이 작품은 하리리 가족 재단과 TJ 마텔 재단에 의해 지원되었다.

1. 수확 및 수술 표본에서 인간의 전립선 암 조직의 처리 <ol><li> 로스웰 파크 메모리얼 연구소 (RPMI) 10 % 소 태아 혈청 (FBS) 및 1 % 페니실린 / 스트렙토 마이신이 보충 된 1640 배지 15 ㎖를 50 ㎖이 함유 폴리스티렌 원추형 튜브를 준비한다. </li><li> 병리학 서비스 담당자가 프로토콜을 승인 한 기관 심사위원회에서 각각 전립선 및 완화 수술 표본에서 기본 및 전이성 전립선 암 샘플을, 수확. 전이성 샘플 차 수술 중 림프절 해부에서 얻을 수 있습니다, 다른 사람의 사이에서 하나의 전이성 병변의 진단 제거, 동안 고식적 수술 척수 압축 해제 등의 절차, 폐 동안 척추. <ol><li> 단계 1.1에서 첫 번째 50 ML의 폴리스티렌 원뿔 관에 임상 진단을 위해 필요하지 초과 대량 조직을 수확, 4 ℃에서 바로 저장 harvesteD 조직은 적어도 지역 4 ~ 5 ㎠의 두께에서 1-2 cm를 측정해야한다. </li></ol></li><li> 연구소 직원은 세포 현탁액의 조직 학적 검사와 세대를위한 준비에 병리 서비스에서 얻은 대량 조직을 처리합니다. <ol><li> 대량 조직의 처리는 최대한의 세포 생존을 보장하기 위해 수술 적 절제에서 24 시간을 초과 할 수 없습니다. </li><li> 멸균 메스와 집게와 생물 안전 캐비닛에서 작업, 거시적 종양 결절 2-4 mm 두께의 수평 조직 섹션을. 즉시 단계 1.1에서 두 번째 50 ㎖ 폴리스티렌 원뿔 튜브에 섹션을 저장합니다. </li><li> 거시적 결절이 전립선 샘플에서 가시화되지 않을 경우, 후부의 엽 (叶) 및 전환 영역에서 임의의 2~4밀리미터 두께 수평 조직 섹션을 수집합니다. </li></ol></li><li> 멸균 메스와 핀셋을 사용하여 수술 조직의 일부를 분리하고 이전 세대에서 10 % 포르말린에 고정세포 현탁액의 기. 보관 및 전립선 암 조직의 존재를 확인하기 위해이 물질을 조직 학적 분석. </li></ol> 2. 조직 섹션의 세포 현탁액의 생성 <ol><li> 멸균 생물 안전 캐비닛에서 작업, 멸균 1X 인산염 완충 생리 식염수 (PBS) 1 ㎖를 포함하는 60 X 15mm 배양 접시에 조직 섹션을 전송합니다. <ol><li> 기계적으로 멸균 메스와 집게를 사용하여 샘​​플을 씹다. </li><li> 어떤 효소 digestions이 프로토콜에 없습니다. </li></ol></li><li> 멸균 50 ML 원뿔 튜브에 1 ㎖ 현탁액을 전송합니다. <ol><li> 배양 접시에 1 ㎖를 추가하고 조직 절편이 완전히 보통 3 - 4 배, 해리 될 때까지 분쇄 단계를 반복합니다. </li><li> 모든 조직 섹션이 분해 될 때까지 순차적으로이 절차를 반복합니다. </li></ol></li><li> 5 ㎖의 혈청 학적 PI로 50 ml의 폴리스티렌 원뿔 관의 내용을 재현 탁pette 1 분 동안 최대 속도 (약 3,200 RPM)에서 소용돌이. </li><li> 35 μm의 셀 스트레이너를 통해 생성 된 현탁액을 필터 및 제 멸균 50 ML의 폴리스티렌 원뿔 튜브에 수집합니다. </li><li> 10 분 450 XG에서 원심 분리하여 펠렛을 생성합니다. </li><li> , 상층 액을 버린다 적혈구 세포 용해 완충액 5 ㎖로 펠렛을 재현 탁하고, RT에서 5 분을위한 솔루션을 품어. RT 450 XG에 5 분 동안 원심 분리하여 적혈구 세포 용해 버퍼를 제거합니다. </li><li> 상층 액을 버리고 5 % FBS로 보충 된 PBS 1X에 펠렛을 재현 탁하고, 혈구 또는 자동 세포 계수기를 사용하여 실행 가능한 (트리 판 블루 음성) 세포의 수를 정량. 1 × 10 6 세포 / ㎖ 현탁액을 생성하고 더 이상 1 시간 이상 동안 얼음에 보관하지. <ol><li> 생존과 세포의 수율은 종양 표본 사이에 상당히 다를 수 있습니다. 전립선 설정에서 생존 능력 45~70%와 인터넷 사이의 범위 수거시적 종양 결절에서 두꺼운 수평 조직 섹션은 약 3 × 6 가능한 세포를 얻을 것으로 예상 할 수있다 2-4밀리미터을했습니다. </li></ol></li></ol> 3. FACS에 대한 세포의 항체 염색법 <ol><li> 아래 그림과 같이 다섯 15 ML의 폴리스티렌 원뿔 튜브 레이블을 지정합니다. 각각 조혈 및 내피 요소를 제외 CD45 및 CD31 항원 라벨을 사용합니다. <ol><li> 흠없는 </li><li> 의 IgG 2A의 κ-PE + IgG의 1 κ-FITC </li><li> HLAI-PE + IgG의 1 κ-FITC </li><li> 의 IgG 2A의 κ-PE + CD31-FITC + CD45-FITC </li><li> HLAI-PE + CD45-FITC + CD31-FITC </li></ol></li><li> 3.1 단계에있는 5 개의 튜브에 이전 섹션 (단계 2.7)에서 정량화 된 세포 현탁액을 배포합니다. 1:250의 희석에 항체를 추가하고 30 분 동안 얼음에 세포 현탁액을 품어. </li><li> 4 ° C에서 3 분 450 XG에서 세포를 원심 분리하고, 상층 액을 버린다. </li&gt; <li> 멸균 1X PBS 각 펠렛을 재현 탁하여 세포를 씻으 4 ℃에서 3 분 450 XG에서 원심 분리 한 다음 10 % FBS와 보충 </li><li> 10 ㎍ / ㎖의 농도의 4를 함유하는 1X PBS를 1 ㎖ 용액 &#39;,6-diamidino-2-페닐 인돌 (DAPI)로 세포를 재현 탁. </li><li> 12mm X 75mm의 폴리스티렌 튜브에 35 ㎛의 여과기 모자를 통해 최종 솔루션을 필터링합니다. </li></ol> 4. FACS에 의한 전립선 암줄 기세포의 분리 <ol><li> 셀 정렬의 흐름 세포 계수기를 사용한다. </li><li> 튜브에서 # 1, # 2, # 3 세포를 사용하여 보정 컨트롤을 설정하고, # 4. </li><li> 전방 및 측면 산란 매개 변수를 사용하여 파편과 세포의 클러스터를 제외 게이트를 만듭니다. </li><li> 각각 튜브 # 3, # 4 관에서 현탁액을 사용하여 FITC와 PE 게이트를 설정합니다. </li><li> 퍼시픽 블루-A 채널을 사용 가능한 게이트 (DAPI 음성) 세포. </li><li> 관 # 5 폐기는 CD31 양성 및 CD45 양성 세포를 사용하여RPMI 2 ㎖를 10 % FBS와 보충을 포함하는 멸균 15 ML의 폴리스티렌 원뿔 튜브에 모두 HLAI 음성 및 HLAI 양성 인구를 수집합니다. </li><li> 원심 분리기 5 분 450 XG에서 세포 현탁액을 분류하고 10 % FBS와 보충 RPMI의 200 ~ 500 μL에 펠렛을 재현 탁. </li><li> 혈구 또는 자동 셀 카운터를 사용하여 실행 가능한 (트리 판 블루 음) 세포를 정량화. </li></ol>

