Avian Chorioallantoic Membrane Research Articles

Assessment of Endothelial Barrier Functions in Extra Embryonic Vasculature of Chick Embryo as an Alternative Model.

Vascular permeability, a tightly regulated process, is a direct measure of angiogenic and immune responses in the endothelium altered in several acute and chronic diseases such as sepsis, high-altitude pulmonary edema (HAPE), high-altitude cerebral edema (HACE), ischemia, and coronavirus disease 2019 (COVID-19) endotheliitis. Both endogenous and exogenous factors such as cytokines, chemokines, and hormones may affect vascular permeability. The conventional tools available for the measurement of vascular permeability in vitro and in vivo based on collagen-coated Transwell and dye-based spectrophotometric methods are indirect measures of permeability. In this chapter, we present our live in ovo protocols based on dextran-Texas red and avian chorioallantoic membrane assay developed using custom-made equipment to assess leakiness of endothelial cell barrier both in vitro and in vivo. Further, we validate this assay using different stressors such as ischemia and hypoxia known to affect endothelial barrier properties by potentiating actin stress fiber disorganization and disrupting the cell-cell junctions.

Establishment of patient-derived xenografts of retinoblastoma and choroidal melanoma on the avian chorioallantoic membrane.

To develop a viable in vivo chorioallantoic membrane (CAM) model to study the growth and invasion of patient-derived retinoblastoma (RB) and choroidal melanoma (CM) xenografts (PDXs). The study utilizes primary tumor samples instead of cancer cell lines, which provides a more authentic representation of tumors due to conserved morphology and heterogeneity. Fertilized chicken eggs were procured, windowed, and their CAM layers were dropped. On embryonic development day (EDD) 10, freshly cut patient-derived CM and RB tumors were implanted on the CAM layer and the setup was incubated for 7 days. The tumor-embedded CAM layer was harvested on EDD 17, and the extracted tumor samples were subjected to hematoxylin and eosin staining and immunohistochemical analysis to evaluate the extent of tumor invasion. Significant changes in the vascularity around the RB and CM PDXs were observed, indicating an angiogenic environment. The cross-sectional histological view of the tumor implant site revealed the invasion of both the tumors into the CAM mesoderm. Invasion of CM into CAM mesoderm was visualized in the form of pigmented nodules, and that of RB was indicated by synaptophysin and Ki-67 positivity in Immunohistochemistry (IHC). The CAM xenograft model was successfully able to support the growth of CM and RB PDXs and their invasion in CAM, thus presenting as a feasible alternative to mammalian models for studying tumorigenicity and invasiveness of ocular tumors. Moreover, this model can further be utilized to develop personalized medicine by inoculating patient-specific tumors for preclinical drug screening.

Introducing blood flow in kidney explants by engraftment onto the chick chorioallantoic membrane is not sufficient to induce arterial smooth muscle cell development.

ABSTRACTKidney explant cultures are an important tool to gain insights into developmental processes, insights that can be used to develop strategies for engineering kidneys from stem cells. However, explants are not connected to a perfused vascular system. This limits their survival and limits physiological studies, for example of blood filtration, the main function of the kidney. Previous studies have shown that grafting kidneys onto avian chorioallantoic membrane (CAM) can establish perfusion and enable glomerular vascularization, but the realism and maturity of the resultant vasculature has not been examined. Here, we show that vasculature of kidney explants grafted onto CAM is very different from natural kidney vasculature, showing excessive growth of endothelial cells, absence of a hierarchical arterio-venous network and no vascular smooth muscle cell recruitment. The model therefore has serious limits.

