우리는 하버드 의과 대학에서 니콘 이미징 센터, 특별히 제니퍼 워터스, 라라 Petrak과 웬디의 연어, 훈련과 timelapse의 현미경의 사용을 감사드립니다. 우리는 또한 가치있는 토론을 위해 로자 잉과 Achim Besser 감사드립니다. 이 작품은 NIH 그랜트 5,695,837 (M. Iwanicki까지) 및 JSB에 GM064346에 의해 지원되었다; 박사 미리암과 셀던 G. Adelson 의학 연구 재단 (JSB까지)에서 교부금에 의해.

1. 난소암 세포 회전 타원체 형성 <ol><li> RFP-표현하는 난소암 세포는 10 %베이스 중간 (199과 MCDB105의 50:50 혼합물 10 % inactivated 태아 소 혈청과 1% 펜 strep를 포함하는 사용자 정의 세포 배양 매체)에서 배양해 있습니다. 레이블이 지정되지 않은 난소암 세포에 RFP를 표현하기 위해 플라스미드 포함된 RFP와 세포를 transfect 및 RFP를 표현하는 세포를 위해 선택합니다. 또는 바이러스성 벡터는 transiently 형광 단백질을 표현하는 데 사용할 수있는, 또는 세포에 빨간색 형광 세포 추적기 염료 (Invitrogen)로 사전 품어 될 수 있습니다. </li><li> 이전 난소암의 spheroids의 형성에, 그것은 낮은 접착 96 잘 둥근 바닥 문화 요리를 준비할 필요가 있습니다. , 낮은 접착 배양 접시를 생산하기 위해서 30μl 폴리-HEMA 용액 (1 ML 95% EtOH에서 6mg polyhydroxyethylmethacrylate)는 96 잘 코닝 세포 배양 접시의 각도에 추가됩니다. 96 잘 플레이트는 ° C 비 humidified 인큐베이터는 에탄올, 내가 증발시키는 37에 incubated 있습니다각도에 폴리-HEMA의 영화를 eaving. 이 폴리 - HEMA 영화는 세포 현탁액 18 년 성장 강요, 우물의 바닥에 부착로부터 세포를 방지할 수 있습니다. [또는 초저 첨부 파일 문화 플레이트 (코닝)는 폴리-HEMA 코팅 요리 대신 사용할 수 있습니다.] </li><li> 낮은 접착 배양 접시를 준비 후, 난소암 세포, 펠릿 3 분 동안 900 RCF의 탁상 원심 분리기 (Heraeus)의 세포가 뜨는을 대기음와 10 %의 기본 매체에서 다시 중단 한 접시를 trypsinize. </li><li> hemocytometer를 사용하여 세포를 세어보세요. </li><li> 10 %의 기본 중의 50μl 당 100 세포가있다는 것을 이러한 세포의 농도를 조정합니다. </li><li> 96 잘 폴리 - HEMA 코팅 문화 요리의 각도에 균일하게 정지 희석 세포 현탁액의 50μl를 추가합니다. </li><li> 16시간위한 37 ° C 세포 문화 인큐베이터 (시간이 금액은 그것을 위해 걸리는 시간의 양에 따라 증가하거나 감소해야합니다에서 96 잘 접시를 품어각도에 하나의 다세포 회전 타원체를 형성, 함께 클러스터에 난소암 세포를 허용하도록 다세포 spheroids하거나 원하는 실험 조건)을 형성하는 특정 세포 라인. 일부 종양 세포는이 기간 동안 apoptosis을 받다 수 있으니 사전 apoptosis의 유도에 시간을 선택하는 것이 중요합니다. </li></ol> 2. Mesothelial 세포 단일층 형성 <ol><li> 세포 배양 후드에서 미리 코팅 접시의 각도에 5μg fibronectin / ML PBS 솔루션의 2mL를 추가하고 30 분간 실온에서 잠복기에 의한 fibronectin과 6 잘 유리 바닥 MatTek 요리의 우물. MatTek 요리의 유리 바닥의 광학 품질은 고해상도 현미경 이미징을위한 수 있습니다. </li><li> GFP-표현 mesothelial 세포는 10 %의 기본 매체에서 배양해 있습니다. mesothelial 세포 판을 Trypsinize, 3 분 동안 900RPM에서 탁상 원심 분리기 (Heraeus)에 다운 스핀 뜨는을 대기음, 10 %의 자료 매체에 다시 일시 중지합니다.