이 프로토콜은 테스트 및 박사 리차드 하인즈 (MIT)의 실험실에서 처음에 최적화되었습니다. 기금은 NYSTEM 아이디어 수상 (LX)을하고 루트 L. Kirschstein 내셔널 리서치 서비스 수상 (LX)를 제공합니다.

1. 샘플 준비 <ol><li> 성장 요인 또는 FBS 그들의 특정 미디어에 ~ 70 % 합류 (예 : MC - 1 세포를 10 % FBS와 DMEM)에 세포를 성장. 접시에서 미디어를 대기음 부드럽게 1 X PBS (나 2 HPO 4 NaCl 8 G / L, KCl의 0.2 g / L, 1.15 g / L 0.7 H 2 O, KH 2 PO의 0.2 g / L로 여러 번 씻어 4, 산도 7.3). </li><li> 대기음 PBS가와 versene에서 0.05 %의 트립신 (0.014 g / 페놀 빨간색과 1 X PBS, pH를 7.2에서 0.2의 G / L EDTA (에틸렌 다이아 민 테트라 초산) - 나의 L) 2 ML를 추가합니다. 부드럽게 접시에서 세포 분리를 촉진하기 위해 플레이트를 바위. 현미경 세포를 관찰합니다. 그것은 일반적으로 분리하는 세포에 대한 2-5분 걸립니다. </li><li> 트립신 활동을 담금질하고 50mL 팔콘 튜브에 세포를 수집하는 FBS (또는 대두 트립신 억제제)가 포함된 미디어의 충분한 볼륨을 추가합니다. hemacytometer를 사용하여 셀을 계산합니다. 깨끗한 hemacytometer에 세포 현탁액의 10μL를로드합니다. ML 당 세포의 숫자는 10 4 곱한 다섯 제곱의 각 # 세포의 평균 같습니다. </li><li> 1000 세포를 원심 RPM (또는 ~ 200 XG) 3 분 benchtop 원심 분리기 인치 </li><li> 조심스럽게 세포 펠렛을 방해하지 않고 미디어를 제거합니다. ~ 5 × 10 6 세포 / ML의 최종 농도에 도달하는 행크스 밸런스드 버퍼 솔루션 (HBSS)의 적절한 볼륨으로 세포를 Resuspend. </li><li> 팰컨 70 μm의 셀 스트레이너를 통해 필터 세포는 큰 세포 집계를 제외합니다. 표시 5mL 팰컨 문화 관을 통해 직접 필터에 피펫의 끝에 놓습니다. 신속 문화 튜브에 필터를 통해 중지를 꺼냅니다. </li><li> hemacytometer를 사용하여 다시 셀을 카운트 및 2.5 × 10 6 / ML의 최종 농도로 희석. 얼음 세포 보관하십시오. </li><li> 세포의 생존을 결정합니다. trypan 파랑 몇 가지 세포를 혼합하고 hemacytometer를 사용하여 총 전지를 통해 죽은 (파란색) 세포의 백분율을 측정합니다. 세포의 생존 능력은 주사하기 전에 ≥ 90%되어야합니다. </li></ol> 2. 정맥 주사 <ol><li> 장갑을 변경합니다. 부드럽게 immunodeficient 마우스 (누드, NOD - SCID, 또는 NSG, 잭슨 연구소)의 꼬리를 잡고 restrainer 뒤쪽에있는 작은 오프닝 바깥으로 튀어나와있는 슬릿과 꼬리 상대로 다시와 마우스 restrainer으로 당겨 . </li><li> 반지는 마우스의 입을 잡는다 한번 천천히 슬릿을 따라 안쪽 링 슬라이드 및 장소에 잠금. 마우스가 자유롭게 이동 할 수있는 거 아니지만, 호흡의 정상적인 비율이 있어야합니다. </li><li> 주요 꼬리 정맥을 찾아보십시오. 네 개의 주요 혈관은 마우스 꼬리에 존재한다. 꼬리 지느러미 및 복부 양쪽에 혈관이 동맥 있습니다. 정맥 꼬리의 측면 측면에 있습니다. </li><li> 1mL 주사기에 준비 세포의 200 명 이상 μL을 그립니다. 30 G1 / 2 인치 바늘을 부착하고있을 수있는 공기 방울을 밀어. 주사기의 세포의 최종 부피는 200 μL (즉, 5 총 X 10 5 세포)해야합니다. </li><li> 꼬리 정맥에 세포를 주입. <ol class="lalpha"><li> 첫 번째 재판이 실패한다면, 꼬리보다 근위 영역이 두 번째 시도에 사용될 수 꼬리의 말초 월말부터 시작합니다. </li><li> 70 % 에탄올로 꼬리를 닦아주십시오. 바로 꼬리를 당겨. 엄지와 꼬리의 끝을 잡고 검지로 주입을위한 지점을 지원합니다. </li><li> 정맥에 바늘을 넣고 세포를 주입. 바늘과 주사기가 주사시 정맥에 병렬로되어 있는지 확인하십시오, 그렇지 않으면 바늘이 혈관 벽을 통해 찌를와 인접한 꼬리 조직에 세포를 주입합니다. </li><li> 주사 후 바늘을 철회. 