SS18-SSX drives CREB activation in synovial sarcoma

PurposeSynovial sarcoma (SySa) is a rare soft tissue tumor characterized by a reciprocal t(X;18) translocation. The chimeric SS18-SSX fusion protein represents the major driver of the disease, acting as aberrant transcriptional dysregulator. Oncogenic mechanisms whereby SS18-SSX mediates sarcomagenesis are incompletely understood, and strategies to selectively target SySa cells remain elusive. Based on results of Phospho-Kinase screening arrays, we here investigate the functional and therapeutic relevance of the transcription factor CREB in SySa tumorigenesis.MethodsImmunohistochemistry of phosphorylated CREB and its downstream targets (Rb, Cyclin D1, PCNA, Bcl-xL and Bcl-2) was performed in a large cohort of SySa. Functional aspects of CREB activity, including SS18-SSX driven circuits involved in CREB activation, were analyzed in vitro employing five SySa cell lines and a mesenchymal stem cell model. CREB mediated transcriptional activity was modulated by RNAi-mediated knockdown and small molecule inhibitors (666-15, KG-501, NASTRp and Ro 31-8220). Anti-proliferative effects of the CREB inhibitor 666-15 were tested in SySa avian chorioallantoic membrane and murine xenograft models in vivo.ResultsWe show that CREB is phosphorylated and activated in SySa, accompanied by downstream target expression. Human mesenchymal stem cells engineered to express SS18-SSX promote CREB expression and phosphorylation. Conversely, RNAi-mediated knockdown of SS18-SSX impairs CREB phosphorylation in SySa cells. Inhibition of CREB activity reduces downstream target expression, accompanied by suppression of SySa cell proliferation and induction of apoptosis invitro and in vivo.ConclusionIn conclusion, our data underline an essential role of CREB in SySa tumorigenesis and provides evidence for molecular targeted therapies.

Epithelial‐mesenchymal transition (EMT) drives mesothelial fusion during avian chorioallantoic membrane (CAM) formation

Chorioallantoic membrane (CAM) is formed through fusion of the chorion and allantois in amniote early development. The CAM mediates oxygen and calcium uptake in birds and gives rise to fetal part of the placenta in mammals. Molecular mechanism regulating CAM fusion is poorly understood. We carried out cellular and molecular analyses of CAM fusion between E3.5 and E4.5 of chicken development. we show that CAM fusion is preceded by a brief period of contact between chorionic and allantoic mesothelia (two mesoderm‐derived epithelial structures) followed by their EMT and subsequent integration. We identified molecular (EMT‐related and non‐EMT‐related) markers specific to each tissue layer during CAM fusion. We will also discuss our data on canonical (SNAI1 and ZEB2) and non‐canonical (WT1) EMT‐associated transcription factors in driving mesothelial EMT. We propose that avain CAM fusion is a new model for studying mesothelial cell EMT, which is known to play important physiological (e.g., placenta formation) and pathological (e.g., mesothelioma and ovarian cancer) roles in the human.Support or Funding InformationIRCMS, Takeda Science Foundation, JSPS

Focal adhesion kinase confers pro-migratory and antiapoptotic properties and is a potential therapeutic target in Ewing sarcoma.

Oncogenesis of Ewing sarcoma (EwS), the second most common malignant bone tumor of childhood and adolescence, is dependent on the expression of chimeric EWSR1‐ETS fusion oncogenes, most often EWSR1‐FLI1 (E/F). E/F expression leads to dysregulation of focal adhesions (FAs) enhancing the migratory capacity of EwS cells. Here, we show that, in EwS cell lines and tissue samples, focal adhesion kinase (FAK) is expressed and phosphorylated at Y397 in an E/F‐dependent way involving Ezrin. Employing different EwS cell lines as in vitro models, we found that key malignant properties of E/F are mediated via substrate‐independent autophosphorylation of FAK on Y397. This phosphorylation results in enhanced FA formation, Rho‐dependent cell migration, and impaired caspase‐3‐mediated apoptosis in vitro. Conversely, treatment with the FAK inhibitor 15 (1,2,4,5‐benzenetetraamine tetrahydrochloride (Y15) enhanced caspase‐mediated apoptosis and EwS cell migration, independent from the respective EWSR1‐ETS fusion type, mimicking an anoikis‐like phenotype and paralleling the effects of FAK siRNA knockdown. Our findings were confirmed in vivo using an avian chorioallantoic membrane model and provide a first rationale for the therapeutic use of FAK inhibitors to impair metastatic dissemination of EwS.

