Name: chick heart invasion assay for testing the invasiveness of cancer cells and the activity of potentially anti-invasive compounds
Uploaded: 2015-06-06T15:00:00.000+00:00
Duration: 10 min 16 s
Description: Watch this Scientific Journal Video about Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds at JoVE.com

We thank Marleen De Meulemeester for demonstrating the assay technique in the video film. B. I. R. is a Postdoctoral Research Fellow of the Research Foundation &#8211; Flanders (FWO &#8211; Vlaanderen). L.M.M. is a recipient of an Emmanuel van der Schueren grant from the Flemish League against Cancer (Vlaamse Liga tegen Kanker).

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The authors declare that they have no competing financial interests.

PHF&#39;lerin hazırlanması sırasında, fragmanlar süspansiyon kalmak olmayabilir ama damar duvarına yapışır; Bu kültür ortamı hacmini arttırarak aşılabilir. PHF&#39;lerin sayısı çok düşük olduğu ve boyutu çok büyük ise, kültür ortamının düşürür. Bir araya test hücrelerinin hatası sıcaklığında veya mikrobik enfeksiyona dalgalanmalara bağlı olabilir. Alternatif olarak, agrega bir yetersizlik hücrelerinin özgü bir özelliktir olabilir. PHF için agrega bağlanması sırasında, k?...

Invasion kötü huylu tümörlerin özelliğidir. Bu aktivite, çevredeki dokuların tahrip olmasına yol açar, ama aynı zamanda metastaz oluşumunda rol oynadığı gösterilmiştir tesis edilir. Kanser hastalarının invazyon ve metastaz ve etkili anti-invaziv tedaviler ölmektedir beri hala tümör hücrelerinin işgali geliştirilmiştir taklit kıt, laboratuar deneyleri bulunmaktadır. Civciv, kalp deneyin amacı, üç boyutlu olarak tümör istilasını incelemek için ilgili bir organ kültür yöntemi sunmaktır. Tahlil invaziv ve non-invaziv hücreler arasında ayrım ve tümör işgali üzerine test bileşiklerinin etkilerini incelemek için izin verir. tahlilin kullanımı arkasındaki mantık tümörler neoplastik hücreler sürekli olarak stroma (ev sahibi hücreler ve hücre-dışı matris) etkileşim ekosistemlerin olduğu gerçek bir kavramdır ve bu moleküler interaksiyonlarda işgali ile bu ince ayarlanmış 1 olduğu. Yani, deneyde tümör hücreleri Livi ile karşı karşıya kalırlarTümör hücreleri tarafından işgali için alt-tabakalar olarak değil, aynı zamanda stroma hücreleri ve matris elemanlarının farklı bir kaynağı olarak da iş görür ng civciv embriyosu kalp fragmanları 2. Civciv, kalp miyositleri, fibroblastlar ve endotel hücreleri içerir ve hücre dışı matris laminin, fibronektin ve kollajen farklı oluşmaktadır. Bu şekilde, üç boyutlu bir organ kültürü tekniği, hasta tümör istilası karıştığı bir çok selüler ve moleküler etkileşimleri kapsar. civciv kalp testinin ana avantajı stromal etkilerin uygulamasıdır. Bu özellik, bazal membran 3 veya interstisyel matrisin 4 molekülleri oluşan cansız jeller halinde tümör hücrelerinin istilasını dayanmaktadır, in vitro olarak, diğer invazyon deneylerde daha tamamlanır. organ kültür deney bulunan tümör hücreleri ve normal yaşam konak doku arasındaki çatışma kavramı birkaç tarafından getirilmiştirAlmanya&#39;da 7 Wolff ve Schneider Fransa&#39;da 5, Birleşik Krallık 6 easty ve easty ve Schleich dahil yazarlar. Atıf yöntemler üzerinde civciv kalp işgali testinin iki teknik avantajları parçalarının hacmi kolaylıkla standardize olabilir ve organ kültürü sırasında fonksiyonel bütünlük izlenmesine olanak sağlar kasılma, kalmasıdır. Bunlar yumurta içindeki steril içeriğine kesilerek edilebilir, çünkü Ayrıca, kuş embriyolar, tercih edilir. tahlil tümör hücrelerine çevreleyen karmaşık stromal sunarak civciv chorioallantois membran tahlil 8 kavramsal benzerlik vardır. Deney başarılı şekilde 9 ve HCT-8 (Kolon), 10 hücre çizgisi ailesine MCF-7 (meme) ile aynı insan tümörlerinden invaziv ve invazif olmayan hücre varyantları arasında ayırt etmek için uygulanmıştır. teknik, hem de potansiyel olarak anti-invasif bileşikleri test etmek için kullanışlıdır <sup&gt; 11,12. Daha da açıklandığı üzere, küçük organik molekülleri yapı-aktivite ilişkileri kurulması için kullanılabilir. Deney Bununla birlikte, göz önüne kanser invazyonu immünolojik hücrelerin katkısını almaz. Bu tekniğin nedeniyle manipülasyonlar yüksek sayıda, tahlil çalışır sınırlı sayıda (en fazla 30 kültürler) ve uzun turn-around zaman (yaklaşık 1 ay) içinde, yüksek verimli bir analiz sistemi olarak kabul edilemez vurgulanmalıdır.