피가 분사가 성공적으로 간 경우 주사 사이트에서 넘치도록해야합니다. 응고 촉진 사출 사이트에 종이 타월이나 면봉의 깨끗한 부분을 누르면, 유통 정맥에 잔류 시료를 밀어 꼬리 위쪽을 촉진하다. </li><li> restrainer에서 마우스를 해제하고 감옥에 돌아갑니다. 연구실 노트 (예를 들어, 얼마나 많은 시련이 세포를 주입하는 데리고 얼마나 세포 주입되었다) 사출 과정을 기록합니다. </li><li> 로 샘플 준비 섹션에서 설명한 주입 후 세포의 생존을 결정, 9 단계. 이 단계는 세포가 사출 과정에서 살아남는 것을 안심을 제공합니다. 주사 실험의 끝에, 세포의 생존 능력은 감소되지만 80% 이상이어야합니다. </li><li> (옵션) 남은 세포를 스핀 다운하여 한 번 PBS로 알약을 씻어. 미래의 분석 (예를 들어, 유전자 발현이나 knockdowns 확인을 위해 서부 blots)를 -80 OC에서 알약을 고정. </li></ol></li><li> 일반적으로 1 ~ 2 개월 후, 생쥐 해부되며 metastases의 위치는 조잡한 결정됩니다. 그것은 세포가 그들이 이후에 발생되는 최초의 모세관 침대 포함되어 있으므로 폐암이 전이의 기본 사이트입니다순환을 입력합니다. </li><li> PBS, 폐 (또는 metastases을 포함한 다른 조직)의 각 엽과 rinsing 후 해부 현미경 (그림 1)에서 관찰됩니다. 폐 metastases의 총 개수로 폐 양쪽에 감지 metastases의 수를 계산하고 모든 네 개의 엽 (叶)에 폐 metastases의 숫자가 함께 추가됩니다. 폐가가 포르말린 하룻밤에 수정하는 경우 한 폐에 metastases 더 쉽게 감지 , metastases는 인접한 어두운 갈색 폐 조직에 대조적으로 약간 흰 반점으로 apppear 때문에. </li></ol> 3. 룽 Metastases에서 Culturing 셀 <ol><li> 룽 metastases는 주입 생쥐에서 격리됩니다. 각각은 별도 양식되어야합니다. </li><li> 각 전이는 70 μm의 셀 스트레이너에 주사 바늘 뚜껑 (멸균)의 말까지 다진 것입니다. </li><li> 매체 및 문화 접시에있는 필터를 통과 수집된 세포의 여러 MLS와 셀 스트레이너 린스. </li><li> 방해하지 않고 적어도 나흘 동안 37 ° C에서 세포를 품어. </li><li> PBS와 함께 접시에 혈액이나 조직 파편을 멀리 씻으십시오. </li><li> 신선한 매체를 추가하십시오. 처음에는, 암 세포 fibroblast 세포 모두 접시에 성장하지만, 점차, fibroblast 세포 밖으로 죽을 암 세포로 대체됩니다. 암 세포가 어떠한 약물 모성 유전자를 가지고있다면, 해당 항생제로 셀을 선택합니다. </li><li> 파생 세포의 순도는 인간 특정 단백질에 대한 항체를 사용 immunostaining에 의해 평가됩니다. 우리는 안티 인간 vimentin 항체 (NCL - VIM - V9, Novocastra)를 사용합니다. 우리가 파생된 세포도 약물 선택의 부재에서 인간 세포의 99 % 이상 일반적으로 포함되어 있습니다. 새로 파생된 세포는 위에서 설명한 바와 같이, 그들의 전이성 능력을 테스트하기 위해 immunodeficient 생쥐에 다시 주입 수 있습니다. </li></ol> 4. 대표 결과 <ol><li> 거의 폐 metastases가 저조한 전이성 암 세포 라인에서 올 때이 분석의 끝에, 높은 전이성 암 세포 라인은 일반적으로 많은 폐 metastases (그림 1), 1 상승을 제공합니다. </li><li> 이 방법을 사용하여 파생된 세포는 일반적으로 그들이 다시 분석을 사용하여 테스트하는 부모의 라인보다 metastases을 일으키다. 1-3 </li></ol><img src="/files/ftp_upload/1942/1942fig1.jpg" alt="그림 1" /> 암 세포의 꼬리 정맥 주사 후 마우스 폐 그림 1. 대표 이미지. 전이성 인간 흑색증 셀 라인, SM 세포 4 5 X 10 5 immunodeficient 생쥐의 꼬리 정맥에 주입했다. 두 달 후 폐는 고립되었고 metastases은 정상적인 폐 조직 간의 분산 발견되었습니다.