Abstract 1257: The transcription factor CREB is essential in myxoid liposarcoma

Abstract Introduction: Myxoid Liposarcoma (MLS) is an aggressive soft tissue tumor, characterized by a high rate of local recurrence and development of distant metastases. Most MLS are driven by a specific reciprocal t(12;16) translocation, and the resulting chimeric FUS-DDIT3 fusion protein acts as an oncogenic transcription factor. Although MLS display a higher chemosensitivity than other liposarcoma subtypes, the substantial rate of recurrence and metastasis in MLS underlines the urgent need for novel, biology- guided therapeutic strategies. As the mechanisms of FUS-DDIT3 mediating MLS pathogenesis are incompletely understood, we here investigate the functional relevance of the transcription factor CREB in MLS cells in vitro and in vivo. Experimental procedures: In a large cohort of MLS tissue specimens (n=92), CREB (S133) and transcriptional targets such as Rb, Cyclin D1, PCNA and Bcl-xL were analyzed by immunohistochemistry. The functional interplay between CREB and FUS-DDIT3 was analyzed by RNAi-mediated knockdown of the chimeric fusion protein in MLS cells and induced expression of FUS-DDIT3 in human mesenchymal stem cells. CREB-mediated transcriptional activity was modulated by CREB-specific siRNA and treatment with small molecule inhibitors (666-15, KG-501, NASTRp and Ro-318220). The biological effects on MLS cell proliferation were monitored by cell viability, apoptosis and immunoblotting assays. An established myxoid liposarcoma avian chorioallantoic membrane model was used for in vivo confirmation. Results: Elevated CREB phosphorylation (S133) was demonstrated in 60% of MLS tissue specimens by immunohistochemistry. In MLS cells, RNAi-mediated knockdown of FUS-DDIT3 lead to decreased phosphorylation levels of CREB (S133), accompanied by reduced transcriptional activity. Induced expression of FUS-DDIT3 in human mesenchymal stem cells stimulated phosphorylation of CREB (S133) and CREB target gene expression. Phosphorylation of CREB (S133) was induced by IGF-II stimulation and reduced by RNAi-mediated knockdown of IGF-IR. MLS cell viability was significantly reduced by specific RNAi-mediated knockdown of CREB and treatment with small molecule inhibitors (666-15, KG-501, NASTRp and Ro-318220) in vitro and in vivo. Conclusions: Our preclinical study demonstrates the essential role of CREB-mediated transcriptional activity in myxoid liposarcoma tumorigenesis and provides a molecularly based rationale for a novel targeted therapeutic approach. Citation Format: Magdalene Cyra, Sebastian Huss, Miriam Schulte, Ilka Isfort, Susanne Hafner, Thomas Simmet, Thomas Kindler, Pierre Åman, Eva Wardelmann, Wolfgang Hartmann, Marcel Trautmann. The transcription factor CREB is essential in myxoid liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1257.

SS18-SSX-Dependent YAP/TAZ Signaling in Synovial Sarcoma.

Synovial sarcoma is a soft tissue malignancy characterized by a reciprocal t(X;18) translocation. The chimeric SS18-SSX fusion protein acts as a transcriptional dysregulator representing the major driver of the disease; however, the signaling pathways activated by SS18-SSX remain to be elucidated to define innovative therapeutic strategies. Immunohistochemical evaluation of the Hippo signaling pathway effectors YAP/TAZ was performed in a large cohort of synovial sarcoma tissue specimens. SS18-SSX dependency and biological function of the YAP/TAZ Hippo signaling cascade were analyzed in five synovial sarcoma cell lines and a mesenchymal stem cell model in vitro. YAP/TAZ-TEAD-mediated transcriptional activity was modulated by RNAi-mediated knockdown and the small-molecule inhibitor verteporfin. The effects of verteporfin were finally tested in vivo in synovial sarcoma cell line-based avian chorioallantoic membrane and murine xenograft models as well as a patient-derived xenograft. A significant subset of synovial sarcoma showed nuclear positivity for YAP/TAZ and their transcriptional targets FOXM1 and PLK1. In synovial sarcoma cells, RNAi-mediated knockdown of SS18-SSX led to significant reduction of YAP/TAZ-TEAD transcriptional activity. Conversely, SS18-SSX overexpression in SCP-1 cells induced aberrant YAP/TAZ-dependent signals, mechanistically mediated by an IGF-II/IGF-IR signaling loop leading to dysregulation of the Hippo effectors LATS1 and MOB1. Modulation of YAP/TAZ-TEAD-mediated transcriptional activity by RNAi or verteporfin treatment resulted in significant growth inhibitory effects in vitro and in vivo. Our preclinical study identifies an elementary role of SS18-SSX-driven YAP/TAZ signals, highlights the complex network of oncogenic signaling pathways in synovial sarcoma pathogenesis, and provides evidence for innovative therapeutic approaches.

