이 작품은 넉넉한 어린이 암 재단 (볼티모어, MD), 브랜든 리 캐링턴 재단 (실버 스프링, MD), DOD (W81XWH-10-1-0137)와 NIH (R01CA108641)에서 기금에 의해 지원되었다. 우리는 박사 안톤 Wellstein과 경험을 공유하고 제시된 방법을 최적화하기 위해 우리를 돕는 그의 실험실의 구성원을 감사드립니다.

1. 준비하기 <ol><li> 모든 단계는 조직 문화 후드에서 무균 조건 하에서 수행되어야한다. </li><li> xCELLigence 역은 5 % CO 2의 존재에 37 ° C 배양기에 배치됩니다. </li><li> 통로 6보다 바람직하게 낮은 통로 HUVEC 세포를 사용합니다. 또한, 세포가 더 이상 수확의 시간에 합류 % 75 이상 없는지 확인합니다. </li><li> HUVEC 세포는 성장 인자, 보충 교재 및 5 % 태아 소 혈청을 포함하는 EGM-2 총알 키트 (Lonza의 생물 과학)를 재구성 EGM - 2 미디어에서 재배된다. </li><li> 트립신의 모든 흔적은 200xg에서 세포를 회전 PBS로 한번 세척 및 표시 셀 밀도에게 현탁하여 세포 현탁액에서 제거해야합니다. </li><li> 37에서 1 시간 동안 젤라틴 0.1 %로 코트 xCELLigence E-플레이트 16 ° C 또는 하룻​​밤에 ° C. PBS로 접시를 씻고 100μL 재구성 EGM - 2 미디어를 추가 할 수 있습니다. 배경을 측정하는 xCELLigence 시스템에 빈 읽기를 수행세포의 부재 임피던스. </li></ol> 2. HUVEC 단층 생성 <ol><li> 트립신에 의한 HUVEC 세포의 하위 합류 플라스크를 수확. 2.5 X 10 5 세포 / ML의 최종 농도로 재구성 EGM - 2 미디어 세포를 resuspend. </li><li> 100μL EGM-2 미디어를 포함하는 E-플레이트 교정 배경의 각 웰에, HUVEC 세포 현탁액 (2.5 × 10 4 HUVEC 세포)의 100μL 추가 할 수 있습니다. </li><li> 즉시 xCELLigence 악기의 E-플레이트를 설치합니다. </li><li> 프로그램 xCELLigence 소프트웨어가 10 분 간격으로 임피던스 판독을 수행합니다. </li><li> 그들은 단일 층을 형성 때까지 내피 세포로 (그림 2) 셀 인덱스의 병합에 의해 입증 18-21 시간 동안 성장하자. </li></ol> 3. 셀 및 데이터 정규화를 침입의 추가. <ol><li> 일단 안정 HUVEC 단층은 최종 굴을 트립신과를 Resuspend으로 수확 종양 세포가 형성1 X 10 5 세포 / 종양 세포 (예 : RPMI 또는 DMEM 매체 10 % FBS를 포함)을 성장하는 데 사용 미디어 ML의 ITY </li><li> 일시 정지 임피던스 xCELLigence 소프트웨어를 판독하고 역에서 E-플레이트를 제거합니다. </li><li> 흡인에 의해 EGM-2 용지를 제거합니다. 1 × 10 4 세포를 포함하는 종양 세포 현탁액의 100μL를 추가합니다. 