Normal Vascular Endothelial Cells Research Articles

Novel CD123xCD3 Bispecific Igm Antibody, Igm-2537, Potently Induces T-Cell Mediated Cytotoxicity of Acute Myeloid Leukemia Cells In Vivo and in Vitro with Minimal Cytokine Release

Introduction: New therapeutics are needed for the effective treatment of acute myeloid leukemia (AML), as standard chemotherapeutics are poorly tolerated and ~50% of patients relapse primarily due to the incomplete elimination of leukemia stem cells (LSCs). CD123, the IL-3Rα chain, is highly expressed on leukemic blasts and LSCs, and is further increased in patients with poor prognostic factors. Bispecific T-cell engager (TCE) antibodies and CAR-T cells targeting CD123 have shown promising clinical efficacy in AML patients, but cytokine release syndrome limits their therapeutic window and remains a major safety concern. IGM-2537 is a novel pentameric IgM bispecific TCE antibody engineered with ten anti-CD123 binding sites, and an anti-CD3ε single chain Fv domain fused to the joining chain to engage T-cells. Here, we report that IGM-2537 not only potently induces T-cell mediated cytolytic killing of AML cells in vitro and in vivo but also demonstrates minimal cytokine release, and little to no effect on normal hematopoietic progenitor cells and human vascular endothelial cells (HUVECs) with in vitro and ex vivo studies. Additionally, an improved tolerability, excellent pharmacodynamic (PD) response and lower cytokine release was observed with a cynomolgus cross-reactive IgM TCE in monkeys. Materials and Methods:Surface plasma resonance (SPR), cell membrane protein array, and alanine scanning were utilized to characterize human CD123 binding affinity, specificity and epitope mapping, respectively. T-cell dependent cellular cytotoxicity (TDCC) assays were employed to evaluate the potency of IGM-2537 target cytotoxicity, T cell activation and cytokine release. Ex vivo AML colony formation assays were used to assess the activity on AML blasts. Human MV-4-11 and MOLM-13 xenograft tumor models in humanized NSG dKO mice were utilized to determine in vivo anti-tumor activity of IGM-2537. Healthy human PBMCs and bone marrow (BM) samples as well as HUVECs were used in ex vivo assays to address potential safety concerns of IGM-2537 mediated effects on hematopoietic progenitor cells and HUVECs, respectively. Lastly, to confirm that a CD123 directed IgM-based TCE molecule can potentially provide better safety profile in vivo, a cynomolgus-cross reactive IgM TCE comparator was assessed in monkeys for tolerability and PK/PD responses. Results and Conclusions: IGM-2537 bound with high affinity and avidity to CD123. In vitro, IGM-2537 engaged both CD123 and CD3 to induce potent T-cell activation and T-cell mediated cytotoxicity of CD123 + AML cells, and autologous CD123 + basophils and plasmacytoid dendritic cells (pDCs). Though IGM-2537 demonstrated comparable maximal killing activity to a comparator IgG TCE, IGM-2537 demonstrated minimal cytokine release. In ex vivo patient-derived AML or normal BM colony formation assays, IGM-2537 eliminated AML colony forming cells at physiologically relevant effector/target (E/T) ratios but had little or no effect on normal hematopoietic progenitors. In addition, IGM-2537 demonstrated no cytotoxicity against HUVECs regardless of their CD123 expression at basal levels or at maximal levels upregulated by TNF-a and IFN-g, in contrast to a comparator IgG TCE. In vivo, IGM-2537 completely inhibited tumor growth in two AML xenograft tumor models, a subcutaneous MV-4-11 model, and a systemic MOLM-13 model in humanized NSG dKO mice. To further evaluate the potency and safety of the CD123xCD3 IgM bispecific TCE format in vivo, a cynomolgus cross-reactive CD123xCD3 IgM bispecific TCE was evaluated for tolerability and PD responses in cynomolgus monkeys using single doses. All animals tolerated the cross-reactive bispecific IgM TCE well at doses up to 10 mg/kg (the maximal dose level evaluated), a dose 100-fold greater than published doses of a comparator IgG TCE. Complete depletion of CD123 + basophils and substantial reductions of pDCs were seen in blood and BM with minimal to no cytokine induction. In summary, IGM-2537 demonstrated potent in vitro and in vivo T-cell mediated cytotoxicity of AML cell lines with minimal cytokine induction. The encouraging preclinical safety profiles were further supported by little/no effect on normal hematopoietic progenitor cells and HUVECs. These preclinical data support the clinical development of IGM-2537 for the treatment of AML and other hematological malignancies.