여기서 사용 mesothelial 세포들이 취득 당시 이미 GFP를 표현했지만 레이블이 지정되지 않은 mesothelial 세포 cDNA 플라스미드 함유 GFP와 transfecting, 또는 녹색 형광 세포 추적기 염료 (Invitrogen)에 전지를 preinc​​ubating에서 생산 할 수 있습니다. </li><li> 30 분 fibronectin 보육 (단계 2.1) 후, 2mL PBS로 MatTek 요리의 우물을 씻는다. </li><li> 6 잘 MatTek 요리의 각 우물에 잘 당 PBS 및 플레이트 6 x10 5 mesothelial 세포를 기음. mesothelial 세포 접시에 연결하고 단일층를 형성하도록하여 야간 37 ° C 세포 문화 인큐베이터에서 MatTek 요리를 품다. </li></ol> 3. Mesothelial 세포 정리 분석 <ol><li> 96 잘 폴리 - HEMA 코팅 접시에서 난소 암 spheroids를 수집 pipet을 사용하십시오. </li><li> mesothelial 세포 단일층를 포함하는 6 자 MatTek 요리는 잘 하나에서 매체를 기음. 2mL PBS로 한 번 씻는다. 96 잘 P로부터 spheroids 모든 추가MatTek 요리 (몇 군데되지 않습니다 접시 부분에 spheroids 착륙 계정에 몇 군데 실거 spheroids의 ~ 3 배 번호)을 잘 하나 늦었어. </li><li> 최소한 8 시간의 기간 동안 촬영된 영상을 수행할 수 거꾸로 widefield 형광 현미경의 무대에 MatTek 요리를 놓습니다. 한 실험에 여러 회전 타원체 윤일 이벤트와 함께 접시에있는 이미지를 여러 위치로 전동식 스테이지를 사용하십시오. 우리는 통합된 완벽한 초점 시스템과 낮은 [20x-0.75 수치 조리개 (NA)] 배율 / 없음 차동 간섭 대비 (DIC) 광학과 니콘 티-E 바뀌 동력 Widefield 형광 시간 경과 현미경을 사용하여 0.52 NA와 니콘의 할로겐 transilluminator 긴 작동 거리 (LWD) 콘덴서, 니콘 빠르고 (&lt;100 밀리초 스위칭 시간) 여진과 방사 필터 (GFP 예 40분의 480, 엠 50분의 525, RFP-mCherry 예 50분의 575 엠 50분의 640), 셔터 빠르게 전송 그리고 epifluorescence 빛의 경로 스마트 종료ters, 니콘 선형 인코딩된 동력 스테이지, 하마 오카-AG는 전하 결합 소자 (CCD) 카메라, 온도 및 CO 2 제어의 맞춤식 현미경 보육 챔버, 니콘 NIS-요소 AR 소프트웨어 버전 3을 냉각하고, TMC 진동 절연 테이블. </li><li> 난소암 세포 spheroids은 접시의 바닥에 정착하고 mesothelial 세포 단일층에 연결됩니다. 8 시간 GFP, RFP 및 20 단계 이미지 + 회전 타원체 / 단일층 상호 작용을, 10 분마다를 수집합니다. </li><li> RFP-표현하는 난소암 세포 spheroids은 단일층에 구멍을 만드는 GFP-표현 mesothelial 세포 단일층로 침공합니다. 8시간 후에는 요소 소프트웨어 (또는 이미지 J 같은 다른 적합한 소프트웨어)를 사용하여 GFP 이미지의 검은 구멍을 추적하여 구멍의 크기를 측정합니다. 시간 제로 해당 RFP 이미지의 회전 타원체의 크기로 8 시에 구멍 크기를 나누어 초기 회전 타원체 크기로 구멍 크기를 정상화. 이 예에서는충분한, 구멍의 크기는 한 번만 측정되었지만, 그것은 더 윤달의 역학을 이해하는 데 8 시간 실험을하는 동안 여러 번 측정할 수있다. </li></ol> 4. 대표 결과 이 예제에서, 우리는 OVCA433 spheroids를 제어하는​​ talin-1의 감쇠 표현을 가지고 OVCA433 난소암 세포 spheroids의 mesothelial 통관 능력을 비교했다. 각 그룹에서 OVCA433 spheroids는 ZT mesothelial 세포 monolayers을 포함 MatTek 요리가 추가되었습니다. 각 그룹에서 여​​섯 spheroids은 8 시간 동안 매 10 분 (그림 4, 영화 3, 영화 4) 몇 군데 있었다. 