일반적으로, 종양 세포의 1:2.5 비율 : 내피 세포 시드, 분석에 가장 적합합니다. 그러나 비율은 각각의 세포주 최적화 할 수 있습니다. </li><li> 인큐베이터에있는 xCELLigence 시스템에 E-플레이트를 놓고 10 분 간격으로 임피던스 수치를 계속합니다. </li><li> 침공은 종양 세포에 침입하여 내피 세포 접합 및 침투의 수축에 의한 세포 지수의 하락으로 다음 6-12 시간 동안 실시간으로 모니터링 할 수 있습니다. </li><li> 종양 세포를 침입의 추가 시점에 결과를 정상화. xCELLigence 소프트웨어는 시점과 결과를 정상화 허용직접 소프트웨어 창에서 볼 수 있습니다. </li></ol> 4. 대표 결과 : 전이성 및 비 전이성 세포에 의해 HUVEC 단층 침공의 예는 그림 3에 나와 있습니다. K7M2는 전이성 골육종 세포주이다. 이러한 세포는이 세포주 13,14의 침략적 속성을 차지하고있는 골격 링커 단백질 ezrin의 높은 수준을 표현한다. HUVEC 세포가 K7M2 세포와 도전하면 6 시간 이내에 전기 저항의 저하가있다. 전기 저항이 드롭 침입 종양 세포에 의해 HUVEC 단층의 침공을 나타냅니다. K12 셀 라인은, 다른 한편으로는, ezrin 훨씬 낮은 수준을 표현 13 결과적으로 덜 전이성이다. 세포가 준수 또는 HUVEC 단층 (그림 3)를 통해 침입 할 수없는만큼 저항을 덜 가파른 드롭 K12 세포 결과 HUVEC 단층 도전. <imgSRC = &quot;/ files/ftp_upload/2792/2792fig1.jpg&quot;ALT = &quot;그림 1&quot;/&gt; 그림 1. 종양 세포에 의해 내피 세포의 침윤을위한 워크 플로우 프로세스의 개략도. HUVEC 세포는 젤라틴 코팅 E-접시에 도금과 합류 단층을 형성 할 수 있습니다. 단일 층을 형성하고 세포 지수는 내피 세포 단일 층의 침략으로 인해 변경으로 침공이 실시간으로 모니터링되면 악성 세포가 추가됩니다. <img src="/files/ftp_upload/2792/2792fig2.jpg" alt="그림 2"/> 그림 2. HUVEC 단층 형성. HUVEC 세포에 부착 젤라틴 코팅 금 전극에 확산으로 세포 지수의 변화. 합류 단층의 형성 18 개의 시간 후에 세포 색인의 안정화로 표시됩니다. <img src="/files/ftp_upload/2792/2792fig3.jpg" alt="그림 3"/> 그림 3. K7M2 및 K12 골육종 세포에 의해 HUVEC 세포의 침공. CEL의 가파른 감소L-지수는 높은 전이성 K7M2 세포의 도입 몇 시간 내에 발생합니다. K12 세포는 반면에, 덜 전이성하고 효율적으로 K7M2 세포와 같은 내피 세포 단일 층을 통과 할 수 없습니다. 이 HUVEC 단층은 K12 세포와 도전 셀 인덱스에서 덜 가파른 감소에 의해 표시됩니다. 컨트롤 암 세포의 부재에서 HUVEC 단층의 임피던스를 나타냅니다. 실험 세중의에서 수행 하였다.