Human vascularized bile duct-on-a chip: a multi-cellular micro-physiological system for studying cholestatic liver disease

Exploring the pathogenesis of and developing therapies for cholestatic liver diseases such as primary sclerosing cholangitis (PSC) remains challenging, partly due to a paucity of in vitro models that capture the complex environments contributing to disease progression and partly due to difficulty in obtaining cholangiocytes. Here we report the development of a human vascularized bile duct-on-a-chip (VBDOC) that uses cholangiocyte organoids derived from normal bile duct tissue and human vascular endothelial cells to model bile ducts and blood vessels structurally and functionally in three dimensions. Cholangiocytes in the duct polarized, formed mature tight junctions and had permeability properties comparable to those measured in ex vivo systems. The flow of blood and bile was modeled by perfusion of the cell-lined channels, and cholangiocytes and endothelial cells displayed differential responses to flow. We also showed that the device can be constructed with biliary organoids from cells isolated from both bile duct tissue and the bile of PSC patients. Cholangiocytes in the duct became more inflammatory under the stimulation of IL-17A, which induced peripheral blood mononuclear cells and differentiated Th17 cells to transmigrate across the vascular channel. In sum, this human VBDOC recapitulated the vascular-biliary interface structurally and functionally and represents a novel multicellular platform to study inflammatory and fibrotic cholestatic liver diseases.

Apolipoprotein A-I Binding Protein Inhibits the Formation of Infantile Hemangioma through Cholesterol-Regulated Hypoxia-Inducible Factor 1α Activation

Infantile hemangioma (IH) is the most frequent vascular tumor of infancy with unclear pathogenesis, of which disordered angiogenesis is considered to involve IH formation. We previously found that apolipoprotein A-I binding protein (AIBP)-also known as NAXE (NAD (P)HX epimerase)-a regulator of cholesterol metabolism, plays a critical role in the pathological angiogenesis of mammals. Here, we found that AIBP had much lower expression levels in both IH patients' tissues and hemangioma endothelial cells (HemECs), compared to adjacent normal tissues and human dermal vascular endothelial cells (HDMECs), respectively. Knockout of NAXE by CRISPR-Cas9 in HemECs enhanced tube formation and migration, and NAXE overexpression impaired tube formation and migration of HemECs. Interestingly, AIBP suppressed the proliferation of HemECs in hypoxia. We then found that reduced expression of AIBP correlated with increased Hif1α levels in IH patients' tissues and HemECs. Further mechanistic investigation demonstrated that AIBP disrupted Hif1α signaling via cholesterol metabolism under hypoxia. Notably, AIBP significantly inhibited the development of IH in immunodeficient mice. Furthermore, using the validated mouse endothelial cell (EOMA) and NAXE-/- mouse models, we demonstrated that both endogenous AIBP from tumors and AIBP in the tumor microenvironment limit the formation of hemangioma. These findings suggested that AIBP was a player in the pathogenesis of IH and could be a potential pharmacological target for treating IH.

Tumour associated endothelial cells: origin, characteristics and role in metastasis and anti-angiogenic resistance.

Tumour progression and metastasis remain the leading causes of cancer-related death worldwide. Tumour angiogenesis is essential for tumour progression. The vasculature surrounding tumours is not only a transport channel for nutrients, oxygen, and metabolites, but also a pathway for metastasis. There is a close interaction between tumour cells and endothelial cells in the tumour microenvironment. Recent studies have shown that tumour-associated endothelial cells have different characteristics from normal vascular endothelial cells, play an important role in tumour progression and metastasis, and are expected to be a key target for cancer therapy. This article reviews the tissue and cellular origin of tumour-associated endothelial cells and analyses the characteristics of tumour-associated endothelial cells. Finally, it summarises the role of tumour-associated endothelial cells in tumour progression and metastasis and the prospects for their use in clinical anti-angiogenic therapy.

Effects of long non-coding RNA MALAT1-targeting miR-570-3p and miR-34a on the invasion, proliferation, and apoptosis of human retinoblastoma cells