<ol>
	<li>Xu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+changes+in+an+animal+melanoma+model+correlate+with+aggressiveness+of+human+melanoma+metastases.">Gene expression changes in an animal melanoma model correlate with aggressiveness of human melanoma metastases.</a> Mol Cancer Res. 6 (5), 760-760 (2008).</li><li>Clark, E. A., Golub, T. R., Lander, E. S., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+metastasis+reveals+an+essential+role+for+RhoC.">Genomic analysis of metastasis reveals an essential role for RhoC.</a> Nature. 406 (6795), 532-532 (2000).</li><li>Xu, L., Begum, S., Hearn, J. D., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPR56,+an+atypical+G+protein-coupled+receptor,+binds+tissue+transglutaminase,+TG2,+and+inhibits+melanoma+tumor+growth+and+metastasis.">GPR56, an atypical G protein-coupled receptor, binds tissue transglutaminase, TG2, and inhibits melanoma tumor growth and metastasis.</a> Proc Natl Acad Sci U S A. 103 (24), 9023-9023 (2006).</li><li>Kozlowski, J. M., Hart, I. R., Fidler, I. J., Hanna, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+melanoma+line+heterogeneous+with+respect+to+metastatic+capacity+in+athymic+nude+mice.">A human melanoma line heterogeneous with respect to metastatic capacity in athymic nude mice.</a> J Natl Cancer Inst. 72 (4), 913-913 (1984).</li><li>Kang, 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+multigenic+program+mediating+breast+cancer+metastasis+to+bone.">A multigenic program mediating breast cancer metastasis to bone.</a> Cancer Cell. 3 (6), 537-537 (2003).</li></ol>

