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.

PHFs의 제조 동안, 단편 현탁액에 체류하지 않을 수도 있지만, 혈관 벽에 부착; 이는 배양액의 부피를 증가시킴으로써 극복 될 수있다. PHFs 수가 너무 낮고, 그 크기가 너무 큰 경우, 배양액의 부피를 감소시킨다. 집계 테스트 세포의 실패는 온도 나 미생물 감염에 변동 때문일 수 있습니다. 또한, 집계 무능력 세포의 고유 한 특성 일 수있다. PHF에 골재의 부착 동안에, 접착 불량은 반고체 한천 배지 ?...

침략은 악성 종양의 특징이다. 이 활동은 주변 조직의 파괴에 이르게뿐만 아니라 전이 형성에 연루되어 아닙니다. 암 환자가 침윤 및 전이, 효율적인 안티 침습적 치료법으로 사망 때문에 여전히 종양 세포의 침윤이 개발되었다 모방 부족한 실험실 분석이다. 병아리 심장 분석의 목적은 입체적 종양 침윤을 연구하는 중요한 장기 배양 방법을 제공하는 것이다. 분석은 침습적 및 비 침습적 세포를 구별하고, 종양 침윤에 대한 시험 화합물의 효과를 연구 할 수있다. 분석의 사용 배후의 이론적 근거는 종양은 종양 세포가 지속적으로 기질 (숙주 세포 및 세포 외 기질)과 상호 작용 생태계 있다는 실제적인 개념이며, 이들 분자 상호 침입 통해 그 미세 조정 (1)이다. 그래서, 분석에서 종양 세포 livi 직면종양 세포에 의해 침윤 용 기판으로서, 또한 간질 세포와 행렬 요소의 다른 유형의 소스로서 역할뿐만 NG 병아리 배아 심장 단편 2. 병아리 심장 근육 세포, 섬유 아세포 및 내피 세포를 포함하고, 세포 외 매트릭스는 라미닌, 피브로넥틴 및 콜라겐의 상이한 유형으로 구성된다. 이와 같이, 삼차원 기관 배양 기술은 환자의 종양 침윤에 관련 많은 세포와 분자의 상호 작용을 포함한다. 병아리 심장 분석의 주요 장점은 간질 효과를 구현 한 것이다. 이 측면은 기저막 3 간질 매트릭스 4 분자 구성이 아닌 생활 젤로 종양 세포 침공을 기반으로 체외에서 다른 침략 분석보다 더 완료됩니다. 장기 배양 실험에서 발견 된 바와 같이, 종양 세포 및 정상 생활 숙주 조직 사이의 대결의 개념은 여러 가지로 도입 된독일 7 울프와 슈나이더 프랑스 5, 영국 6 Easty과 Easty 및 Schleich을 포함하여 저자. 위에 인용 된 방법 병아리 심장 침윤 분석의 기술적 장점은 두 조각의 양이 용이하게 표준화 될 수 있도록, 그들은 기관 배양 물 중에 기능적 무결성 모니터링을 허용 수축성 남아 있다는 것이다. 그들은 쉽게 계란의 멸균 콘텐츠 해부 될 수 있기 때문에 또한, 조류 배아 바람직하다. 분석은 종양 세포로 둘러싸 복잡한 기질을 제공하여 병아리 chorioallantois 막 세이 8 컨셉 유사성을 갖는다. 분석은 성공적 9 및 HCT-8 (결장) 10 세포주 패밀리 MCF-7 (유방)과 같은 인간의 종양 침입 및 비 침습성 세포 변형을 구별하기 위해 적용되어왔다. 기술뿐만 아니라 잠재적으로 항 - 침습 화합물을 테스트하는 데 유용 <suP&gt; (11, 12). 상기 한 바와 같이, 그것은 작은 유기 분자의 구조 - 활성 관계의 확립을 위해 사용될 수있다. 분석은, 그러나, 계정으로 암 침공에 면역 세포의 기여를 고려하지 않습니다. 이는 기술이 때문에 조작의 높은 번호, 분석 런 제한된 숫자 (최대 30 배양)과 긴 턴어라운드 시간 (약 1 개월)의, 높은 처리량 분석 시스템으로 간주 될 수 없다는 것을 강조한다.

<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

병아리 심장 분석의 목적은 입체적 종양 침윤을 연구하는 중요한 장기 배양 방법을 제공하는 것이다. 분석은 침습적 및 비 침습적 세포를 구별하고, 종양 침윤에 대한 시험 화합물의 효과에 대한 연구를 가능하게 할 수있다. 암 세포 - 중 집계 또는 단일 세포로 - 배아 병아리 마음의 조각에 직면하고 있습니다. 며칠 또는 몇 주 동안 정지에서 기관 배양 후 직면 문화는 고정 및 조직 학적 분석을 위해 파라핀에 포함. 암세포 및 정상 조직 사이의 입체적인 상호 작용은 다음 헤 마톡 실린 - 에오신으로 또는 심장 조직의 에피토프 또는 대향하는 암 세포에 대한 면역 조직 화학 염색을 한 후 염색 직렬 섹션에서 재구성된다. 분석은 암세포 침윤이 암세포 및 그들의 이웃 기질 호스트 소자 (근육 섬유 모세포, endoth 분자 사이의 상호 작용의 결과 인 것으로 최근의 개념과 일치elial 세포, 세포 외 매트릭스 성분 등). 여기서,이 기질은 환경 생체 조직 절편 같은 암세포에 제공된다. 분석의 관련성에 대한 지원은 여러 양태이다. 프로그레시브 직업 및 교체 시간과 숙주 조직의 공간 및 침입과 대향 세포의 생체 내에서 비 - 침입 일반적 분석의 결과와 상관 관계 : 분석에서 내습 암 침윤의 기준에 따른다. 또한, 생체 내에서 세포의 침입 패턴은 병리학 자에 의해 정의 된 바와 같이, 분석에서 조직 학적 이미지에 반영된다. 많은 잠재적으로 항 - 침습 유기 동족체 화합물과 얻어진 결과의 정량 구조 - 활성 관계 (QSAR) 분석은 임상에서 사용되는 플라보노이드 및 칼콘, 공지 된 항 - 전이성 약물 구조 - 활성 관계 (예를 들어, 미세 소관 억제제의 연구를 허용 )뿐만 아니라 분석에서 침입을 억제한다. HoweveR, 분석은 암 침공 계정 면역 학적 기여를 고려하지 않습니다.