<ol>
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이 논문의 출판 비용은 친절 로슈 응용 과학에 의해 제공되었다.

실시간 HUVEC의 침공 분석은 모니터링 침략을위한 간단하면서도 강력한 방법입니다. 그것은 엔드 포인트 분석에 의존하는 기존의 다른 기술에 비해 상당한 장점입니다 실시간으로 결과를 제공합니다. 분석은 시험 약물, 항체, 리간드와 전이 억제제 또는 촉진제로 단백질을 수정할 수 있습니다. 암 / 내피 세포 간 이야기에 다른 에이전트의 효과뿐만 아니라 평가 될 수있다. 이러한 문화 미디어의 성장 요인과 약물 등 외인성 에이전트를 도입 할 때, 그것은 그들이 HUVEC 단층의 무결성에 영향을주지 않도록하는 것이 중요합니다. HUVEC 단층의 파괴는 세포를 침입의 부재에서 외생 에이전트를 소개하고, 세포 지수의 변화가없는 것을 확인하여 테스트 할 수 있습니다. 따라서, 적절한 제어는 실험 설계의 일부로 포함되어야한다. 그들은 conf의 형태로 다른 세포 라인에서 여러 가지 셀 인덱스 곡선을 보여luent 단층. 곡선뿐만 아니라, 최대 셀 인덱스 달성의 형태는 셀 타입 별입니다. HUVEC 세포의 16-18 시간 후 이상 세포의 인덱스에 다른 안정화 한 다음, 4-6시간 파종 한 후 세포 색인의 평 평화 특성 과도을 보여줍니다. 따라서, 세포가 종양 세포의 추가 전에 적어도 18 시간 동안 합류 단층을 형성하게하는 것이 중요합니다. 세포 지수는 전극 / 세포 계면에서 이온 환경에 의존하기 때문에, 이러한 세포 형태와 세포 간 유착의 품질 등 다양한 요소가 적용 잘 하단의 영역뿐만 아니라, 세포 인덱스에 영향을 미친다. 따라서, 종양 세포의 침략에 발생하는 셀 인덱스의 감소, 내피 세포 접합 및 후속 종양 세포 침공의 철회를 포함 여러 가지 요인의 함수이다. xCELLigence 악기는 세 가지 다른 구성으로 제작되어 실시간으로 세포 분석기(RTCA) SP, RTCA MP 및 RTCA DP. RTCA MP 악기는 96 - 웰 E-플레이트를 동시에 여섯까지 저장할 수있는 반면 RTCA SP 악기 하나 96 - 웰 E-플레이트를 보유하고 있습니다. 이 마약과 화합물의 높은 처리량 검사 할 수 있습니다. 이 문서에 나와있는 실험은 3 개의 16도 E-접시를 보유 할 수 있습니다 RTCA DP 악기를 사용하여 수행됩니다. 각 E-플레이트 보유 스테이션은 독립적으로 여러 사용자가 동시에 실험을 실행할 수있는 운영 할 수있다. RTCA DP 악기는 또한 CIM 플레이트를 사용하여 마이그레이션을 연구하는 데 사용할 수 있습니다. 이들은 8um의 평균 기공 크기와 폴리에틸렌 테레 프탈레이트 (PET) 막으로 구분 상단 및 하단 챔버 16 - 웰 플레이트입니다. 전자 센서는 상부 챔버의 다공성 멤브레인의 아래쪽에 있습니다. 하부 챔버의 상부 챔버의 세포에 대한 화학 주성을위한 저수지 역할을합니다. 그러나 CIM 플레이트에 HUVEC 침공 분석의 주요 장점은 이전에 종양 세포 침공 전내피 세포의 침공은 종양과 내피 세포 사이의 크로스 토크에 달려들, 기공 크기에 의해 제한되지 않습니다. HUVEC 침공 분석도 응용 생물 물리학 (트로이, NY)에 의해 제조 ECIS Z 기기에서 수행 할 수 있습니다. ECIS Z 악기 배열 전극 구성의 차이, xCELLigence 악기와 유사한 기술을 사용합니다. 우리는 성공적으로 8 잘 ECIS 배열을 사용하여 HUVEC 침공 분석을 수행했습니다. 2.5 × 10 5 1 X 10 5 세포를 각각 : 그것은 잘 바닥 면적 도금으로 내피와 종양 세포의 큰 숫자를 요구하는 ECIS 배열에있는 큰 점에 유의하는 것이 중요합니다.

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr> 
 <tr>
 	<td>xCELLigence RTCA DP station</td>
 <td>Roche	</td>
 <td>05469759001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>RTCA control unit</td>
 <td>Roche</td>
 <td>05454417001</td>
 <td>Notebook with preinstalled RTCA software</td>
 </tr>
 <tr>
 	<td>E-Plate 16</td>
 <td>Roche</td>
 <td>05469830001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>HUVEC cells</td>
 <td>Lonza</td>
 <td>CC-2517</td>
 <td></td>
 </tr>
 <tr>
 	<td>EGM-2 media</td>
 <td>Lonza</td>
 <td>CC-3156</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>EGM-2 bullet kit</td>
 <td>Lonza</td>
 <td>CC-4176</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>0.1% Gelatin in 
sterile H2O
</td>
 <td>Millipore</td>
 <td>MCPROTO045</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

a real-time electrical impedance based technique to measure invasion of endothelial cell monolayer by cancer cells