<ol>
	<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144, 646-674 (2011).</li><li>Magee, J. A., Piskounova, E., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells:+impact,+heterogeneity,+and+uncertainty.">Cancer stem cells: impact, heterogeneity, and uncertainty.</a> Cancer Cell. 21, 283-296 (2012).</li><li>Nguyen, L. V., Vanner, R., Dirks, P., Eaves, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells:+an+evolving+concept.">Cancer stem cells: an evolving concept.</a> Nat. Rev. Cancer. 12, 133-143 (2012).</li><li>Valent, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cell+definitions+and+terminology:+the+devil+is+in+the+details.">Cancer stem cell definitions and terminology: the devil is in the details.</a> Nat. Rev. Cancer. 12, 767-775 (2012).</li><li>Visvader, J. E., Lindeman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells:+current+status+and+evolving+complexities.">Cancer stem cells: current status and evolving complexities.</a> Cell Stem Cell. 10, 717-728 (2012).</li><li>Lapidot, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell+initiating+human+acute+myeloid+leukaemia+after+transplantation+into+SCID+mice.">A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.</a> Nature. 367, 645-648 (1994).</li><li>Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J., Clarke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+tumorigenic+breast+cancer+cells.">Prospective identification of tumorigenic breast cancer cells.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 3983-3988 (2003).</li><li>Singh, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+human+brain+tumour+initiating+cells.">Identification of human brain tumour initiating cells.</a> Nature. 432, 396-401 (2004).</li><li>Collins, A. T., Berry, P. A., Hyde, C., Stower, M. J., Maitland, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+tumorigenic+prostate+cancer+stem+cells.">Prospective identification of tumorigenic prostate cancer stem cells.</a> Cancer Res. 65, 10946-10951 (2005).</li><li>Domingo-Domenech, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+acquired+docetaxel+resistance+in+prostate+cancer+through+depletion+of+notch-+and+hedgehog-dependent+tumor-initiating+cells.">Suppression of acquired docetaxel resistance in prostate cancer through depletion of notch- and hedgehog-dependent tumor-initiating cells.</a> Cancer Cell. 22, 373-388 (2012).</li><li>Patrawala, L., Calhoun-Davis, T., Schneider-Broussard, R., Tang, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+organization+of+prostate+cancer+cells+in+xenograft+tumors:+the+CD44+alpha2beta1++cell+population+is+enriched+in+tumor-initiating+cells.">Hierarchical organization of prostate cancer cells in xenograft tumors: the CD44+alpha2beta1+ cell population is enriched in tumor-initiating cells.</a> Cancer Res. 67, 6796-6805 (2007).</li><li>Qin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PSA(-/lo)+prostate+cancer+cell+population+harbors+self-renewing+long-term+tumor-propagating+cells+that+resist+castration.">The PSA(-/lo) prostate cancer cell population harbors self-renewing long-term tumor-propagating cells that resist castration.</a> Cell Stem Cell. 10, 556-569 (2012).</li><li>Siegel, R., Naishadham, D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2013.">Cancer statistics, 2013.</a> CA Cancer J. Clin. 63, 11-30 (2013).</li><li>Patrawala, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+purified+CD44++prostate+cancer+cells+from+xenograft+human+tumors+are+enriched+in+tumorigenic+and+metastatic+progenitor+cells.">Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells.</a> Oncogene. 25, 1696-1708 (2006).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 프로토콜은 신선한 인간의 전립선 암 조직에서 암줄 기세포의 분리에 대해 설명합니다. 몇 가지 중요한 고려 사항은이 프로토콜의 성공적인 결과에 영향을 미친다. 가능한 전립선 암 세포의 높은 숫자의 회복 수술 견본 조심 총 평가에 의존한다. 거시적 종양 결절이 관찰 10을 처리 할 때 우리의 경험에서, 종양 형성 전립선 세포의 성공적인 분리가 가장 확실하다....