Purpose: To investigate the expression of lncRNA MALAT1-targeting miR-570-3p and miR-34a and its effects on the invasion, proliferation, and apoptosis of human retinoblastoma cells.
 Methods: MiR-34a, miR-570-3p, and IncRNA MALAT1 in a human normal retinal vascular endothelial cell line (ACBRI-181), human retinoblastoma cell line (SO-Rb50), human normal retinal tissue and human retinoblastoma tissue were determined. Luciferase assay was used to verify the targeting relationship between LncRNA MALAT1 and miR-570-3p and miR-34a. while cell invasion, cell apoptosis and cell proliferation were assessed by Transwell assay, flow cytometry, and tumor pellet-forming assay, respectively.
 Results: LncRNA MALAT1 in SO-Rb50 cell line and human retinoblastoma tissue line were significantly up-regulated, while the expression levels of miR-34a and miR-570-3p were significantly down-regulated (p < 0.05). Luciferase assay results showed that lncRNA MALAT1 targeted miR-570-3p and miR-34a. The invasion and proliferation of SO-Rb50 cells in the miR-570-3p inhibitor and miR-34a inhibitor groups were significantly increased, while the apoptosis of SO-Rb50 cells was significantly decreased (p < 0.05). However, the invasion and proliferation of SO-Rb50 cells in sh-MALAT1 group were significantly decreased, while apoptosis significantly increased. However, compared with sh-MALAT1 group alone, the invasion and proliferation of SO-Rb50 cells in co-transfected sh-MALAT1+miR-570-3p inhibitor + miR-34a inhibitor group w significantly increased, but apoptosis significantly decreased (p < 0.05).
 Conclusion: LncRNA MALAT1 negatively regulates miR-570-3p and miR-34a to promote the invasion and proliferation of human retinoblastoma SO-Rb50 cells and inhibit apoptosis. These findings may be of significance in developing suitable therapies for human retinoblastoma

Hsa_circ_0001017 promotes cell proliferation, migration and invasion in osteosarcoma by sponging miR-145-5p

BackgroundCircular RNAs (circRNAs) have displayed important roles in the development and progression of various cancers. However, the functions of the majority of circRNAs in osteosarcoma (OS) remain unknown.MethodsCircular RNA microarray analysis was performed in three OS cell lines (Saos-2, U2OS and MG63) and normal vascular endothelial cells. The co-differentially expressed circRNAs (CDECs) were identified in OS cell lines with the criterion of FDR (false discovery rate) < 0.05 and |fold change (FC)|> 2. Quantitative real-time PCR was used to validate the expression levels of selected CDECs. A series of functional assays, including MTT assay, flow cytometry and transwell assay were conducted in OS cells. The interaction between circRNA and miRNAs was confirmed by luciferase reporter assay and RNA immunoprecipitation assay.ResultsA total of 241 CDECs, including 75 upregulated and 166 downregulated CDECs, were identified in three OS cell lines compared with normal vascular endothelial cells. PCR validation showed that hsa_circ_0000704, hsa_circ_0001017 and hsa_circ_0005035 were all highly expression in the three OS cell lines, compared with osteoblast cell lines (HECC, hFOB1.19 and HFF-1). Functionally, overexpression of circ_0001017 significantly promoted the cell proliferation, migration and invasion and decreased apoptosis in U2OS cells. Knockdown of circ_0001017 obtained the opposite results. Circ_0001017 may downregulate miR-145-5p through direct binding. Furthermore, the expression of miR-145-5p was negatively regulated by circ_0001017 in OS cells. In addition, further functional studies indicated that miR-145-5p inhibitor eliminated the effects caused by si-circ_0001017 in OS cells.ConclusionsIn conclusion, our study suggested that circ_0001017 may be a novel oncogenic factor during the progression and development of OS by targeting miR-145-5p.

Succinate dehydrogenase-deficient renal cell carcinoma:a clinicopathological, ultrastructural and molecular analysis

Objective: To investigate the clinicopathological features, immunophenotype, ultrastructure, genetic alterations and prognosis of succinate dehydrogenase-deficient renal cell carcinoma (SDH RCC). Methods: A total of 11 SDH RCCs, diagnosed from 2010 to 2019, were selected from the Department of Pathology of Nanjing Jingling Hospital, Nanjing University School of Medicine for clinicopathologic, immunohistochemical (IHC), ultrastructural investigation and follow-up. The molecular features of seven cases were analyzed by the panel-targeted DNA next generation sequencing (NGS). Results: There were seven males and four females, with ages ranging from 24 to 62 years (mean 41.4 years, median 41 years). Microscopically, SDH RCC was mainly composed of solid and tubular structures with local cystic change. Four cases showed nested or trabecular structure distributed in a loose hypocellular connective tissue or around scar, similar to oncocytoma. The neoplastic cells demonstrated flocculent eosinophilic cytoplasm with typical intracytoplasmic vacuoles. Immunohistochemically, eight cases were negative for SDHB; three cases showed focal and weak expression, whereas normal renal tubular and vascular endothelial cells demonstrated strong cytoplasmic staining. NGS of DNA targeted-panel detected pathogenic mutations of SDHB gene in seven cases (including three cases with equivocal IHC expression of SDHB), without any mutations in other SDH related genes. There were four cases of SDHB missense mutation, one case of frameshift mutation, one case of splicing mutation, and one case of acquired stop codon mutation. Conclusions: SDH RCC is a distinct variant of RCCs with genetic tendency or with hereditary cancer syndrome. NGS is recommended to detect the related gene mutations for a definitive diagnosis. The patients should be closely followed up.

