissues (P<0.05). BAI‑1 expression levels were negatively correlated with those of VEGF (r=‑0.661, P<0.001), mutant p53 (r=‑0.406, P=0.002) and with the MVD (r=‑0.675, P<0.001). BAI‑1 may be involved in the negative regulation of BTCC microvascular proliferation, and its expression may be associated with a reduction in p53 mutations.

Effect of microRNA-1 on hepatocellular carcinoma tumor endothelial cells.

To investigate the effect of microRNA-1 (miR-1) on tumor endothelial cells (TECs) of human hepatocellular carcinoma (HCC). MiR-1 specific short hairpin RNA (shRNA) was synthesized and cloned into a recombinant lentiviral vector. TECs were then infected by the miRNA-1-shRNA recombinant lentivirus. TECs were divided into three groups: a control (CON) group consisting of normal TECs without lentiviral infection, a negative control (NC) group consisting of normal TECs infected with a negative control virus, and a micro-down (MD) group consisting of normal TECs infected with the miR-1-inhibition virus containing the target gene. Silencing of miR-1 expression was quantified via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The proliferation of TECs was detected using MTT (Thiazolyl Blue Tetrazolium Bromide) assay; the observations were continued for 5 d, and the optical density value at 490 nm was detected every day. Apoptosis was detected via flow cytometry using Annexin V-APC single staining. The migration and invasion of TECs were detected using transwell assays. Lentiviral miR-1 shRNA was successfully transduced into TECs, and specifically silenced the expression of miR-1. The results of qRT-PCR showed that the expression of miR-1 was significantly decreased in the MD group (2(-ΔΔCt) = 0.57 ± 0.14) compared with the CON group (2(-ΔΔCt) = 1) and the NC group (2(-ΔΔCt) = 1.05 ± 0.13) (P < 0.01). The results of MTT assay showed that the cell proliferation was all significantly inhibited in the MD group in the 5 days compared with the CON and NC groups (P < 0.01). The results of flow cytometry showed that the apoptosis was significantly increased in the MD group (6.32% ± 0.33%) compared with the CON group (2.03% ± 0.30%) and the NC group (2.18% ± 0.15%) (P < 0.01). The ability of cell migration was significantly inhibited in the MD group (62.0 ± 5.48) compared with the CON group (99.8 ± 3.11) and the NC group (97.2 ± 3.70) (P < 0.01). The ability of invasion of TECs was also significantly inhibited in the MD group (29.8 ± 2.39) compared with the CON group (44.6 ± 3.36) and the NC group (44.4 ± 5.17) (P < 0.01). MiR-1 might be a potential tumor activator. Inhibiting its expression could decrease proliferation, induce apoptosis, and inhibit the migration and invasion of TECs of human HCC.

TRPV4 channel activation selectively inhibits tumor endothelial cell proliferation

TRPV4 channel activation selectively inhibits tumor endothelial cell proliferation

Antagonism of Ang-Tie2 and Dll4-Notch signaling has opposing effects on tumor endothelial cell proliferation, evidenced by a new flow cytometry method

Sustained angiogenesis is essential for tumor growth as it provides the tumor with a network of blood vessels that supply both oxygen and essential nutrients. Limiting tumor-associated angiogenesis is a proven strategy for the treatment of human cancer. To date, the rapid detection and quantitation of tumor-associated endothelial cell (TAEC) proliferation has been challenging, largely due to the low frequency of endothelial cells (ECs) within the tumor microenvironment. In this report, we address this problem using a new multiparametric flow cytometry method capable of rapid and precise quantitation of proliferation by measuring bromodeoxyuridine (BrdUrd) uptake in mouse TAECs from established human tumor xenografts. We determined the basal proliferation labeling index of TAECs in two human tumor xenografts representing two distinct histologies, COLO 205 (colorectal cancer) and U-87 (glioblastoma). We then investigated the effects of two large-molecule antiangiogenic agents targeting different biochemical pathways. Blocking angiopoietin-Tie2 signaling with the peptide-Fc fusion protein, trebananib (AMG 386), inhibited proliferation of TAECs, whereas blocking Dll4-Notch signaling with an anti-Dll4-specific antibody induced hyperproliferation of TAECs. These pharmacodynamic studies highlight the sensitivity and utility of this flow cytometry-based method and demonstrate the value of this assay to rapidly assess the in vivo proliferative effects of angiogenesis-targeted agents on both the tumor and the associated vasculature.

Abstract 1022: Development and preclinical testing of AMG 780, a fully human antibody targeting angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2)