확산 spheroids하여 단일층에서 생산되는 구멍 측정되었으며 각 그룹에서 6 순위는 평균되었다. talin 한 최저의 spheroids 만든 평균 통관 영역 제어 spheroids 만든 평균 면적보다 훨씬 작은 것을 그림 4는 쇼, talin는 O에 의해 mesothelial 통관을 위해 필요하다고 제안VCA433 난소 암 spheroids. <img alt="그림 1" src="/files/ftp_upload/3888/3888fig1.jpg" /> 그림 1. 난소 암 전이. 기본 난소 종양은 난소 표면 상피 또는 나팔관에서 두 개발할 수 있습니다. 종양 세포 / 클러스터는 일차 종양에서 끊다과 복막 구멍에 수집합니다. 종양 세포는 다음 다세포 spheroids을 형성 종합 수 있습니다. Spheroids 후 복막 캐비티에 즐비한 mesothelial 세포 monolayers에 첨부합니다. mesothelial 세포 spheroids은 기본 지하 막에 액세스할 수 있도록 첨부된 난소암의 회전 타원체 수렁에서 제외됩니다. 영화 1. 난소 암 전이 있습니다. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_1_Lower_Quality.mp4">동영상을 보려면 여기를 클릭하십시오</a> . <img alt="그림 2" src="/files/ftp_upload/3888/3888fig2.jpg" /> 그림 2. </str긴 사연이&gt; Mesothelial 세포 라인 인간 복막 조직의 표면과 난소암 세포를 이식 수렁에서 제외됩니다. <img alt="그림 3" src="/files/ftp_upload/3888/3888fig3.jpg" /> 그림 3. Mesothelial 정리 분석. 난소 암 spheroids는 100을 잠복기에 의해 형성되는 RFP-표현 16시간위한 37 ° C에서 폴리 - HEMA 코팅 96 잘 둥근 바닥 배양 접시에 잘마다 난소암 세포를. 폴리-HEMA는 세포 현탁액에 남아 당 잘 단일 클러스터를 형성하기 위해 서로에 충실 수 있도록 문화 접시에 부착로부터 세포를 방지할 수 있습니다. Mesothelial 세포 monolayers는 fibronectin 코팅 여섯 잘 MatTek 요리에 잘 당 6x10 5 mesothelial 세포를 도금 및 16 시간 동안 37 ° C에서 번호판을 잠복기에 의해 준비가되어 있습니다. spheroids 그런 다음 mesothelial 단일층로 MatTek 요리로 전송되고 두 세포 집단은 니콘 티-E 나를 사용하여 8 시간 10 분 간격으로 몇 군데있다nverted 동력 Widefield 형광 촬영된 현미경과 요소 소프트웨어. 영화 2. Mesothelial 정리 분석가. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_2_lower_quality.mp4">동영상을 보려면 여기를 클릭하십시오</a> . <img alt="그림 4" src="/files/ftp_upload/3888/3888fig4.jpg" /> OVCA433 spheroids의 talin 한 표현의 그림 4. 감쇄량은 mesothelial 통관 능력을 감소시킵니다. 과 및 talin 1 감쇠 표현없이 OVCA433 spheroids (빨간색)은에 연결하고 ZT mesothelial 단일층 (녹색)으로 나눠야 허용되었다. 두 세포 집단은 니콘 티-E 바뀌 동력 Widefield 형광 촬영된 현미경과 요소 소프트웨어를 사용하여 8 시간 10 분 간격으로 몇 군데 있었다. 그래프는 talin 한 감쇠를 보여줍니다크게 mesothelial 세포 통관 (수단에 녹색 막대로 Quantile 플롯)을 낮춥니다. 동영상 3. 제어 mesothelial 단일층 (녹색)에 침입하는 OVCA433 spheroids이 (빨간색). <a href="http://www.jove.com/files/ftp_upload/3888/Movie_3.mov">동영상을 보려면 여기를 클릭하십시오</a> . OVCA433 spheroids의 talin 한 표현 (빨간색)의 영화 4. 감쇄량은 (녹색) 통관 능력을 mesothelial 낮춥니다. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_4.mov">동영상을 보려면 여기를 클릭하십시오</a> .