A comparison expression analysis of CXCR4, CXCL9 and Caspase-9 in dermal vascular endothelial cells between keloids and normal skin on chemotaxis and apoptosis

This present study was designed to explore key biological characteristics and biomarkers associated with dermal vascular endothelial cells of keloids. GSE121618 dataset was downloaded in the Gene Expression Omnibus (GEO) Database, including the KECs group and NVECs group. Through GEO2R, we have screened the differentially expressed genes (DEGs) and performed gene ontology (GO), Gene Set Enrichment Analysis (GSEA), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Then, we constructed a protein–protein interaction (PPI) network and analyzed hub genes via the Search Tool for the Retrieval of Interacting Genes (STRING) Online Database and Cytoscape software. Furthermore, experiments were performed to validate the expression of selected genes, including H&E staining, immunohistochemical staining, Western blot, and RT-qPCR. A total of 1040 DEGs were selected with GEO2R online tools. Most of the enriched pathways and processes focus on cell migration, tube development, chemotaxis, cell motility, and regulation of apoptosis. With the assistance of STRING and Cytoscape, hub genes were selected. In our validation experiments of RT-qPCR, the mRNA expression of selected genes has significant differences between different groups in tissue and cell experiments. As was shown in immunohistochemical staining, the proteins of CXCR4, CXCL9, and Caspase-9 had higher expression levels in tissue samples of the Keloid group than the Normal skin group. Western blot and RT-qPCR in dermal vascular endothelial cell experiments were consistent with the aforementioned results. This study has provided a deeper analysis of the pathogenesis of dermal vascular endothelial cells in keloids. Genes of CXCR4, CXCL9, and Caspase-9 may influence the processes of inflammatory responses and vascular endothelial cell apoptosis to exert crucial effects in the development of keloids. Abbreviations: GEO: gene expression omnibus; DEGs: differentially expressed genes; KVECs: keloid vascular endothelial cells; NVECs: normal skin vascular endothelial cells; GO: gene ontology; KEGG: Kyoto encyclopedia of genes and genomes; PPI: protein protein interaction; BP: biological process; CC: cellular component; MF: molecular function; GSEA: gene set enrichment analysis; STRING: search tool for the retrieval of interacting genes; MCODE: molecular complex detection

A cancer-specific anti-podocalyxin monoclonal antibody (60-mG2a-f) exerts antitumor effects in mouse xenograft models of pancreatic carcinoma

Overexpression of podocalyxin (PODXL) is associated with progression, metastasis, and poor outcomes in several cancers. PODXL also plays an important role in the development of normal tissues. For antibody-based therapy to target PODXL-expressing cancers using monoclonal antibodies (mAbs), cancer-specificity is necessary to reduce the risk of adverse effects to normal tissues. In this study, we developed an anti-PODXL cancer-specific mAb (CasMab), named as PcMab-60 (IgM, kappa) by immunizing mice with soluble PODXL, which is overexpressed in LN229 glioblastoma cells. The PcMab-60 reacted with the PODXL-overexpressing LN229 (LN229/PODXL) cells and MIA PaCa-2 pancreatic cancer cells in flow cytometry but did not react with normal vascular endothelial cells (VECs), whereas one of non-CasMabs, PcMab-47 showed high reactivity for not only LN229/PODXL and MIA PaCa-2 cells but also VECs, indicating that PcMab-60 is a CasMab. Next, we engineered PcMab-60 into a mouse IgG2a-type mAb, named as 60-mG2a, to add antibody-dependent cellular cytotoxicity (ADCC). We further developed a core fucose-deficient type of 60-mG2a, named as 60-mG2a-f, to augment its ADCC activity. In vivo analysis revealed that 60-mG2a-f exerted antitumor activity in MIA PaCa-2 xenograft models at a dose of 100 μg/mouse/week administered three times. These results suggested that 60-mG2a-f could be useful for antibody-based therapy against PODXL-expressing pancreatic cancers.

