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).

<ol>
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The authors declare that they have no competing financial interests.

たPHFの準備中に、断片は懸濁液中に滞在しないかもしれないが、血管壁に付着します。これは、培養培地の体積を増加させることによって克服することができます。たPHFの数が少なすぎるとそのサイズが大きすぎる場合には、培地の容積を減少させます。集約する試験細胞の障害は、温度または微生物感染に対する変動に起因する可能性があります。あるいは、集約することができないこと?...

侵略は、悪性腫瘍の特徴です。この活動は、周囲の組織の破壊につながるだけでなく、転移形成に関与しているだけでなく。癌患者は、浸潤および転移によって死亡し、効率的な抗侵襲治療はまだ不足しているので、腫瘍細胞の浸潤を模倣する実験用アッセイが開発されています。ニワトリ心臓アッセイの目的は、三次元で腫瘍浸潤を研究するための関連する器官培養方法を提供することです。アッセイは、侵襲的および非侵襲的な細胞とを区別し、腫瘍浸潤に対する試験化合物の効果を研究することを可能にすることができます。 アッセイの使用の理論的根拠は、腫瘍は、腫瘍細胞が継続的に間質（宿主細胞と細胞外マトリックス）と相互作用し、かつ、これらの分子相互作用を介して侵入を微調整1である生態系であり、実際の概念です。そのため、アッセイにおいて腫瘍細胞をliviに直面しています腫瘍細胞による浸潤のための基質として、また間質細胞とマトリックス要素の異なるタイプの供給源として働くだけでなく、ngのニワトリ胚の心臓断片2、。ニワトリの心臓は、筋細胞、線維芽細胞および内皮細胞を含む、細胞外マトリックスはラミニン、フィブロネクチンおよびコラーゲンの異なる種類で構成されています。このように、三次元器官培養技術は、患者の腫瘍の浸潤に関与する多くの細胞および分子間相互作用を覆います。 ニワトリの心臓アッセイの主な利点は、間質効果の実装です。この局面は、基底膜3または間質マトリックス4分子からなる非生物ゲルに腫瘍細胞の浸潤に基づいて、in vitroで他の浸潤アッセイよりも完全です。器官培養実験に見られるように、腫瘍細胞と正常な生体宿主組織との間の対立の概念は、いくつかによって導入されましたイギリス6、およびドイツ7のシュライヒでウルフとシュナイダーフランス5で、イースティとイースティ含む作者。引用された方法に比べて、ニワトリの心臓浸潤アッセイの二つの技術的利点は、フラグメントの量を簡単に標準化することができること、およびそれらが器官培養中に機能の完全性の監視を可能に収縮、残っていることです。それらは容易に卵の無菌内容から切除することができるのでさらに、鳥類の胚が好ましいです。アッセイは、腫瘍細胞に周囲の複雑な間質を提供することにより、ニワトリ漿尿膜の膜アッセイ8に概念的な類似性を有しています。 アッセイは、正常にかかる9およびHCT-8（大腸）10細胞株ファミリーMCF-7（乳房）と同様のヒト腫瘍の浸潤性および非浸潤性細胞の変異体を区別するために適用されています。技術は、同様に、潜在的に抗浸潤化合物を試験するのに有用です<suP&gt; 11,12。さらに説明されるように、それは小有機分子の構造 - 活性関係を確立するために使用することができます。アッセイは、しかし、アカウントに癌浸潤に対する免疫学的細胞の寄与を取ることはありません。それは技術があるため操作の数が多い、検定実行の限られた数（最大30文化）と長いターンアラウンドタイム（約1ヶ月）の、ハイスループット分析システムとみなすことはできないことを強調すべきです。