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+culture+of+human+ovarian+surface+epithelial+cells:+a+potential+model+for+ovarian+carcinogenesis.">Organotypic culture of human ovarian surface epithelial cells: a potential model for ovarian carcinogenesis.</a> In Vitro Cell Dev. Biol. Anim. 34, 636-639 (1998).</li><li>Roberts, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+molecular+and+cellular+events+during+neoplastic+progression:+a+mouse+syngeneic+ovarian+cancer+model.">Sequential molecular and cellular events during neoplastic progression: a mouse syngeneic ovarian cancer model.</a> Neoplasia. 7, 944-956 (2005).</li><li>Okada, T., Okuno, H., Mitsui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+in+vitro+assay+system+for+transendothelial+tumor+cell+invasion:+significance+of+E-selectin+and+alpha+3+integrin+in+the+transendothelial+invasion+by+HT1080+fibrosarcoma+cells.">A novel in vitro assay system for transendothelial tumor cell invasion: significance of E-selectin and alpha 3 integrin in the transendothelial invasion by HT1080 fibrosarcoma cells.</a> Clin. Exp. Metastasis. 12, 305-314 (1994).</li><li>Zervantonakis, I. K., Kothapalli, C. R., Chung, S., Sudo, R., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+studying+heterotypic+cell-cell+interactions+and+tissue+specimen+cultures+under+controlled+microenvironments.">Microfluidic devices for studying heterotypic cell-cell interactions and tissue specimen cultures under controlled microenvironments.</a> Biomicrofluidics. 5, 13406-1310 (2011).</li><li>Brandt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-extravasation+model+--+selection+of+highly+motile+and+metastatic+cancer+cells.">3D-extravasation model -- selection of highly motile and metastatic cancer cells.</a> Semin. Cancer Biol. 15, 387-395 (2005).</li><li>Condeelis, J., Segall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+cell+movement+in+tumours.">Intravital imaging of cell movement in tumours.</a> Nat. Rev. Cancer. 3, 921-930 (2003).</li><li>Dai, J., Ting-Beall, H. P., Hochmuth, R. M., Sheetz, M. P., Titus, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+I+contributes+to+the+generation+of+resting+cortical+tension.">Myosin I contributes to the generation of resting cortical tension.</a> Biophys. J. 77, 1168-1176 (1999).</li><li>Laferriere, J., Houle, F., Taher, M. M., Valerie, K., Huot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transendothelial+migration+of+colon+carcinoma+cells+requires+expression+of+E-selectin+by+endothelial+cells+and+activation+of+stress-activated+protein+kinase-2+(SAPK2/p38)+in+the+tumor+cells.">Transendothelial migration of colon carcinoma cells requires expression of E-selectin by endothelial cells and activation of stress-activated protein kinase-2 (SAPK2/p38) in the tumor cells.</a> J. Biol. Chem. 276, 33762-33772 (2001).</li><li>Dong, C., Slattery, M. J., Rank, B. M., You, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+characterization+and+micromechanics+of+tumor+cell+chemotactic+protrusion,+locomotion,+and+extravasation.">In vitro characterization and micromechanics of tumor cell chemotactic protrusion, locomotion, and extravasation.</a> Ann. Biomed. Eng. 30, 344-355 (2002).</li></ol>