Discovery of new fluorescent thiazole\u2013pyrazoline derivatives as autophagy inducers by inhibiting mTOR activity in A549 human lung cancer cells

A series of fluorescent thiazole–pyrazoline derivatives was synthesized and their structures were characterized by 1H NMR, 13C NMR, and HRMS. Biological evaluation demonstrated that these compounds could effectively inhibit the growth of human non-small cell lung cancer (NSCLC) A549 cells in a dose- and time-dependent manner in vitro and inhibit tumor growth in vivo. The structure–activity relationship (SAR) of the compounds was analyzed. Further mechanism research revealed they could induce autophagy and cell cycle arrest while had no influence on cell necrosis. Compound 5e inhibited the activity of mTOR via FKBP12, which could be reversed by 3BDO, an mTOR activator and autophagy inhibitor. Compound 5e inhibited growth, promoted autophagy of A549 cells in vivo. Moreover, compound 5e showed good selectivity with no influence on normal vascular endothelial cell growth and the normal chick embryo chorioallantoic membrane (CAM) capillary formation. Therefore, our research provides potential lead compounds for the development of new anticancer drugs against human lung cancer.

Mechanism of FGF7 gene silencing in regulating viability, apoptosis, invasion of retinoblastoma cell line HXO-Rb44 and angiogenesis.

To explore the mechanism of fibroblast growth factor 7 (FGF7) gene silencing in regulating viability, apoptosis, invasion of retinoblastoma (RB) cell line HXO-Rb44 and angiogenesis. Human normal retinal vascular endothelial cells ACBRI-181 was set as the normal group. The cultured RB cell lines HXO-Rb44 were divided into three groups: the blank group (without plasmid transfection), negative control group (transfection of FGF7 plasmid), and the si-FGF7 group (transfection of FGF7 siRNA plasmid). Quantitative Real Time-Polymerase Chain Reaction and Western blot were used to measure the mRNA and protein expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietin-2 (Ang-2), and proliferating cell nuclear antigen (PCNA) in each group. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, transwell invasion assay, and flow cytometry were used, respectively, to assess cell viability, invasive capability, and cell apoptosis in each group. The mRNA and protein expression of FGF7, Bcl-2, VEGF, bFGF, Ang-2, and PCNA were significantly decreased, and the mRNA and protein expression of Bax were significantly increased in the si-FGF7 group than in the blank group (all p<0.05). Compared with the blank group, the si-FGF7 group had significantly decreased cells invasive capability, cell viability at 48 h and 72 h and proliferation, and significantly increased apoptosis rate (all p<0.05). FGF7 gene silencing can inhibit the viability and invasion of RB cells and the expression of angiogenesis-related factors and can promote RB apoptosis.

Downregulation of CKS1B restrains the proliferation, migration, invasion and angiogenesis of retinoblastoma cells through the MEK/ERK signaling pathway.

Retinoblastoma (RB) is a common neoplasm that is exhibited in individuals globally. Increasing evidence demonstrated that cyclin-dependent kinase regulatory subunit 1B (CKS1B) may be involved in the pathogenesis of various tumor types, including multiple myeloma and breast cancer. In the present study, the hypothesis that CKS1B down-regulation would effectively inhibit the proliferation, invasion and angiogenesis of RB cells through the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway was examined. Initial investigation of the expression profile of CKS1B in RB and adjacent retina tissues was performed using reverse transcription-quantitative polymerase chain reaction and western blot analysis. A total of three RB cell lines, SO-RB50, Y79 and HXO-RB44, were examined for selection of the cell line with the highest expression of CKS1B, and human normal retinal vascular endothelial cells (ACBRI-181) were also evaluated. CKS1B short hairpin RNA (shRNA) sequences (shRNA CKS1B-1, shRNA CKS1B-2 and shRNA CKS1B-3) and negative control shRNA sequences were constructed and transfected into cells at the third generation to evaluate the role of shCKS1B and the MEK/ERK signaling pathway in RB. Furthermore, the effect of shCKS1B on cell proliferation, migration, invasion, apoptosis and angiogenesis was investigated. CKS1B was determined to be highly expressed in RB tissue, compared with adjacent retina tissue. SO-RB50 and HXO-RB44 cells treated with shRNA CKS1B-1 and shRNA CKS1B-2 were selected for the present experiments. Activation of the MEK/ERK signaling pathway increases the expression of MEK, ERK, B-cell lymphoma 2, proliferating cell nuclear antigen, cyclin D1, vascular endothelia growth factor and basic fibroblast growth factor, enhances cell proliferation, migration, invasion and lumen formation, and decreases apoptosis. Following silencing CKS1B, the aforementioned conditions were reversed. The key observations of the present study demonstrated that shCKS1B can inhibit the proliferation, invasion and angiogenesis of RB cells by suppressing the MEK/ERK signaling pathway. Thus, CKS1B represents a potential research target in the development of therapeutics for RB.