<table><tbody><tr><td>Ringer&#039;s salt solution</td><td>Braun</td><td>CEO123</td><td></td></tr><tr><td>Bacto-agar</td><td>Becton Dickinson</td><td>214010</td><td></td></tr><tr><td>Tris-(hydroxymethyl)-aminomethaan Analar</td><td>VWR</td><td>103157P</td><td></td></tr><tr><td>BSA : albumin from bovine serum, cohn V fract.</td><td>Sigma</td><td>A4503-500G</td><td></td></tr><tr><td>MEM-Rega 3</td><td>Life Technologies&amp;nbsp;</td><td>19993013</td><td></td></tr><tr><td>Gyrotory shakers</td><td>New Brunswick Scientific</td><td>G10 and G33</td><td></td></tr><tr><td>Erlenmeyer flasks 50 ml</td><td>Novolab</td><td>92717</td><td></td></tr><tr><td>Glass Petri dishes</td><td>Novolab</td><td>68516</td><td></td></tr><tr><td>Iridectomy scissors</td><td>Rumex</td><td>11-0625</td><td></td></tr><tr><td>Macroscope with calibrated ocular grid</td><td>Wild</td><td>157702</td><td></td></tr><tr><td>Paraffin wax</td><td>International Medical products</td><td>8599956</td><td></td></tr><tr><td>Eosin Y</td><td>Sigma</td><td>230251-25g</td><td></td></tr><tr><td>Harris&#039; hematoxylin</td><td>Sigma</td><td>HHS32-1L</td><td></td></tr><tr><td>Coverslipping resin (Tissue-Tek)</td><td>Sakura</td><td>4494</td><td></td></tr><tr><td>Paraffin melting apparatus</td><td>GFL</td><td>1052</td><td></td></tr><tr><td>Microtome for paraffin sectioning</td><td>Reichert</td><td></td><td></td></tr><tr><td>Stppers for Erlemeyer flasks with gas in- and outlets</td><td>Novolab</td><td>2602421 and 260243</td><td></td></tr><tr><td>Diaminobenzidine</td><td>Sigma</td><td>D8001</td><td></td></tr><tr><td>REAX rocking table</td><td>Heidolph</td><td>54131</td><td></td></tr><tr><td>24-well tissue dishes</td><td>VWR</td><td>NUNC142475</td><td></td></tr><tr><td>Ophtalmological enucleation spoon</td><td>Rumex</td><td>16-060</td><td></td></tr><tr><td>Sharp forceps</td><td>Rumex</td><td>4-111T</td><td></td></tr><tr><td>Blunt forceps</td><td>Nickel-Electro LTD</td><td>7112</td><td></td></tr><tr><td>Erlenmeyer flasks 5 ml</td><td>Novolab</td><td>211901</td><td></td></tr><tr><td>Microscope slides washed and degreased</td><td>International Medical products</td><td>8613908</td><td></td></tr></tbody></table>

chick heart invasion assay for testing the invasiveness of cancer cells and the activity of potentially anti-invasive compounds