전이는 암 환자에서 사망의 주요 원인입니다. 그들의 주요 loci, 유통에 자신의 항목 (intravasation), 순환 (넘쳐 흐름)에서 ​​자신의 종료, 그리고 먼 장기에 생존과 성장에서 암 세포의 분리 : 그것은 네 개의 주요 단계를 포함한다. 인간의 전이는 느린 과정으로 간주하고 종종 지연 년 이후 각성입니다. 적시에 자사의 진행을 연구하기 위해 위의 비교적 신속하게 실험적인 분석은 immunodeficient 생쥐에서 설립되었습니다. 4 설립 이래, 그것이 다른 전이성 능력을 가진 세포를 분리하기 위해 효과적으로 사용되고 있습니다, 최대 - 아래 - 규제하는 동안 아르 유전자를 식별 전이 1,2,5, 그리고 전이 동안 이러한 유전자의 인과 관계 역할을 테스트 3. 이 분석에서 가장 어려운 단계는 꼬리 정맥에 세포를 주입하는 것입니다. 그것은 주입 기술을 마스터하는 방법을 걸립니다. 비록 경험이 연구자를위한, 성공적인 주사를 달성하기 전에 몇 가지 실험은 매우 일반적인 수 있습니다. 마우스 꼬리에 정맥 바로 그들의 중간에 아주 얇고 삽입 바늘 아르 쉬운 일이 아닙니다. 램프를 사용하여 간단히 꼬리를 데워 부드럽게 주사하기 전에 꼬리를 마사지하면 혈관의 팽창을 유도하고 주사를 용이하게 수 있습니다. 대부분의 연구자에 대한 일반적인 실수는 혈관이 꼬리에 깊은되고 따라서 피부에 너무 깊이 세포를 주입 있다고 가정하는 것입니다. 혈관 실제로 피부의 표면에 매우 가까이 있으며, 따라서 하나는 가능 한한 shallowly 주입하려고한다. 바늘이 성공적으로 정맥 안으로 삽입하는 경우 주사는 아무런 저항도 함께 원활하게 이동해야합니다. 바늘은 정맥에 삽입 아니라 인접 조직에 주입 샘플 신속하게 유체 압력을 구축하고 샘플의 나머지의 전달을 방지할 수있는 경우. 이러한 실패 시험의 각 따라서 몇 가지 샘플을 잃게됩니다. 이 손실을 최소화하기 위해 경험이 연구자는 바늘이 샘플의 최소 금액을 주입하고 유체 압력의 저항을 평가하여 정맥 또는하지의 여부를 예측하려고합니다. 그가 저항을 느끼는 경우, 바늘은 정맥에해서는 안됩니다 그는 다음 바늘을 뽑아 다시 시도합니다. 아무 저항을 생각하지 않으면 바늘이 혈관에해야하며 시료의 모든 한번에 주입됩니다. 후자의 경우, 하나는 종종 그것이 몸쪽으로 그것의 방법을 만들면서 혈액을 이동시켜 생기죠, 정맥 최대 샘플 &quot;골인&quot;를 볼 수 있습니다. 그것이 크게 세포의 전이성 가능성에 따라 다릅니다 이후 분석은 각 세포주에 대한 경험적으로 결정되어야 종료할 때. 생쥐는 주사 후 다른 시간 시점에서 해부되고 폐 전이의 범위가 결정됩니다. 그것은 양적 결과를 제공하며 후보 유전자 섭동 의한 metastases의 증가 또는 감소를위한 공간을 떠난 이후 미세 감지 폐 metastases의 셀 수있는 숫자 결과가 일반적으로 선택되는 시간의 길이. 긍정적인 제어로 높은 전이성 세포 라인을 (예 : 인간 흑색증 셀 라인, A375, 또는 높은 전이성 파생 상품 등)를 사용하여, 처음으로이 분석을 수행하는 연구자를위한 것이 좋습니다. 또한, 마우스의 나이와 성별은 크게 결과에 영향을, 그것이 다른 세포 라인의 전이성 능력을 비교하는 경우 일관된 연령 및 성별을 유지하는 것이 좋다는 것이다. 위에서 언급한 요인에 의해 도입된 변화로 인해 적어도 다섯 생쥐는 어떤 통계적으로 의미있는 결론에 도달하는 각 세포주에 대한 주입해야합니다.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10313-021</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30088.03</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14175-095</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S9888</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Mallinckrodt Chemicals</td>
<td>6838-04</td>
<td>&nbsp;</td>
<tr>
<td>Na2 HPO4.7H2O</td>
<td>&nbsp;</td>
<td>Mallinckrodt Chemicals</td>
<td>7896-04</td>
<td>&nbsp;</td>
<tr>
<td>KH2PO4</td>
<td>&nbsp;</td>
<td>Mallinckrodt Chemicals</td>
<td>7100-12</td>
<td>&nbsp;</td>
<tr>
<td>Phenol red</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P3532</td>
<td>&nbsp;</td>
<tr>
<td>EDTA-Na</td>
<td>&nbsp;</td>
<td>EMD Biosciences</td>
<td>4010</td>
<td>&nbsp;</td>
<tr>
<td>Trypan Blue</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15250-061</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin 2.5%</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15090-046</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>&nbsp;</td>
<td>Ultrapure</td>
<td>200-CSGP</td>
<td>&nbsp;</td>
<tr>
<td>50 ml Falcon Tube</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89039-656</td>
<td>&nbsp;</td>
<tr>
<td>Microcentrifuge Tubes</td>
<td>&nbsp;</td>
<td>Axygen Scientific</td>
<td>MCT-175-C</td>
<td>&nbsp;</td>
<tr>
<td>Falcon 70&#181;m Cell Strainer</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>5ml Falcon Culture Tube</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>60818-576</td>
<td>&nbsp;</td>
<tr>
<td>Hemacytometer</td>
<td>&nbsp;</td>
<td>Hausser Scientific</td>
<td>15170-208</td>
<td>&nbsp;</td>
<tr>
<td>1 ml syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>329650</td>
<td>&nbsp;</td>
<tr>
<td>30 &frac12; gauge needle</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>305106</td>
<td>&nbsp;</td>
<tr>
<td>Mouse restrainer</td>
<td>&nbsp;</td>
<td>Plas Labs Inc.</td>
<td>551-BSRR</td>
<td>&nbsp;</td>
<tr>
<td>Surgical scissors</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-940</td>
<td>&nbsp;</td>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-902</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>SMZ1500</td>
<td>&nbsp;</td>
<tr>
<td>Benchtop Centrifuge</td>
<td>&nbsp;</td>
<td>Thermoscientific</td>
<td>75003491</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