도 5에 제시된 조직 학적 단면은 성공적인 분석의 숫자의 최종 결과를 나타낸다. 문화의 소리 조직학가 가능한 세포를 표시하고 종양 세포와 정상 조직 사이의 상호 작용을 해석 할 수 있습니다. 또한, 정상 조직에서 어떠한 면역 반응은 면역 거부 시스템이 개발되기 전에, 예를 들어 사용 된 병아리 배아의 정확한 기간을 확인하는 관찰 할 수 없다. 배양 기간 동안 (총) 오염의 부재를 나타?...

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

여기서는 입체적 정상 조직 절편에 살아있는 종양 세포의 침윤을 연구 프로토콜을 제시한다. 이 기관 배양 기술은 주로 시험 관내에서 잠재적으로 항 - 침습 약물을 시험에 적용된다.

테스트를위한 병아리 심장 침략 분석 암 세포의 침윤과 잠재적 안티 침략 화합물의 활동

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 조회수.  University of Ghent.  여기서는 입체적 정상 조직 절편에 살아있는 종양 세포의 침윤을 연구 프로토콜을 제시한다. 이 기관 배양 기술은 주로 시험 관내에서 잠재적으로 항 - 침습 약물을 시험에 적용된다. 

비디오: 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.

위쪽으로 이동: 간단 하 고 유연한 생체 외에서 3 차원 침공 분석 실험 프로토콜

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

연구

암 연구학

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.

암 세포에 의해 내피 세포 단층의 침략을 측정하는 실시간 전기 임피던스 기반 기술

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

의학

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF - β - 유발 침략을 측정하는 회전 타원체 분석

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

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.

모델링 및 3 차원 이미징 단체 셀 침략

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

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.

종양 발생 세포 행동의 척도로 시험관 내 침략 분석에서 마우스 유방 종양 세포의 사용

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 ...

유전학

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.

신속하고 정량적인 병아리 Chorioallantoic 멤브레인 모델을 사용하여 전이성 레귤레이터의 발견

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

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of cancer cell populations typically generate a single endpoint measurement that does not distinguish between cancer cell subpopulations with different invasive potential. Also, the tumor microenvironment consists of different resident stromal and immune cells that alter and participate in the invasive behavior of cancer cells. Invasion into tissues also plays a role in immune cell subpopulations fending off microorganisms or eliminating diseased cells from the parenchyma and endothelial cells during tissue remodeling and angiogenesis. Real-Time Cellular Analysis (RTCA) that utilizes impedance biosensors to monitor cell invasion was a major step forward beyond endpoint measurement of invasion: this provides continuous measurements over time and thus can reveal differences in invasion rates that are lost in the endpoint assay. Using current RTCA technology, we expanded dual-chamber arrays by adding a further chamber that can contain stromal and/or immune cells and allows measuring the rate of invasion under the influence of secreted factors from co-cultured stromal or immune cells over time. Beyond this, the unique design allows for detaching chambers at any time and isolating of the most invasive cancer cell, or other cell subpopulations that are present in heterogeneous mixes of tumor isolates tested. These most invasive cancer cells and other cell subpopulations drive malignant progression to metastatic disease, and their molecular characteristics are important for in-depth mechanistic studies, the development of diagnostic probes for their detection, and the assessment of vulnerabilities. Thus, the inclusion of small- or large-molecule drugs can be used to test the potential of therapies that target cancer and/or stromal cell subpopulations with the goal of inhibiting (e.g., cancer cells) or enhancing (e.g., immune cells) invasive behavior.

We describe an approach to detect and capture invasive cell subpopulations in real-time. The experimental design uses Real-Time Cellular Analysis by monitoring changes in the electric impedance of cells. Invasive cancer, immune, endothelial or stromal cells in complex tissues can be captured, and the impact of co-cultures can be assessed.

공동 배양물로부터의 침습적 세포 하위집단의 실시간 검출 및 포획

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of ...

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.

체외에서 FluoroBlok 종양의 침략 분석

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 ...

생물학

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.

수정<em&gt; 시험관</em&gt; 침공 분석은 호르몬, 중재 암 세포 침략의 사이토 카인 및 / 또는 성장 인자의 잠재적 인 역할을 확인하는

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.

암 세포 회전 타원체 분석은 3D 설정에 침략을 평가하기 위해

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