Expression and clinical significances of miR-215 and RUNX1 in retinoblastoma

Objective To investigate the expression and clinical significances of miR-215 and runt-related protein1 (RUNX1) in retinoblastoma (RB), and to study the regulation effect of miR-215 on RUNX1 in the retinoblastoma cell line PMC-RB. Methods The expressions of miR-215 and RUNX1 in the tumor tissue, non tumor tissues adjacent to cancer, human RB cell line FMC-RB and human normal retinal vascular endothelial cell line ATCC of RB patients were detected by quantitative real-time polymerase chain reaction (qRT-PCR). miR-215 mimics (miR-215-mimic), miR-NC, si-RUNX1 and si-NC were transfected into FMC-RB cell line respectively. Cell proliferation, migration and invasion ability were measured respectively, thus detecting the regulation effect of miR-215 on RUNX1. Results The expression of miR-215 in RB tissues was significantly lower than that in non tumor tissues adjacent to cancer, while the mRNA expression of RUNX1 was higher than that in non tumor tissues adjacent to cancer (P<0.05). The expression of miR-215 in PMC-RB cells was lower than that in ATCC, while the mRNA expression of RUNX1 was higher than that in ATCC (P<0.05). The expression of miR-215 and RUNX1 in RB tumor tissues were closely related to the clinicopathological features of optic nerve infiltration, tumor tissue differentiation and lymph node metastasis (P<0.05). Cell proliferation, migration and invasion in miR-215-mimic group were significantly lower than those in miR-NC group (P<0.05). In transfected 3′ untranslated region (3′UTR)-Wt cells, the luciferase activity in miR-215-mimic group was lower than that in miR-NC group (P<0.05); the expression level of RUNX1 protein in transfected miR-215-mimic cells was lower than that in transfected miR-NC cells (P<0.05). Cell proliferation, migration and invasion in si-RUNX1 group were all lower than those in si-NC group (P<0.05). There was a negative correlation between mRNA expression level of miR-215 and RUNX1 in RB tumor tissues. Conclusions During the occurrence and development of RB, the down-regulation of miR-215 expression can promote malignant progression of tumor by targeting RUNX1. miR-215 can be used as a biological markers and therapeutic target for RB diagnosis. Key words: Retinoblastoma; MicroRNAs; Core binding factor alpha 2 subunit; Cell line, tumor

Abstract 4661: Magnetic nanoparticles (MNPs) for cancer drug delivery: The value of in vitro modeling

Abstract BACKGROUND: Magnetic nanoparticles (MNPs) have attracted great interest for use as delivery vehicles for cancer and other diseases due to their ability to be functionalized and localized using external magnets. In the presence of a magnetic field, individual MNPs form aggregates, which in turn can be made to spin and surface walk, in response to rotation of the field. Here, we present data from the use of a in vitro model system, which is useful in mimicking the various conditions and distances that MNP-drug combinations may encounter in vivo. METHODS: A sterilizable acrylic tray was designed, which has 1/8th inch wide lanes and can be used with or without the addition of cultured cells. The tray is compatible with standard plate readers, and the lanes can be modified to mimic various conduits within the body. In studies described here, glioma cell lines and normal vascular endothelial cells were used in the tray. In order to test the effect of fluid viscosity on MNP velocity in response to the rotating magnetic field, lanes were filled with 1mL of PBS, culture medium, serum, or whole blood. Pre-magnetized and unmagnetized MNPs were aliquoted into the lanes at volumes from 10 - 100 uL. T-PA and trypan blue were used as a model drugs. Velocities of MNP aggregates were determined by videography at five different tray positions relative to the magnet: centered, offset, push, pull, and below. Particle adhesion to cells could be quantified using ImageJ. RESULTS: Using the model system, greater MNP velocities were achieved with pre-magnetization, and larger aliquots. For example, 100ul aliquots had a total average velocity 1.27± 0.21 times that of 20ul. MNP velocity was found to be inversely -related to fluid viscosity, but particles could be moved magnetically even through whole blood. Test drugs could be successfully delivered by convection, even without prior binding to MNPs. MNP velocity also varied according to magnet position, with speeds up to 0.75 +/- 0.05 cm/sec in the pull (fastest) position. In the offset position, a distance of 20 cm above the magnet produced the greatest velocity. MNPs moved more slowly over confluent monolayers of endothelial or glioma cells (with greater adhesion), but a mean velocity of 0.25 cm/sec +/- 0.03 cm was typically observed. CONCLUSIONS: In vitro modeling is extremely helpful in predicting the behavior of particles intended for clinical use in magnetically-enhanced drug delivery. The velocity and cell surface adhesion of MNP aggregates can be quantified, and the effect of factors - such as fluid viscosity, cell type, and position with respect to the magnet - analyzed in order to better understand the advantages and limitations of this technology. Citation Format: Sebastian P. Pernal, Alexander J. Willis, Herbert H. Engelhard. Magnetic nanoparticles (MNPs) for cancer drug delivery: The value of in vitro modeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4661.