악성 세포의 전이성 확산은 호스트에서 먼 사이트에 교통 수단으로 혈류를 입력 할 내피 층을 지하 막, 혈관 내피 세포에 종양 세포의 부착, 혈관 접합의 철회 및 종양 세포의 마지막 침략과 이동의 저하를 필요로 1-3. 일단 순환 시스템, 암 세포 벽을 모세와 전이성 종양 4,5 형성하는 주변 조직에 extravasate을 준수합니다. 종양 세포 - 내피 세포 상호 작용의 다양한 구성 요소는 암 세포와 인간 제대 정맥 내피 세포 (HUVEC)의 단층에 도전하여 체외에서 복제 할 수 있습니다. 전자 및 위상 대비 현미경으로 수행 연구의 이벤트 체외 순서에서 상당히 생체 전이성 과정 6을 대표하는 것이 좋습니다. 여기, 우리는 모니터링 및 실시간 invas의 정량화 전기 임피던스 기반 기술을 설명악성 종양 세포에 의해 내피 세포의 이온. Giaever 및 Keese 먼저 세포의 인구는 전극 7,8의 표면에서 자랄 때 임피던스의 변동을 측정하는 방법을 설명합니다. 로슈에 의해 제조 xCELLigence 악기, 세포가 잘 하단에있는 면적의 약 80 %를 커버 골드 미세 전극 배열로 덮여 문화 접시에 부착 확산으로 전기 임피던스 변화를 측정하기 위해 유사한 기술을 사용합니다. 세포 부착 및 전극 표면에 확산으로, 그것은 9-12 전기 임피던스의 증가로 연결됩니다. 임피던스는 세포에 의해 덮여 잘 조직 문화의 총 면적에 비례 차원 매개 변수 되나 세포 인덱스로 표시됩니다. 따라서, 세포 지수는 세포 부착, 확산, 형태학 및 세포 밀도를 모니터링하는 데 사용할 수 있습니다. 이 문서에서 설명 침공 분석은 변화에 근거전극 / 세포 계면에서의 전기 임피던스, 악성 세포의 인구로 HUVEC 단층 (그림 1)를 통해 침입. 내피 분기점의 장애, 종양 세포에 의해 내피 세포의 단층 및 교체의 철회는 임피던스 큰 변화로 이어집니다. 이러한 변화는 직접적으로 종양 세포의 침입 용량 상관 관계, 즉, 매우 공격적인 세포에 의해 침략 세포 임피던스와 그 반대의 큰 변화로 이어집니다. 내피 세포 종양 세포의 상호 작용이 더 밀접하게 생체 과정에서 모방, 2) 데이터의 실시간 얻을 수있다 : 1)이 기술은 보이든 챔버와 마트 리젤의 분석으로 침략을 측정하는 기존의 방법에 비해 두 가지 이점을 제공합니다 시간과 같은 다른 방법에 대한 엔드 포인트 분석이 아닌, 더 쉽게 정량화 할 수있다.

Watch this Scientific Journal Video about A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells at JoVE.com

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

이 문서에 설명<em&gt; 체외에서</em내피 세포의 단일 층을 통해 침입 감시 암 세포&gt; 기술. 데이터가 잘 하단에 전극의 표면 임피던스의 변화의 함수로 실시간으로 획득됩니다.

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 ...

a-real-time-electrical-impedance-based-technique-to-measure-invasion

Research

JoVE Journal

Medicine

27.2K 조회수.  Georgetown University. 이 문서에 설명 체외에서 기술. 데이터가 잘 하단에 전극의 표면 임피던스의 변화의 함수로 실시간으로 획득됩니다.

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

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Intravital 이미징을 사용하여 실시간으로 종양의 Nanoparticle의 이해 평가

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

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의학

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

구강 편평 세포 암종의 Orthotopic 마우스 모델에서 종양 세포 침략의 다중 광자 이미징

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

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.

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

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 ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

배양 세포에서 암과 연관된 신호 전달을 심문위한 멀티 플렉스 루시 페라 기반 스크리닝 플랫폼

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

정량 실시간 PCR을 사용하여 뇌척수액의 마이크로 RNA 프로파일

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

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 ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

실시간으로 중피 세포의 난소 암 회전 타원체의 부착과 침략의 평가

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

3 차원 (3D) 종양 회전 타원체 침공 분석

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

다중 매개 변수 형광 활성화 된 셀 정렬하여 인간의 림프 내피 세포의 분리

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...