종양 내 이질 암 (1)의 특징으로 간주됩니다. 실제로, 종양 내 이성의 몇 가지 메커니즘이 유전자 변이와 미세 환경과의 상호 작용을 포함하여 기술되었다. 또한, 일부 암은 직렬 이식 분석 2-5 종양 개시 용량과 자기 갱신의 특성을 나타내는 암 줄기 세포 (암줄 기세포)의 모집단으로 셀룰러 계층 구조를 포함 할 수있다. 초기에 혈액 6, 유방 7 뇌 8 악성 종양에 설명, 암줄 기세포는 전립선 암 9-12뿐만 아니라 다른 종양 유형 2-5에서 연구되고있다. 암줄 기세포는 일반적으로 이종 인구 2-5 내에서 세포 부분으로 간주됩니다. 따라서 암줄 기세포의 기능 및 분자 특성은 대량 집단에서 자신의 농축에 달려있다. 따라서, 지난 20 년간 여러 가지 사항이 충족CSC 농축 hodologies는 일반적으로 유동 세포 계측법에 의해 표시 인구의 분리를 포함하는 고안되었다. 또한, 암줄 기세포의 연구에 중요한 고려 사항은 인체 조직의 계층 적 조직 등의 문화 또는 면역 결핍 생쥐의 일련 계대으로 실험 조작에 의해 중단 될 수 있다는 것이다. 그 결과, 인간의 조직으로부터의 직접적인 CSC 추가 절연은 CSC 분야에서 중요한 방법으로 떠오르고있다. 전립선 암은 암 이환율 및 미국 및 세계 (13) 주변의 사망의 주요 원인이다. 따라서, 전립선 암줄 기세포의 분리 및 생물학적 특성은 상당한 관심입니다. 전립선 암줄 기세포는 이전 세포주, 환자 유래의 이종 이식, 낮은 통로 환자 파생 된 서스펜션 문화 9,10,12,14에서 풍성하게한다. 우리는 최근에 직접적으로 인간의 수술들에서 전립선 암줄 기세포의 분리를보고자신의 HLAI 음성 세포 표면 표현형 (10)의 미덕 amples. 여기서 자세하게는 이들 세포의 분리를 위해 구현 된 절차를 우리. 전립선 종양 수술 표본에서 수확 세포 현탁액으로 만들어집니다. 세포는 다음 HLAI에 대한 항체뿐만 아니라 간질과 생존 마커를 사용하여 염색하고, 암줄 기세포는 형광 활성화 된 셀 정렬 (FACS)에 의해 격리됩니다. 고립 된 암줄 기세포는 가능한 세포를 필요로하는 분석에 사용할 수 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="779">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RPMI</td>
			<td style="width:195px;">Gibco Life Technologies</td>
			<td style="width:187px;">11875-093</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Fetal bovine serum</td>
			<td style="width:195px;">Gibco Life Technologies</td>
			<td style="width:187px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Penicillin Streptomycin</td>
			<td style="width:195px;">Gibco Life Technologies</td>
			<td style="width:187px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">PBS</td>
			<td style="width:195px;">Corning cell gro</td>
			<td style="width:187px;">21-031-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">60 mm-15 mm cell culture dish</td>
			<td style="width:195px;">Corning</td>
			<td style="width:187px;">3295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">35 &#181;m Cell Strainer</td>
			<td style="width:195px;">BD Falcon</td>
			<td style="width:187px;">352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">50 ml conial tube</td>
			<td style="width:195px;">Crystalgen</td>
			<td style="width:187px;">23-2263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">15 ml conical tune</td>
			<td style="width:195px;">Crystalgen</td>
			<td style="width:187px;">23-2265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Red blood cell lysing buffer</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:187px;">R7757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">HLA class I (W6/32) PE antibody</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:187px;">ab43545</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">CD31 FITC antibody</td>
			<td style="width:195px;">ebioscience</td>
			<td style="width:187px;">11-0319-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">CD45 FITC antibody</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:187px;">ab27287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">IgG2a&#954; PE antibody</td>
			<td style="width:195px;">BD Pharminogen</td>
			<td style="width:187px;">555574</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">IgG1&#954; FITC antibody</td>
			<td style="width:195px;">BD Pharminogen</td>
			<td style="width:187px;">551954</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">DAPI</td>
			<td style="width:195px;">Invitrogen&#160;</td>
			<td style="width:187px;">d3571</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">12 x 75 mm polystyrene tubes with cell strainer cap</td>
			<td style="width:195px;">BD Falcon&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:187px;">352235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Vortex Mixer</td>
			<td style="width:195px;">Crystalgen</td>
			<td style="width:187px;">CG-BV1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">10% Neutral Buffer Formalin</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:187px;">RBBP-0480</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of cancer stem cells from human prostate cancer samples