Phosphatidylinositol-3-kinase (PI3K)/Akt Signaling is Functionally Essential in Myxoid Liposarcoma.

Myxoid liposarcoma (MLS) is an aggressive soft-tissue tumor characterized by a specific reciprocal t(12;16) translocation resulting in expression of the chimeric FUS-DDIT3 fusion protein, an oncogenic transcription factor. Similar to other translocation-associated sarcomas, MLS is characterized by a low frequency of somatic mutations, albeit a subset of MLS has previously been shown to be associated with activating PIK3CA mutations. This study was performed to assess the prevalence of PI3K/Akt signaling alterations in MLS and the potential of PI3K-directed therapeutic concepts. In a large cohort of MLS, key components of the PI3K/Akt signaling cascade were evaluated by next generation seqeuncing (NGS), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). In three MLS cell lines, PI3K activity was inhibited by RNAi and the small-molecule PI3K inhibitor BKM120 (buparlisib) in vitro An MLS cell line-based avian chorioallantoic membrane model was applied for in vivo confirmation. In total, 26.8% of MLS cases displayed activating alterations in PI3K/Akt signaling components, with PIK3CA gain-of-function mutations representing the most prevalent finding (14.2%). IHC suggested PI3K/Akt activation in a far larger subgroup of MLS, implying alternative mechanisms of pathway activation. PI3K-directed therapeutic interference showed that MLS cell proliferation and viability significantly depended on PI3K-mediated signals in vitro and in vivo Our preclinical study underlines the elementary role of PI3K/Akt signals in MLS tumorigenesis and provides a molecularly based rationale for a PI3K-targeted therapeutic approach which may be particularly effective in the subgroup of tumors carrying activating genetic alterations in PI3K/Akt signaling components.

The avian chorioallantoic membrane as an alternative tool to study medullary thyroid cancer.

Preclinical trials of medullary thyroid cancer (MTC) therapeutics require both in vitro and in vivo analyses. Human tumour xenografted rodent models, which are considered the ‘gold standard’ to study and validate the efficacy and toxicity of lead compounds before translation to clinical trials, are very expensive, subject to organismal variability and ethical controversies. The avian chorioallantoic membrane (CAM) assay provides an alternative versatile, cost-effective and ethically less objectionable short-term, in vivo model for reliable screening of drugs. In this work, we grafted two MTC cell lines and patient-derived MTC tumour samples onto the avian CAM and characterised the resulted tumours histologically and immunohistochemically. Our findings provide the evidence that the CAM assay is a suitable model for studying the pathophysiology of MTC and can even be used as in vivo system for drug testing.

A short-term in vivo model for Merkel Cell Carcinoma.

In vivo tumor models are essential for studying the biology of cancer, identifying tumor targets and evaluating antitumor drugs. Considering the request for the minimisation of animal experiments and following the “3R”‐rule (“replacement,” “refinement,” “reduction”), it has become crucial to develop alternative experimental models in cancer biology. Several studies have already described the avian chorioallantoic membrane (CAM) model as an alternative to rodents, suitable to investigate growth, progression and metastasis of various types of cancer. In the present work, we grafted three Merkel cell carcinoma (MCC) cell lines onto the avian CAM and monitored tumor growth and development of solid tumor nodules. Morphology of xenograft was characterised histologically and immunohistochemically. Our results demonstrate CAM assay as a useful tool to study MCC pathophysiology.