우리는 공개 할게 없다.

여기에 제시된 &quot;Mesothelial 정리 분석은&quot;훌륭한 공간과 시간적 자세히, 난소암 다세포 spheroids 및 mesothelial 세포 monolayers의 상호 작용을 모니터링하기 위해 촬영된 현미경을 사용합니다. 이전에는 8-14 몇몇 그룹은 난소암 세포가 부착하고 mesothelial 세포 monolayers에 침공 표시하도록 끝점 assays를 사용했다. 이 분석은 독특한 점에서이 두 세포 인구의 역학은 검정에 걸쳐 모니터링할 수 있...

<table border="1"><tbody><tr><td> 시약 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> OVCA433 난소 암 세포 </td><td></td><td></td><td> 박사 데니스 Slamon의 선물 </td></tr><tr><td> Mesothelial 셀을 ZT </td><td></td><td></td><td> 박사 탄의 선물 Ince </td></tr><tr><td> 중형 199 </td><td> Gibco </td><td> 19,950 </td><td></td></tr><tr><td> MCDB105 </td><td> 세포 응용 주식 회사 </td><td> 117-500 </td><td></td></tr><tr><td> FBS-더위 inactivated </td><td> Gibco </td><td> 10,082 </td><td></td></tr><tr><td> 펜-Strep </td><td> Gibco </td><td> 15,070 </td><td></td></tr><tr><td> 96 잘 접시 </td><td> 코닝 Costar </td><td> 3799 </td><td></td></tr><tR&gt; <td> Polyhydroxyethylmethacrylate (폴리-HEMA) </td><td> 시그마 알드리치 </td><td> 192066-25G </td><td> 폴리 - HEMA 솔루션을 95 % EtOH의 1ml에 6mg 폴리 - HEMA 분말을 용해 </td></tr><tr><td> EtOH </td><td> Pharmco-aaper </td><td> 111ACS200 </td><td> DH 2 0에서 95 %까지 희석 </td></tr><tr><td> 세포 배양 후드 </td><td> Nuaire </td><td> NU-425-300 </td><td></td></tr><tr><td> 조직 문화의 인큐베이터 </td><td> 써모 과학 </td><td> 3110 </td><td></td></tr><tr><td> 폴리 - HEMA 플레이트위한 인큐베이터 </td><td> Labline 악기 </td><td> 제국 III 305 </td><td></td></tr><tr><td> 탁상 원심 분리기 </td><td> Heraeus </td><td> 75003429 / 01 </td><td></td></tr><tr><td> 6 잘 유리 바닥 요리 </td><td> MatTek 사. </td><td> P06G-1.5-20-F </td><td></td></tr><tr><td> Fibronectin </td><td> 시그마 </td><td> F1141-1MG </td><td></td></tr><tr><td> PBS </td><td> Cellgro </td><td> 21-040 - 이력서 </td><td></td></tr><tr><td> Timelapse 현미경 : </td><td></td><td></td><td></td></tr><tr><td> 현미경 </td><td> 니콘 </td><td></td><td> 통합된 완벽한 초점 시스템과 티-E 바뀌 동력 형광 촬영된 현미경 </td></tr><tr><td> 렌즈 </td><td> 니콘 </td><td></td><td> 20X-0.75 수치 apeture </td></tr><tr><td> 할로겐 transilluminator </td><td> 니콘 </td><td></td><td> 0.52 없음 긴 작동 거리 응축기 </td></tr><tr><td> 여진과 방사 필터 </td><td> 니콘 주택 채도 단일 패스 필터 </td><td></td><td> GFP 예 40분의 480, 엠 50분의 525 RFP-mCherry 예 50분의 575 엠 50분의 640 </td></tr><tr><td> 전송 및 광 경로를 Epifluoresce </td><td> 셔터 </tD&gt; <td></td><td> 스마트 셔터 </td></tr><tr><td> 선형 인코딩 동력 무대 </td><td> 니콘 </td><td></td><td></td></tr><tr><td> 냉각 충전 - 결합 장치 카메라 </td><td> 하마 마츠 </td><td> 오카-AG </td><td></td></tr><tr><td> 온도 및 CO 2 제어 현미경 보육 챔버 </td><td> 주문품의 </td><td></td><td></td></tr><tr><td> 진동 절연 표 </td><td> TMC </td><td></td><td></td></tr><tr><td> 국정원 - 요소 소프트웨어 </td><td> 니콘 </td><td></td><td> 버전 3 </td></tr></tbody></table>

in vitro mesothelial clearance assay that models the early steps of ovarian cancer metastasis