<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

ニワトリ心臓アッセイの目的は、三次元で腫瘍浸潤を研究するための関連する器官培養方法を提供することです。アッセイは、侵襲的および非侵襲的な細胞とを区別し、腫瘍浸潤に対する試験化合物の効果の研究を可能にすることができます。癌細胞は - のいずれかの凝集体または単一細胞として - ニワトリ胚の心臓の断片に直面しています。数日または数週間のための懸濁液中の器官培養した後に直面している培養物を固定し、組織学的分析のためにパラフィンに包埋しました。癌細胞と正常組織との間の三次元相互作用は、その後、ヘマトキシリン​​ - エオシンで、または心臓組織内のエピトープまたは対向癌細胞に対する免疫組織化学的染色後の染色連続切片から再構築されます。アッセイは、癌浸潤は、癌細胞とその隣接する間質ホスト要素（筋線維芽細胞、endoth間の分子間相互作用の結果であることを最近の概念と一致していますelial細胞、細胞外マトリックス成分、等）。ここで、この間質環境は、生体組織片などの癌細胞に提供されます。アッセイの妥当性への支援の側面が複数あります。時間と宿主組織の空間の漸進的占領と交換、および直面細胞の生体内での侵襲性および非侵襲性は、一般的に、アッセイの結果と相関する：アッセイにおける侵入は癌浸潤の基準に従うものです。さらに、 インビボでの細胞の浸潤パターンは、病理学者によって定義されるように、アッセイでの組織学的画像に反映されています。多数の潜在的抗浸潤有機同族化合物を用いて得られた結果の定量的構造活性関係（QSAR）分析は、診療所で使用されるフラボノイドおよびカルコン、および既知の抗転移薬のための構造-活性関係（ 例えば、微小管阻害剤の研究を可能にしました）ならびにアッセイに侵入を阻害します。 HoweveR、アッセイは、アカウントに癌浸潤に対する免疫学的貢献を取ることはありません。

図5に示すように、組織切片は、成功した多数のアッセイの最終結果を示しています。培養物の組織学的音、生細胞を示し、腫瘍細胞と正常組織との間の相互作用を解析することを可能にします。さらに、正常組織からの免疫反応は、免疫拒絶システムが開発される前に、例えば、使用されるニワトリ胚の正確な年齢を確認する、観察することができません。培養期間中（グロス）の汚...

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

ここでは、三次元での正常組織片をリビングへの腫瘍細胞の浸潤を研究するためのプロトコルを提示します。この器官培養技術は、主にin vitroで 、潜在的に抗浸潤薬をテストするために適用されます。

テストのためのひよこ心臓浸潤アッセイ癌細胞の浸潤性および潜在的に抗浸潤化合物の活性

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 ビュー.  University of Ghent. ここでは、三次元での正常組織片をリビングへの腫瘍細胞の浸潤を研究するためのプロトコルを提示します。この器官培養技術は、主にin vitroで 、潜在的に抗浸潤薬をテストするために適用されます。 

ビデオ: 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.

上に移動: シンプルかつ柔軟なIn Vitro三次元侵略試金のプロトコル

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

がん研究

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.

癌細胞による内皮細胞単層の侵入を測定するリアルタイム電気インピーダンス基づく手法

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

医学

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.

モデリングとイメージング、3次元の集団細胞浸潤

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.

住セルイメージ投射アッセイ研究神経膠腫脳腫瘍幹細胞の遊走と浸潤に

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.

腫瘍性細胞行動の尺度としてのインビトロ侵血アッセイにおけるマウス乳腺腫瘍細胞の使用

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 ...

遺伝学

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;

がん細胞の移動と浸潤のインピーダンスベースリアルタイム測定

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.

迅速かつ定量的なチック絨毛管内膜モデルを用いた転移性調節因子の発見

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.

アン試験管内でフルオロブロックの腫瘍の浸潤アッセイ

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 ...

生物学

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.

変更された<em&gt;インビトロ</em媒介する癌細胞浸潤におけるホルモン、サイトカインおよび/または成長因子の潜在的な役割を決定するために&gt;浸潤アッセイ

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.

3Dの設定で侵略を評価するために、癌細胞スフェロイドアッセイ

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