Short-term culture of adult bovine ovarian tissues: chorioallantoic membrane (CAM) vs. traditional in vitro culture systems

BackgroundA suitable culture system is important for follicle growth in adult bovine ovarian tissue. This study aimed to assess the avian chorioallantoic membrane (CAM) for short-term culture of adult bovine ovarian tissues compared with a traditional in vitro culture system.MethodsOvarian cortical tissues (1–2 mm3), collected from slaughtered adult cows, were randomly assigned to control, CAM or in vitro culture groups. In the control group, ovarian tissues were fixed with paraformaldehyde without culture. In CAM and in vitro culture groups, the ovarian tissues were cultured for up to 5 days and then fixed. Ovarian tissues were examined on culture days 0, 1, 3 and 5 for angiogenesis, follicle morphology and growth. In all groups, primordial and growing (healthy and atretic) follicle densities were determined.ResultsIn the CAM culture, the avian blood vessel density increased (p < 0.01) over time with a decline (p < 0.001) in the bovine blood vessel density. Healthy primordial, atretic primordial and healthy growing follicle densities were higher (p < 0.05) in CAM-cultured ovarian tissues than in vitro-cultured tissues. Regardless of the culture system, the density of healthy primordial follicles decreased (p < 0.001) over time with an increase in healthy growing follicles on day 3 (p < 0.01) and an increase in atretic (primordial and growing) follicles during the 5-day culture period (p < 0.001). The proportions of healthy primordial and atretic growing follicles were also affected by culture day (p < 0.001).ConclusionsThe CAM culture in chick embryos supported the bovine ovarian tissue grafts for 3 days demonstrating that CAM can be used as a satisfactory short-term culture system to assess ovarian tissue health, and to study follicle activation and development.

Nerve Growth Factor-Induced Angiogenesis: 2. The Quail Chorioallantoic Membrane Assay.

The avian chorioallantoic membrane (CAM) is a simple, highly vascularized extraembryonic membrane, which performs multiple functions during embryonic development. Therefore, the models of chicken and quail assays represent robust experimental platforms to study angiogenesis, which reflects perturbation of the entire vascular tree. This experimental approach, when combined with fractal morphometry, is sensitive to changes in vascular branching pattern and density. Nerve growth factor is a neurotrophin promoting angiogenesis in CAM models. Here, we provide a detailed protocol of the quail CAM, shell-less model, to study nerve growth factor effects on blood capillary sprouting. The quail CAM assay may be beneficial in investigations of cellular and molecular aspects of neurotrophin-induced angiogenesis and for developing novel anti-angiogenesis and anticancer therapies.

FUS–DDIT3 Fusion Protein-Driven IGF-IR Signaling is a Therapeutic Target in Myxoid Liposarcoma

Purpose: Myxoid liposarcoma is an aggressive disease with particular propensity to develop hematogenic metastases. Over 90% of myxoid liposarcoma are characterized by a reciprocal t(12;16)(q13;p11) translocation. The resulting chimeric FUS-DDIT3 fusion protein plays a crucial role in myxoid liposarcoma pathogenesis; however, its specific impact on oncogenic signaling pathways remains to be substantiated. We here investigate the functional role of FUS-DDIT3 in IGF-IR/PI3K/Akt signaling driving myxoid liposarcoma pathogenesis.Experimental Design: Immunohistochemical evaluation of key effectors of the IGF-IR/PI3K/Akt signaling axis was performed in a comprehensive cohort of myxoid liposarcoma specimens. FUS-DDIT3 dependency and biological function of the IGF-IR/PI3K/Akt signaling cascade were analyzed using a HT1080 fibrosarcoma-based myxoid liposarcoma tumor model and multiple tumor-derived myxoid liposarcoma cell lines. An established myxoid liposarcoma avian chorioallantoic membrane model was used for in vivo confirmation of the preclinical in vitro results.Results: A comprehensive subset of myxoid liposarcoma specimens showed elevated expression and phosphorylation levels of various IGF-IR/PI3K/Akt signaling effectors. In HT1080 fibrosarcoma cells, overexpression of FUS-DDIT3 induced aberrant IGF-IR/PI3K/Akt pathway activity, which was dependent on transcriptional induction of the IGF2 gene. Conversely, RNAi-mediated FUS-DDIT3 knockdown in myxoid liposarcoma cells led to an inactivation of IGF-IR/PI3K/Akt signaling associated with diminished IGF2 mRNA expression. Treatment of myxoid liposarcoma cell lines with several IGF-IR inhibitors resulted in significant growth inhibition in vitro and in vivoConclusions: Our preclinical study substantiates the fundamental role of the IGF-IR/PI3K/Akt signaling pathway in myxoid liposarcoma pathogenesis and provides a mechanism-based rationale for molecular- targeted approaches in myxoid liposarcoma cancer therapy. Clin Cancer Res; 23(20); 6227-38. ©2017 AACR.

