Investigation of Angiogenesis and Wound Healing Potential Mechanisms of Zinc Oxide Nanorods

The angiogenesis process is an essential issue in tissue engineering. Zinc oxide nanorods are biocompatible metals capable of generating reactive oxygen species (ROS) that respond to induced angiogenesis through various mechanisms; however, released Zn (II) ions suppress the angiogenesis process. In this study, we fabricated green ZnO nanorods using albumin eggshell as a bio-template and investigate its angiogenic potential through chorioallantoic membrane assay and excision wound healing assay. This study demonstrated that angiogenesis and wound healing processes depend on pro-angiogenic factors as VEGF expression due to ZnO nanorods' exiting. Angiogenesis induced via zinc oxide nanorods may develop sophisticated materials to apply in the wound healing field.

The CAM Model for CIC-DUX4 Sarcoma and Its Potential Use for Precision Medicine

(1) Background: CIC-DUX4 sarcoma is a rare mesenchymal small round cell tumor which belongs to rare cancers that occupy a significant percentage of cancer cases as a whole, despite each being rare. Importantly, each rare cancer type has different features, and thus there is a need to develop a model that mimics the features of each of these cancers. We evaluated the idea that the chicken chorioallantoic membrane assay (CAM), a convenient and versatile animal model, can be established for the CIC-DUX4 sarcoma. (2) Methods: Patient-derived cell lines of CIC-DUX4 were applied. These cells were transplanted onto the CAM membrane and tumor formation was examined by H&E staining, immunohistochemistry and Western blotting. The CAM tumor was transferred onto a fresh CAM and was also used to form organoids. Retention of the fusion gene was examined. (3) Results: H&E staining as well as molecular characterization demonstrated the formation of the CIC-DUX4 tumor on the CAM membrane. Expression of cyclin D2 and ETV4 was identified. The CAM tumor was transferred to a fresh CAM to form the second-generation CAM tumor. In addition, we were successful in forming tumor organoids using the CAM tumor. Retention of the fusion gene CIC-DUX4 in the CAM, second-generation CAM, and in the CAM-derived organoids was confirmed by RT-PCR. (4) Conclusions: The CAM assay provides a promising model for CIC-DUX4 sarcoma.

P16.12 Proangiogenic effect of fibroblast activation protein positive stromal cells derived from human glioblastomas

BACKGROUND Increased expression of fibroblast activation protein (FAP) is characteristic for several cancer types including human glioblastomas (GBM). FAP is expressed on both cancer as well as stromal cells which were demonstrated in extracranial tumors to maintain a microenvironment permissive for tumor growth and thus to contribute to tumor progression. FAP is considered a potential diagnostic and therapeutic target and several approaches for its targeting have been recently developed. In this work, we investigated the role of FAP+ stromal cells in glioblastoma angiogenesis. MATERIAL AND METHODS Expression of FAP and other phenotypic markers was assessed by immunocytochemistry and immunohistochemistry. FAP+ stromal cells and primary microvascular endothelial cells were isolated using magnetic activated cell sorting (MACS). Genetic aberrations were assayed by comparative genomic hybridization/single-nucleotide polymorphism analysis. Angiogenesis was evaluated using a 3D spheroid-based sprouting assay and a chorioallantoic membrane assay. A cytokine array was used to analyze soluble mediators released by FAP+ stromal cells. RESULTS FAP+ stromal cells were predominantly localized around activated CD105+ endothelial cells and their quantity positively correlated with glioblastoma vascularization. FAP+ stromal cells derived from human GBMs had a mesenchymal phenotype, were non-tumorigenic and in most cases lacked cytogenetic aberrations characteristic of GBMs. Conditioned media derived from FAP+ stromal cells induced angiogenic sprouting of both macrovascular HUVEC as well as microvascular primary endothelial cells derived from human GBMs. In a chorioallantoic membrane assay, admixture of FAP+ stromal cells to glioma cells was associated with increased angiogenesis and more frequent occurrence of hemorrhages. Cytokine array revealed a significant disbalance between several proangiogenic and antiangiogenic mediators compared to normal pericytes, and an increased Angiopoietin 2/1 ratio in conditioned media from FAP+ stromal cells. CONCLUSION Our results bring new evidence that GBM associated FAP+ stromal cell promote angiogenesis by changing the balance between proangiogenic and antiangiogenic mediators and thus they may contribute to glioblastoma progression. ACKNOWLEDGEMENT Supported by Progres Q28/1LFUK and grant LM2015064 of the EATRIS-CZ and the Center for Tumor Ecology (CZ.02.1.01/0.0/0.0/16_019/0000785).

Targeting the Endothelin-1 Receptors Curtails Tumor Growth and Angiogenesis in Multiple Myeloma

The endothelin-1 (ET-1) receptors were recently found to mediate pro-survival functions in multiple myeloma (MM) cells in response to autocrine ET-1. This study investigated the effectiveness of macitentan, a dual ET-1 receptor antagonist, in MM treatment, and the mechanisms underlying its activities. Macitentan affected significantly MM cell (RPMI-8226, U266, KMS-12-PE) survival and pro-angiogenic cytokine release by down-modulating ET-1-activated MAPK/ERK and HIF-1α pathways, respectively. HIF-1α silencing abrogated the ET-1 mediated induction of genes encoding for pro-angiogenic cytokines such as VEGF-A, IL-8, Adrenomedullin, and ET-1 itself. Upon exposure to macitentan, MM cells cultured in the presence of the hypoxia-mimetic agent CoCl2, exogenous ET-1, or CoCl2 plus ET-1, down-regulated HIF-1α and the transcription and release of downstream pro-angiogenic cytokines. Consistently, macitentan limited significantly the basal pro-angiogenic activity of RPMI-8226 cells in chorioallantoic membrane assay. In xenograft mouse models, established by injecting NOG mice either via intra-caudal vein with U266 or subcutaneously with RPMI-8226 cells, macitentan reduced effectively the number of MM cells infiltrating bone marrow, and the size and microvascular density of subcutaneous MM tumors. ET-1 receptors targeting by macitentan represents an effective anti-proliferative and anti-angiogenic therapeutic approach in preclinical settings of MM.

Evolving applications of the egg: chorioallantoic membrane assay and ex vivo organotypic culture of materials for bone tissue engineering

The chick chorioallantoic membrane model has been around for over a century, applied in angiogenic, oncology, dental and xenograft research. Despite its often perceived archaic, redolent history, the chorioallantoic membrane assay offers new and exciting opportunities for material and growth factor evaluation in bone tissue engineering. Currently, superior/improved experimental methodology for the chorioallantoic membrane assay are difficult to identify, given an absence of scientific consensus in defining experimental approaches, including timing of inoculation with materials and the analysis of results. In addition, critically, regulatory and welfare issues impact upon experimental designs. Given such disparate points, this review details recent research using the ex vivo chorioallantoic membrane assay and the ex vivo organotypic culture to advance the field of bone tissue engineering, and highlights potential areas of improvement for their application based on recent developments within our group and the tissue engineering field.