Stromal cells in breast cancer as a potential therapeutic target.

Breast cancer in the United States is the second most commonly diagnosed cancer in women. About 1 in 8 women will develop invasive breast cancer over the course of her lifetime and breast cancer remains the second leading cause of cancer-related death. In pursuit of novel therapeutic strategies, researchers have examined the tumor microenvironment as a potential anti-cancer target. In addition to neoplastic cells, the tumor microenvironment is composed of several critical normal cell types, including fibroblasts, vascular and lymph endothelial cells, osteoclasts, adipocytes, and immune cells. These cells have important roles in healthy tissue stasis, which frequently are altered in tumors. Indeed, tumor-associated stromal cells often contribute to tumorigenesis, tumor progression, and metastasis. Consequently, these host cells may serve as a possible target in anti-tumor and anti-metastatic therapeutic strategies. Targeting the tumor associated host cells offers the benefit that such cells do not mutate and develop resistance in response to treatment, a major cause of failure in cancer therapeutics targeting neoplastic cells. This review discusses the role of host cells in the tumor microenvironment during tumorigenesis, progression, and metastasis, and provides an overview of recent developments in targeting these cell populations to enhance cancer therapy efficacy.

Dimer-monomer equilibrium of human HSP27 is influenced by the in-cell macromolecular crowding environment and is controlled by fatty acids and heat

Small heat shock protein 27 (HSP27) is an essential element of the proteostasis network in human cells. The HSP27 monomer coexists with the dimer, which can bind unfolded client proteins. Here, we evaluated the in-cell dimer-monomer equilibrium and its relevance to the binding of client proteins in a normal human vascular endothelial cell line. When cells were treated with a membrane-permeable crosslinker, the protein existed primarily as a free monomer (27 kDa) with a markedly smaller percentage of dimer (54 kDa), hetero-conjugates, and minor smear-like bands. When the protein was crosslinked in a cell-free lysate, two of the hetero-conjugates that were crosslinked in live cells were also detected, but the dimer and other complexes were absent. However, when cells were pretreated with fatty acid (FA) and/or heat (42.5 °C), dissociation of the dimer was selectively prevented and two types of covalently linked dimers were increased. These changes occurred most prominently in cells treated with docosahexaenoic acid (DHA) and heat, which appeared to intensify the heat resistance of the cell. Both the formation of covalently linked dimers and heat resistance were prevented by N-acetylcysteine. By contrast, nearly all of the free monomers in the lysate converted to disulfide bond-linked dimers by a simple, long incubation at 4 °C. These results strongly suggest that the monomer-dimer equilibrium of HSP27 was inversed between the in-cell and cell-free systems. Temperature- and amphiphile-regulated dimerization was restricted probably due to the low hydration of the in-cell crowding environment.

Synthesis and in vitro experiments of carcinoma vascular endothelial targeting polymeric nano-micelles combining small particle size and supermagnetic sensitivity

Objective: To construct carcinoma vascular endothelial-targeted polymeric nanomicelles with high magnetic resonance imaging (MRI) sensitivity and to evaluate their biological safety and in vitro tumor-targeting effect, and to monitor their feasibility using clinical MRI scanner.Method: Amphiphilic block copolymer, poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-PCL) was synthesized via the ring-opening polymerization of ε-caprolactone (CL) initiated by poly(ethylene glycol) (PEG), in which cyclic pentapeptide Arg-Gly-Asp (cRGD) was conjugated with the terminal of hydrophilic PEG block. During the self-assembly of PEG-PCL micelles, superparamagnetic γ-Fe2O3 nanoparticles (11 nm) was loaded into the hydrophobic core. The cRGD-terminated γ-Fe2O3-loaded polymeric micelles targeting to carcinoma vascular endothelial cells, were characterized in particle size, morphology, loading efficiency and so on, especially high MRI sensitivity in vitro. Normal hepatic vascular endothelial cells (ED25) were incubated with the resulting micelles for assessing their safety. Human hepatic carcinoma vascular endothelial cells (T3A) were cultured with the resulting micelles to assess the micelle uptake using Prussian blue staining and the cell signal intensity using MRI.Results: All the polymeric micelles exhibited ultra-small particle sizes with approximately 50 nm, high relaxation rate, and low toxicity even at high iron concentrations. More blue-stained iron particles were present in the targeting group than the non-targeting and competitive inhibition groups. In vitro MRI showed T2WI and T2 relaxation times were significantly lower in the targeting group than in the other two groups.Conclusion: γ-Fe2O3-loaded PEG-PCL micelles not only possess ultra-small size and high superparamagnetic sensitivity, also can be actively targeted to carcinoma vascular endothelial cells by tumor-targeted cRGD. It appears to be a promising contrast agent for tumor-targeted imaging.