MP71-19 ESTABLISHING AN EXPERIMENTAL MODEL FOR TARGETING RENAL CELL CARCINOMA USING MESENCHYMAL STEM CELLS IN THE AVIAN CHORIOALLANTOIC MEMBRANE

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I1 Apr 2016MP71-19 ESTABLISHING AN EXPERIMENTAL MODEL FOR TARGETING RENAL CELL CARCINOMA USING MESENCHYMAL STEM CELLS IN THE AVIAN CHORIOALLANTOIC MEMBRANE Zhihao (James) Xu, Hua Chen, Desmond Pink, Katia Carmine-Simmen, John Lewis, and Ronald Moore Zhihao (James) XuZhihao (James) Xu More articles by this author , Hua ChenHua Chen More articles by this author , Desmond PinkDesmond Pink More articles by this author , Katia Carmine-SimmenKatia Carmine-Simmen More articles by this author , John LewisJohn Lewis More articles by this author , and Ronald MooreRonald Moore More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1465AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Metastatic renal cell carcinoma (mRCC) is a lethal urologic disease with 10 year survival less than 5 percent. Recent developments in cell signalling and small molecule therapy for mRCC have impacted progression free survival (PFS) only. High-dose IL-2 immunotherapy can produce durable complete responses (CR) but the response rate is low (16%), highlighting the need for novel therapy. Mesenchymal stem cells (MSCs) have the capacity to mobilize to sites of inflammation. Recently, MSCs were reported to traffic to murine RCC lung metastases via inflammatory signals, opening the potential of delivering tumor site-specific treatment. We hypothesize that human MSCs could target human RCC engrafted in the chorioallantoic membrane (CAM) of the avian embryo. In this project, we investigated potential use of the avian CAM model to study MSC RCC homing. This immunodeficient model is ideal for imaging tumor growth, and cell trafficking. METHODS We first transduced 786-O RCC lines to express green fluorescent protein (GFP), blue fluorescent protein (BFP), or mApple respectively using lentiviral-based vectors. Successful transduction was selected using blasticidin antibiotic treatment. Concurrently, human adipose derived MSCs were isolated from patient perirenal fat, cultured, and characterized using flow cytometry. For the avian CAM model, fluorescent 786-O cells were initially injected into the CAM to establish a viable tumor. The chicken egg vascular endothelium was fluorescently labelled with lectin. MSCs, labelled with a fluorescent plasma membrane stain, were injected IV and observed using intravital imaging via confocal microscopy. RESULTS At 6 weeks post MSC isolation, flow cytometry results indicated that the patient adipose derived primary culture was CD73, CD90, CD105, CD44, CD166 positive and CD45, CD19, CD106, CD146 negative, consistent with MSC expression. All three fluorescent 786-O cell lines were observed by fluorescent microscopy to establish viable and vascularized tumors in the CAM, 7 days post injection. The injected commercial and patient-derived human MSCs were observed to be in proximity to the RCC tumor consistently over periods of 18 hrs and 3 hrs respectively. However, MSCs were not observed to specifically traffic to the RCC tumor. CONCLUSIONS Our results demonstrated that both viable human RCC tumors and human MSCs were established and imaged in the avian CAM model. The stage is set for future studies exploring MSC tumor homing. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e923 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Zhihao (James) Xu More articles by this author Hua Chen More articles by this author Desmond Pink More articles by this author Katia Carmine-Simmen More articles by this author John Lewis More articles by this author Ronald Moore More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

A new in vivo magnetic resonance imaging method to noninvasively monitor and quantify the perfusion capacity of three-dimensional biomaterials grown on the chorioallantoic membrane of chick embryos.