암 줄기 세포 (CSC) 모델은 상당히 지난 20 년간 재 방문하고있다. 이 시간 동안 암줄 기세포를 확인하고 직접 고립 된 인간의 조직에서 순차적으로 일반적으로 유동 세포 계측법에 의해 세포와 분별의 모집단의 항체 라벨링을 통해, 면역 결핍 쥐에 전파되었다. 그러나, 전립선 암의 유일한 임상 적 특징은 상당히 신선한 인간 종양 샘플에서 전립선 CSC 추가의 연구를 한정 하였다. 우리는 최근 자신의 HLA 클래스 I (HLAI) 음성 표현형의 미덕에 의해 직접 인체 조직에서 전립선 암줄 기세포의 분리를보고했다. 전립선 암 세포는 수술 표본에서 수확 기계적으로 분해된다. 세포 현탁액은 생성과 형광 복합 HLAI과 기질 항체가 표시되어 있습니다. HLAI 부정적인 세포의 모집단은 마지막으로 유동 세포 계수기를 사용하여 격리됩니다. 이 프로토콜의 주요 제한은 자주 현미경 및 다 초점 본성전립선 절제 표본의 기본 암. 그럼에도 불구하고, 고립 된 라이브 전립선 암줄 기세포는 면역 결핍 마우스에 이식하여 분자 특성 및 기능 검증에 적합하다.