난소암은 미국 1의 암 관련 사망의 다섯 번째 주요 원인이다. 치료에 긍정적인 초기 대응에도 불구하고 난소암과 여성의 70~90% 새로운 metastases을 개발하며, 재발은 종종 두 치명적이다. 그것은 중급과 늦은 무대 난소암 더 나은 치료법을 개발하기 위해 보조 metastases가 발생하는지 이해하기 위해, 따라서 필요합니다. 악성 세포가 일차 종양이 사이트에서 분리 및 복막 캐비티 전체에 보급하면 난소암의 전이가 발생합니다. 전파 세포가 복막 구멍 3 (그림 1, 영화 1) 내의 장기에 다세포 클러스터, 또는 spheroids 역시 남아 맨몸으로, 아니면 임플란트를 형성할 수 있습니다. 복막 캐비티 내의 장기의 모든 mesothelial 세포를 4-6 (그림 2) 단일, 연속, 레이어로 채워져 있습니다. 그러나 mesothelial 세포 수렁에서 결석입니다같은 excised 인간 종양 조직 섹션 3,5-7 (그림 2)의 전자 현미경 연구에 의해 밝혀 복막 종양 유출. 이것은 mesothelial 세포를 알 수없는 프로세스에 의해 종양이 대량 수렁에서 제외됩니다 것을 제안합니다. 체외 실험에서 이전은 기본 난소암 세포 8 mesothelial 세포보다는 세포외 기질에 더 효율적으로 첨부하고, 더 최근의 연구는 그 기본 복막 mesothelial 세포가 실제로 난소암 세포 부착 및 침입 (같은 접착 및 침략에 비해에 장벽을 제공 보여주 것을 증명 ) mesothelial 세포에 9,10을 취재되지 않은 기판에. 이것은 mesothelial 세포가 난소 암 전이에 대한 장벽 역할을하는 것이 좋습니다 것이다. 난소암 세포가이 장벽을 위반하고있다 mesothelium를 제외하는 세포와 분자 메커니즘은 최근까지 알려지지 않았다. 여기 일을 설명시험 관내 분석을위한 전자 방법론 해당 모델 생체내 (그림 3, 영화 2) 난소암 세포 spheroids과 mesothelial 세포 사이의 상호 작용. 우리의 프로토콜은 mesothelial monolayers 8-16과 난소 종양 세포의 상호 작용 분석을위한 앞에서 설명한 방법에서 적응되었고, 먼저 난소 종양 세포의 배제를 추진 마이 오신과 견인 힘을 인테 종속 활성화를 활용 보여주는 리포트에서 설명되었다 종양 회전 타원체 17 세 이하의 mesothelial 세포. 이 모델은 상호 작용의 공간적 및 시간적 정보를 제공하고, 실시간으로 두 세포 인구를 모니터링하는 데 시간이 경과 형광 현미경을 이용합니다. mesothelial 세포가 녹색 형광 단백질 (GFP)을 표현하는 동안 난소 암 세포는 빨간 형광 단백질 (RFP)를 표현. RFP-표현하는 난소암 세포 spheroids하면 GFP-표현 mesothelial 단일층에 첨부해 주시기 바랍니다. spheroids 확산은 침공, 그리고옆으로 단일층에 구멍을 만드는 mesothelial 세포를 강제로. 이 구멍은 GFP 이미지의 부정적인 공간 (블랙)와 같은 시각이다. 구멍의 지역 그리고 양적 통제와 난소암 및 / 또는 mesothelial 세포 실험 집단 간의 클리어런스 활동의 차이를 분석하는 측정 수 있습니다. 이 분석은 난소암 세포 (조건 회 회전 타원체 X 20-30 spheroids 당 100 세포)만이 소수의 필요 때문에 귀중한 일차 종양 세포 샘플을 사용하여이 분석을 수행 가능합니다 이상입니다. 또한이 분석은 쉽게 높은 처리량 검사에 대해 적용할 수 있습니다.

Watch this Scientific Journal Video about In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis at JoVE.com

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

여기에 설명된 mesothelial 통관 분석은 fluorescently 표시된 셀 및 촬영된 비디오 현미경 시각화하고 양적 난소암 다세포 spheroids 및 mesothelial 세포 monolayers의 상호 작용을 측정하기 위해 활용합니다. 이 분석은 난소 암 전이의 초기 단계를 모델.

체외 Mesothelial 정리 분석에서는 그것이 난소 암 전이의 초기 단계 모델

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

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In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

14.4K 조회수.  Harvard Medical School. 여기에 설명된 mesothelial 통관 분석은 fluorescently 표시된 셀 및 촬영된 비디오 현미경 시각화하고 양적 난소암 다세포 spheroids 및 mesothelial 세포 monolayers의 상호 작용을 측정하기 위해 활용합니다. 이 분석은 난소 암 전이의 초기 단계를 모델.

비디오: In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

실험 전이 분석

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

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MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

종양의 4D 라이브 세포 이미징을위한 MAME 모델 : Microenvironment 상호 작용 영향을 미치는 악성 진행

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

자기 공명 영상, 체적 계산의 Tomography 및 초음파를 이용한 유방암의 뼈 전이의 누드 쥐 모델에서 Angiogenesis의 멀티 모달 이미징

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

3 차원 Heterotypic<em&gt; 체외에서</em난소 암 개시 및 진행하는 동안 Stromal - 상피 조직의 상호 작용&gt; 모델링

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

뼈 전이의 모델

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

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 ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

난소 암 종양 시작 세포의 생체 농축에

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

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 ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

생체 내 및 생체 외 복막 지방이에 밀키 스팟 구조의 난소 암 전이성 식민지를 연구하는 접근 방식

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

복강 내 난소 암 전이의 정량

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...