Adequate vascularization in biomaterials is essential for tissue regeneration and repair. Current models do not allow easy analysis of vascularization of implants in vivo, leaving it a highly desirable goal. A tool that allows monitoring of perfusion capacity of such biomaterials noninvasively in a cheap, efficient, and reliable in vivo model would hence add great benefit to research in this field. We established, for the first time, an in vivo magnetic resonance imaging (MRI) method to quantify the perfusion capacity of a model biomaterial, DegraPol(®) foam scaffold, placed on the embryonic avian chorioallantoic membrane (CAM) in ovo. Perfusion capacity was assessed through changes in the longitudinal relaxation rate before and after injection of a paramagnetic MRI contrast agent, Gd-DOTA (Dotarem(®); Guerbet S.A.). Relaxation rate changes were compared in three different regions of the scaffold, that is, at the interface to the CAM, in the middle and on the surface of the scaffold (p<0.05). The highest relaxation rate changes, and hence perfusion capacities, were measured in the interface region where the scaffold was attached to the CAM, whereas the surface of the scaffold showed the lowest relaxation rate changes. A strong positive correlation was obtained between relaxation rate changes and histologically determined vessel density (R(2) = 0.983), which corroborates our MRI findings. As a proof-of-principle, we measured the perfusion capacity in different scaffold materials, silk fibroin either with or without human dental pulp stem cells. For these, three to four times larger perfusion capacities were obtained compared to DegraPol; demonstrating that our method is sensitive to reveal such differences. In summary, we present a novel in vivo method for analyzing the perfusion capacity in three-dimensional-biomaterials grown on the CAM, enabling the determination of the perfusion capacity of a large variety of bioengineered materials.

Avian area vasculosa and CAM as rapid in vivo pro-angiogenic and antiangiogenic models.

Angiogenesis, the development of new blood vessels from preexisting ones, is driven by coordinated signaling pathways governed by specific molecules, hemodynamic forces, and endothelial and periendothelial cells. The processes involve adhesion, migration, and survival machinery within the target endothelial and periendothelial cells. Factors that interfere with any of these processes may therefore influence angiogenesis either positively (pro-angiogenesis) or negatively (antiangiogenesis). The avian area vasculosa (AV) and the avian chorioallantoic membrane (CAM) are two useful tools for studying both angiogenesis and antiangiogenesis since they are amenable to both intravascular and topical administration of target, agents, are relatively rapid assays, and can be adapted very easily to study angiogenesis-dependent processes, such as tumor growth. Both models provide a physiological setting that permits investigation of pro-angiogenic and antiangiogenic agent interactions in vivo.

Influence of physically crosslinked gelatins on the vasculature in the avian chorioallantoic membrane

Gelatins functionalized with desaminotyrosine (DAT) or desaminotyrosyl tyrosine (DATT) form physically crosslinked hydrogels, due to the interactions between the introduced aromatic moieties and gelatin triple helices, whose extent depends on the thermal treatment of the material. The G-modulus of these hydrogels can be tailored to the range of the natural extracellular matrix by adjusting the degree of crosslinking. While these gelatin-based materials have been shown to be not angiogenic, the aim of the study was to evaluate whether these biomaterials influence the regulation of blood vessels when positioned on the chorionallantoic membrane (CAM) of fertilized eggs. The results clearly indicate that the DAT-functionalized gelatin led to an increase of the diameter of the blood vessels in the CAM, which at the same time is probably associated with an increased blood flow in these CAM vessels. The vessel diameters of the four groups (DAT-functionalized gelatin, DATT-functionalized gelatin, plain gelatin, control group without gelatin, each n = 10) differed significantly (p < 0.0001). Vessels in the CAM exposed to the DAT-functionalized gelatin showed with 36.4 μm ± 3.4 μm the largest mean diameters compared to the mean diameters of the samples exposed to DATT gelatin (16.0 μm ± 0.8 μm; p < 0.05) and the plain gelatin (21.2 μm ± 1.0 μm; p < 0.05), which both did not differ significantly from the vessels of the control group. The biocompatibility of the materials in vitro motivates the exploration of their application as matrix in local drug-release systems with short half-life times (one hour up to several days).