Abstract Targeting the angiopoietins with trebananib (a peptibody that inhibits the interaction between the endothelial cell-selective Tie2 receptor and its ligands Ang1 and Ang2) in combination with paclitaxel was associated with clinical benefit in a phase 3 ovarian cancer clinical trial (TRINOVA 1). Herein, we describe the generation of AMG 780, a fully human IgG2 antibody targeting Ang1 and Ang2. We compared AMG 780 to trebananib in affinity/neutralization ELISA assays and in vivo studies assessing inhibition of tumor growth and endothelial cell proliferation. AMG 780 is a derivative of an Ang2-binding antibody (Ab X). Ab X was generated by panning a human phage display Fab library (Dyax Corporation) against recombinant Ang2. The Ab X Fab was affinity matured by individually substituting every possible amino acid at every position in the light and heavy chain complementarity determining regions (CDRs). Resulting phage clones were interrogated for improved Ang1- and Ang2-binding activity. Clones with improved potency were converted to full antibodies. The most improved heavy chain clones were paired with the most improved light chain clones. The resulting IgGs were tested for neutralization of the interaction between the angiopoietins and their receptor, Tie2, as measured by homogenous time-resolved fluorescence (HTRF). Three resulting fully human IgG2 antibodies (AMG 780, Ab Y, and Ab Z) and the parental clone (Ab X) with potent Ang2 inhibitory activities (IC50 [nM], 0.05-0.10) and a range of Ang1 inhibitory activities (IC50 [nM], 0.31-547.00) were selected for xenograft studies. Mice were implanted with 5x106 Colo205 (human colon carcinoma) cells. When tumors were approximately 200 mm3, 300 µg of each of the four antibodies intraperitoneally or 14 µg of trebananib subcutaneously were administered twice weekly. For viable tumor fraction analyses, treated tumors at the end of the xenograft study were harvested and processed for paraffin histology. To examine endothelial cell proliferation, treated tumors were enzyme digested and stained with anti-CD31, anti-CD45, and anti-BrdU antibodies. The percentage of BrdU-positive tumor-associated endothelial cells (CD31high/CD45neg) was determined by flow cytometry. Relative to an isotope control antibody, all four IgG2 antibodies and trebananib inhibited tumor growth (P &amp;lt; 0.0001), viable tumor fraction (P &amp;lt; 0.0001), and tumor endothelial cell proliferation (P &amp;lt; 0.002). Tumor xenografts in those animals receiving AMG 780 exhibited the most pronounced inhibition in tumor growth (66%), viable tumor fraction (81%), and endothelial cell proliferation (84%). The inhibitory activities of AMG 780 (IC50 [nM]: Ang1, 4.5; Ang2, 0.06) were similar to those of trebananib (IC50 [nM]: Ang1, 3.5; Ang2, 0.03). Based on these findings, AMG 780 was selected for clinical development and currently is being evaluated in a first-in-human phase 1 trial. Citation Format: James V. Bready, Kyung Lee, Rick Jacobsen, Kevin Graham, Juan Estrada, Stephen A. Kaufman, Dongyin Yu, Angela Coxon, Jon Oliner. Development and preclinical testing of AMG 780, a fully human antibody targeting angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2). [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 1022. doi:10.1158/1538-7445.AM2014-1022

Growth inhibition of hepatocellular carcinoma tumor endothelial cells by miR-204-3p and underlying mechanism.

To investigate the mechanism by which miR-204-3p inhibits the growth of hepatocellular carcinoma (HCC) tumor endothelial cells (TECs). Flow cytometry was used to identify HCCTECs and analyze their purity. Differentially expressed miRNAs in HCC TECs as compared to normal hepatic sinusoidal endothelial cells (HSECs) were examined using the HmiOA v4 Human miRNA OneArray microarray. miR-204-3p showed the most significant decrease in expression and was further studied. Over-expression of miR-204-3p was achieved using lentiviral transduction into TECs of HCC. The biological changes in HCC TECs before and after transduction were detected using MTT and apoptosis assays. The association between miR-204-3p and fibronectin 1 (FN1) was determined using the dual luciferase activity assay. Changes in FN1 protein expression before and after transduction were detected using Western blot analysis. Microarray results showed that compared to normal HSECs, 15 miRNAs were differentially expressed in HCC TECs, including 6 miRNAs with increased expression and 9 miRNAs with decreased expression. Among them, miR-204-3p showed the most significant decrease in expression (log2 = -1.233477, P = 0.000307). Over-expression of miR-204-3p in HCC TECs via lentiviral transduction significantly inhibited the proliferation of HCC TECs and promoted apoptosis. Results from the dual luciferase activity experiment showed that the luciferase intensity in the wild type FN1 group was significantly inhibited (P < 0.05), while that in the mutant FN1 group was not obviously affected. This observation indicated that FN1 was one of the potential targets of miR-204-3p. After over-expression of miR-204-3p in HCC TECs, Western blot analysis showed that the expression of FN1 protein was significantly inhibited. MiR-204-3p acts on its potential target gene, FN1, and inhibits its expression, thus blocking the adhesion function of FN1 in promoting the growth of TECs.

Stem Cell Niches in Glioblastoma: A Neuropathological View

Glioblastoma (GBM) stem cells (GSCs), responsible for tumor growth, recurrence, and resistance to therapies, are considered the real therapeutic target, if they had no molecular mechanisms of resistance, in comparison with the mass of more differentiated cells which are insensitive to therapies just because of being differentiated and nonproliferating. GSCs occur in tumor niches where both stemness status and angiogenesis are conditioned by the microenvironment. In both perivascular and perinecrotic niches, hypoxia plays a fundamental role. Fifteen glioblastomas have been studied by immunohistochemistry and immunofluorescence for stemness and differentiation antigens. It has been found that circumscribed necroses develop inside hyperproliferating areas that are characterized by high expression of stemness antigens. Necrosis developed inside them because of the imbalance between the proliferation of tumor cells and endothelial cells; it reduces the number of GSCs to a thin ring around the former hyperproliferating area. The perinecrotic GSCs are nothing else that the survivors remnants of those populating hyperproliferating areas. In the tumor, GSCs coincide with malignant areas so that the need to detect where they are located is not so urgent.