experimental metastasis assay

전이는 암 환자에서 사망의 주요 원인입니다. 전이의 메커니즘을 이해하기 위해서, 실험적인 전이 분석은 immunodeficient 마우스를 사용하여 설립되었습니다. 이 문서는 샘플 준비, 정맥 주사, 그리고 폐 metastases에서 culturing 세포를 포함하여이 분석에 관련된 절차를 delineates. 간단히, 인간의 암 세포의 미리 정해진 숫자는 준비가되어있다<em&gt; 체외에서</em&gt; 직접 자신의 꼬리 정맥을 통해 immunodeficient 생쥐의 순환에 주입. 세포의 소수의 순환에 난기류를 생존과 같은 폐 등 내부 장기에 metastases로 성장합니다. 주입 생쥐는 일정 기간 후 해부를하고 있습니다. metastases의 조직 분포는 해부 현미경으로 결정됩니다. 특정 조직에 metastases의 수를 계산하고 직접 주입 암 세포의 전이성 능력을 상호 있습니다. 일어 metastases는 절연 및 양식 아르<em&gt; 체외에서</em&gt;로 immunodeficient 생쥐에 다시 주입했을 때 종종 부모의 라인보다 더 향상된 능력을 보여주 전이성 세포 라인. 이러한 높은 전이성 파생 상품은 전이성 진행을 조절 유전자 또는 분자 경로를 확인하는 유용한 도구가됩니다.

Watch this Scientific Journal Video about Experimental Metastasis Assay at JoVE.com

Experimental Metastasis Assay

이 문서는 인간의 암 세포 라인의 전이성 잠재력을 결정하는 데 사용되는 실험적인 전이 분석의 절차를 설명합니다.

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 ...

Nanyang Technological University

Research

JoVE Journal

Medicine

20.2K 조회수.  University of Rochester Medical Center. 이 문서는 인간의 암 세포 라인의 전이성 잠재력을 결정하는 데 사용되는 실험적인 전이 분석의 절차를 설명합니다.

비디오: Experimental Metastasis Assay

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

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 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

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

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

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 ...

연구

의학

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 ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

실험 인간 폐렴의 운송

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

<em&gt; OVO에서</em육종을위한 Xenograftmodel으로&gt; CAM-분석

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

인간의 전립선 암 전이의 동소 쥐과 모델

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

차 종양과 폐 전이을 연구하기 위해 MEF 세포 격리

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

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

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 between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

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 ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

&quot;환자와 같은&quot;동소 이식 동계 마우스 모델 간세포 암의 전이

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms 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 ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

정량 실시간 PCR에 의해 마우스 유방 종양 모델에서 폐 전이의 평가

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...