Abstract 4217: Engaging CD151 inhibits cell migration and metastasis through a novel mechanism involving the cell adhesion molecule ALCAM/CD166

Abstract We recently demonstrated that actively promoting the formation of cell surface complexes by engaging the tetraspanin CD151 with a monoclonal antibody (mAb 1A5) inhibits metastasis by immobilizing the tumor cells. Tetraspanins influence cellular behavior via the regulation of associated partner molecules such as integrins, cell adhesion molecules, and members of the immunoglobulin superfamily. Although CD151 is able to associate with and regulate the activity of laminin-binding integrins we demonstrate that CD151 does not associate with α3α1 in the presence of mAb 1A5. Using a 2-color flow cytometric approach we mapped the domains of CD151 required for antigen recognition by mAb 1A5 and determined that 1A5 maps to the integrin 194QRD196 motif of CD151, using this information we further performed CD151 co-immunoprecipitations and demonstrated that 1A5 recognizes CD151 not associated with α3α1.To determine the identity of the protein(s) associated with the antibody-engaged CD151 and also possibly responsible for the anti-migratory, anti-metastatic phenotype we used a proteomic approach. Using tandem mass spectrometry on CD151-associated proteins co-immunoprecipitated by mAb 1A5, we detected in excess of 200 proteins. This strategy identified several putative partners of CD151. Further analysis of the CD151 associated proteins led to our discovery of the novel CD151-associated protein ALCAM/CD166, a member of the immunoglobulin superfamily. Using an RNAi-mediated knockdown approach we demonstrate that ALCAM/CD166 is required for the CD151 complex to regulate tumor cell mobility in vitro. Conversely, CD151 is required for ALCAM to control motility. Spontaneous metastasis assays using the avian chorioallantoic membrane and subcutaneous murine xenografts demonstrate thatALCAM/CD166 is indeed required for the engaged tetraspanin to inhibit tumor cell dissemination in vivo. Further mechanistic analysis of this complex revealed that the CD151/ALCAM protein complex promotes activation of the small GTPase Rap1 via activated protein kinase C. Collectively, we present data, which demonstrate that, the ALCAM-containing CD151 complex can control metastasis. This novel complex reveals how cell-cell interactions can control migration through the regulation of cell-matrix adhesions. These results also demonstrate mechanistically that tetraspanins can integrate signaling between physically distinct molecular functions and therefore behave as a molecular clutch that controls matrix adhesion in response to cell-cell interactions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4217. doi:1538-7445.AM2012-4217

Physically crosslinked gelatins functionalized with tyrosine moieties do not induce angiogenesis or thrombus formation in the developing vasculature in the avian chorioallantoic membrane

Gelatins functionalized with desaminotyrosine or desaminotyrosyl tyrosine form physically crosslinked polymer networks due to the interactions between the introduced aromatic moeties. In the swollen state, their mechanical properties can be tailored in a range similar to the elasticity of soft tissues. The aim of this study was to evaluate their potential as biomaterials by determining whether these materials - in comparison to plain gelatin - induce bleedings, thrombotic processes, or angiogenesis. These investigations were performed using the hen's egg chorioallantoic membrane (HETCAM) assay. These results indicate that the gelatin-based hydrogels did not possess angiogenic effects and also did not induce bleedings, thrombotic processes or vessel destruction (avascular zones). The biocompatibility of the materials in vitro motivates the exploration of their application as matrix in local drug-release systems with short half-life times (1 hour up to several days).