Civciv, kalp deneyin amacı, üç boyutlu olarak tümör istilasını incelemek için ilgili bir organ kültür yöntemi sunmaktır. Deney invaziv ve invazif olmayan hücreler arasında ayrım ve tümör invazyonu üzerinde test bileşiklerinin etkileri etüdüne olanak sağlamıştır olabilir. Kanser hücreleri - ya agrega veya tek hücreleri gibi - embriyonik civciv kalp parçaları ile karşı karşıya kalırlar. Birkaç gün veya hafta içinde süspansiyon içinde organ kültürü sonra, karşı karşıya kültürler sabitlenmiş ve histolojik analiz için parafin içine gömülmüştür. kanser hücreleri ve normal doku arasında üç boyutlu etkileşimi, daha sonra hematoksilen-eozin ile ya da kalp dokusunda, epitoplar ya da karşı karşıya kanser hücreleri için immünohistokimyasal boyama sonrası lekelenmiş dizi bölgelerinin arasında yeniden yapılmıştır. Deney kanseri istilası kanser hücreleri ve komşu stromal ana elemanlarının (miyofibroblastlar, endoth arasındaki moleküler etkileşimlerin sonucu olduğu son kavramı ile tutarlıdırElial hücreler, hücre dışı matris bileşenleri, vs.). İşte, bu stromal ortamı yaşayan bir doku parçası olarak kanser hücrelerine sunulmaktadır. Testin alaka Destekleyici yönleri birden vardır. Ilerici işgal ve yedek zamanında ve ev sahibi dokusunun alanı ve invaziv ve karşı karşıya hücrelerin in vivo non-invaziv genel tahlilin sonucu ile ilişkilidir: tahlilde Invasion kanser işgali kriterlerine uygundur. Ayrıca, in vivo hücre istilası desen, patologlar tarafından tanımlanan deneyde histolojik görüntü yansıtılır. Çok sayıda potansiyel olarak anti-invasif organik konjener bileşiklerle elde edilen sonuçlar kantitatif yapı-aktivite ilişkisi (QSAR) analizi, klinikte kullanılan flavonoidler ve chalcone ve bilinen bir anti-metastatik ilaçlar için yapı-aktivite ilişkilerinin (örneğin, mikrotübül inhibitörleri çalışma izin ) ve deneyde istilasını inhibe eder. However, tahlil kanser işgali hesap immünolojik katkıları dikkate almaz.

Şekil 5&#39;de gösterildiği gibi, histolojik bölümler başarılı deneylerde, bir dizi sonucunu göstermektedir. kültürlerin ses histoloji canlı hücreleri belirtir ve tümör hücreleri ve normal doku arasındaki etkileşimi yorumlamak için izin verir. Bundan başka, normal doku ile ilgili hiç bir bağışıklık reaksiyonu immün red sisteminin oluşturulması önce, örneğin, kullanılan piliç embriyoları doğru yaş teyit olan gözlenebilir. Kültür döneminde (brüt) kontaminasyon yokluğunu ...

Watch this Scientific Journal Video about Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds at JoVE.com

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

Burada, üç boyutta normal doku parçaları canlı olarak tümör hücrelerinin istilasını incelemek için bir protokol mevcut. Bu organ kültürü tekniği, özellikle in vitro koşullarında potansiyel anti-invasif ilaçlar test etmek için uygulanır.

Test Chick Kalp Invasion Testi Kanser hücrelerinin invazivliğini ve Potansiyel Anti-invaziv Bileşikler Etkinlik

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

chick-heart-invasion-assay-for-testing-invasiveness-cancer-cells

Research

JoVE Journal

Medicine

9.0K Görüntüleme.  University of Ghent.  Burada, üç boyutta normal doku parçaları canlı olarak tümör hücrelerinin istilasını incelemek için bir protokol mevcut. Bu organ kültürü tekniği, özellikle in vitro koşullarında potansiyel anti-invasif ilaçlar test etmek için uygulanır. 

Video: Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Yukarı doğru hareket: Bir basit ve esnek Vitro üç boyutlu işgali tahlil iletişim kuralı

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Kanser Araştırmaları

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

Kanser Hücreleri Endotel Hücre Tek tabakalı Invasion Tedbir Gerçek zamanlı Elektrik Empedans Tabanlı Tekniği

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

Tıp

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modelleme ve Görüntüleme 3-Boyutlu Toplu Hücre Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Canlı hücre görüntüleme deneyleri için çalışma Glioblastoma Beyin tümör kök hücre göç ve işgali

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

Onkojenik hücre davranışının bir ölçüsü olarak bir In vitro Invazif tahlil içinde Mouse mammary tümör hücrelerinin kullanımı

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

Genetik

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line.&#160;

Empedans tabanlı Gerçek Zamanlı Kanser Hücre Göçü ve İstilası Ölçümü

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

Hızlı ve Nicel Intravital Chick Chorioallantoic Membran Modeli Kullanılarak Metastatik Regülatörlerin Keşfi

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

Bir In vitro FluoroBlok Tümör Invasion Testi

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Biyoloji

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Bir Modifiye<em&gt; In vitro</em&gt; Invasion Deneyi Hormonlar, Açan Kanser Hücre Invasion Sitokinlerin ve / veya Büyüme Faktörleri Potansiyel Rolü belirlemek için

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

Bir Kanseri Hücre Sfero Testi 3D ortamda Invasion Değerlendirmek için

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...