The Polysaccharide Extracted from Umbilicaria esculenta Inhibits Proliferation of Melanoma Cells through ROS-Activated Mitochondrial Apoptosis Pathway.

Melanoma is one of the most aggressive skin cancers with an increasing rate of morbidity. Umbilicaria esculenta is an edible lichen and its main component of extracts-polysaccharide (PUE) has shown significant antitumor effects in a variety of cancer types such as stomach adenocarcinoma. However, whether it has an anti-melanoma effect and the underlying mechanism has not been revealed. In this article, we showed that PUE extracted from Umbilicaria esculenta could inhibit the growth of A875 and A375 melanoma cells but without obvious toxicity to normal vascular endothelial cells. The generation of reactive oxygen species (ROS) in A875 cells was significantly elevated when treated with PUE for 24h. In addition, the expression of caspase-3 and -9 also increased as compared to the controlled group which resulted in the apoptosis of A875 melanoma cells. In the meantime, when pre-treated with N-acetylcysteine (NAC), the ROS scavenger, PUE induced apoptosis and cell death could be reversed via suppression of elevated generation of ROS and ROS-mediated caspase-9 expression. In summary, our study demonstrated that PUE extracts from Umbilicaria esculenta have a potent anti-melanoma effect through the induction of ROS and caspases-3 and -9. It could provide a promising strategy of melanoma therapy with the components from the extracts of natural and edible plants such as lichen Umbilicaria esculenta.

Synthesis, antitumor activity, and cytotoxicity of 4-substituted 1-benzyl-5-diphenylstibano-1H-1,2,3-triazoles

Trisubstituted 5-organostibano-1H-1,2,3-triazoles (3a–f) were synthesized by the Cu-catalyzed azide-alkyne cycloaddition of various ethynylstibanes (1) with benzylazide (2) in the presence of CuBr (5 mol%) under aerobic conditions. The reaction of 5-stibanotriazoles with HCl afforded C5-unsubstituted 1,2,3-triazoles (4a–f). The antitumor activity of trisubstituted 5-organostibano-1H-1,2,3-triazoles (3a–f) and their 5-unsubstituted 1,2,3-triazoles (4a–f) were evaluated in several tumor cell lines. All 5-stibanotriazoles (3a–f) exerted an excellent antitumor activity. On the contrary, 5-unsubstituted 1,2,3-triazoles (4a–f) without a diphenylantimony group in the molecule exhibited very low antitumor activity compared with 5-stibanotriazoles (3a–f). In compounds of both the series, the substituted 4-butyl group appeared to decrease antitumor activity. However, results suggested that organometal (antimony) in the molecule was required for greater antitumor activity. In addition, all 5-stibanotriazoles (3a–f), but not all 5-unsubstituted 1,2,3-triazoles (4a–f), exhibited cytotoxicity in normal vascular endothelial cells derived from bovine aorta. Among the compounds (3b–e) that exhibited excellent antitumor activity, those with 4-methylphenyl (3b) and 1-cyclohexenyl (3e) showed relatively low cytotoxicity to vascular endothelial cells. Together, these results suggest that trisubstituted 5-organostibano-1H-1,2,3-triazoles, including compounds 3b and 3e, may serve as potential anticancer therapeutic drugs in the future.

PI3K/AKT/Afadin signaling pathway contributes to pathological vascularization in glioblastomas.

Glioblastomas are brain tumors with extensive vascularization that are associated with tumor malignancy. The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is activated in endothelial cell tumors, although its exact function in glioblastoma neovascularization is poorly characterized. The present study identified that endothelial cells derived from human glioblastomas exhibit increased permeability and motility compared with normal brain vascular endothelial cells. Furthermore, the phosphorylation of AKT was significantly induced in glioblastoma-derived endothelial cells and glioblastoma vessels. To the best of our knowledge, the present study demonstrated for the first time that the cell-cell adhesion junction protein Afadin is phosphorylated and re-localized in glioblastoma-derived endothelial cells, and the phosphorylation and re-localization of Afadin is PI3K/AKT pathway-dependent. AKT-mediated phosphorylation and re-localization of Afadin may be critically involved in the modulation of brain endothelial permeability and migration. Therapies targeting the PI3K/AKT/Afadin pathway may therefore be beneficial for reducing the angiogenic potential of glioblastoma.