<img alt="그림 1" fo:content-width="5in" fo:src="/files/ftp_upload/51332/51332fig1highres.jpg" src="/files/ftp_upload/51332/51332fig1.jpg" /> 그림 1. 인간의 전립선 암 종양의 수확과 인간 전립선 암의 특정 집단을 설명 줄거리 유동 세포 계측법. (A) 종양 결절 세포 현탁액을 생성하기 위해 수확. 처리 된 샘플의 종양의 콘텐츠는 종래의 헤 마톡 실린 및 에오신 염색을 이용하여 확인된...

Watch this Scientific Journal Video about Isolation of Cancer Stem Cells From Human Prostate Cancer Samples at JoVE.com

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

직접 인체 조직에서 암 줄기 세포 (암줄 기세포)의 분리는 생물학적 특성 분석을위한 필수입니다. 또한 어려운 단계를 문제 해결에 대한 팁을 제공하면서이 원고는, 인간의 조직에서 전립선 암줄 기세포의 분리를위한 방법을 설명합니다.

인간의 전립선 암 샘플에서 암 줄기 세포의 분리

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

isolation-of-cancer-stem-cells-from-human-prostate-cancer-samples

Research

JoVE Journal

Medicine

14.0K 조회수.  Icahn School of Medicine at Mount Sinai.  직접 인체 조직에서 암 줄기 세포 (암줄 기세포)의 분리는 생물학적 특성 분석을위한 필수입니다. 또한 어려운 단계를 문제 해결에 대한 팁을 제공하면서이 원고는, 인간의 조직에서 전립선 암줄 기세포의 분리를위한 방법을 설명합니다. 

비디오: Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

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 ...

연구

의학

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

인간 치과 펄프의 줄기 세포의 분리, 특성화 및 비교 차별화 두 가지 방법을 사용하여 영구 치아에서 파생

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

기본 Preneoplastic 유방 상피 세포의 시간 경과 영상은 유방암의 유전자 변형 마우스 모델에서 파생

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

환자 유래 세포 배양 및 CD133의 절연<sup&gt; +</sup흑종에서&gt; Putative 암 줄기 세포

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

설치류 비뇨 부비동 간엽 및 인간 줄기 세포의 조직 재조합을 사용하여 인간의 전립선 상피의 형성

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

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  ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

인간 세포주에서 Chemoresistant 난소 암 줄기 세포에 대한 심화 학습

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

췌장암 새로운 치료 전략을 개발하기위한 전지 특성 줄기 공부

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

마우스 종양 모델에서 종양 세포 순환의 분리 및 전파

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).