우리는 세포 계측법 시설의 사용을위한 SF 4206 ICORE을 (캉 대학교) 감사합니다. JC는 헌법위원회 데 방사선 방호 디 EDF와 노르망디의 헌법위원회 지역에서 박사 후 장학금을 받았다. Comités 드 난 Orne의 등 뒤 칼바도스 - 해당 데이터는 리그 국립 contre 르 암에 의해 투자 프로젝트의 일환이었다.

1. C12FDG, Bafilomycin A1, 및 세포 배양의 준비 <ol><li> 20 mm의 최종 농도로 디메틸 설폭 사이드 (DMSO)의 C 12 FDG 가루를 녹여. -20 ° C.에서 나누어지는 및 저장 DMSO는 적절한 안전 조건을 사용해야합니다. </li><li> 0.1 mm의 최종 농도로 DMSO에 bafilomycin A1 가루를 녹여. 에서 나누어지는 및 저장 - 20 ° C. Bafilomycin는 적절한 안전 조건을 사용해야합니다. </li><li> 37 10 % 소 태아 혈청과 1 % 항생제, ° C, 5 % CO 2를 포함하는 미디어, RPMI 8226 세포주 또는 부착하거나되지 않은 다른 세포 라인을 육성. 실험에 필요한 세포의 수를 추정한다. 유동 세포 계측법 분석 중에 이벤트의 충분한 수를 기록하기 위해, 5 × 10 5 세포가 필요합니다. 노화 세포의 비율을 결정하는 세 가지 세포 샘플이 필요합니다 : 레이블 샘플, 치료 세포가 C 12 FDG와 염색 처리 된 세포는 염색C 12 FDG. </li><li> 세포가 세포 증식 곡선의 로그 상에 있도록 씨앗 세포를 24 시간 전에 실험을 수행. </li></ol> 2. 노화의 유도 <ol><li> chemotherapeutical 약물을 추가하여 암 세포의 노화를 유도. 약물 농도와 치료 기간은 시험 세포 유형에 적응해야한다. 약 10 6 세포 / ML에 세포의 농도를 50 nm의 독소루비신의 최종 농도에서 48 시간 처리를 시작합니다. (대조군) 치료 샘플을 포함하는 것을 잊지 마십시오. </li></ol> 3. Bafilomycin A1 치료 <ol><li> 이전 단계에서 사용 chemotherapeutical 약이 세포 사멸로 이어질 수도로, 판 블루 (V / V)로 혼합 세포에 의해 세포 생존을 확인합니다. Malassez 챔버를 입력하고 파란색 셀 (죽은 세포)와 백혈구 (살아있는 세포)의 수를 계산합니다. 생존의 비율이 70 % 미만인 경우 죽은 세포는 REM 수 있습니다보장하기 위해 적절한 장비를 사용하여 oved 죽은 세포 편견 후속 분석을하지 것이다. </li><li> 문화 미디어를 제거하고 prewarmed 신선한 배양액으로 교체합니다. bafilomycin A1 100 nm의 최종 농도로 세포를 처리합니다. Bafilomycin A1은 리소좀의 산성 산도를 중화하는 데 사용됩니다. 37 ° C, 5 % CO 2에서 1 시간을 품어. </li></ol> 4. C 12 FDG 염색법 <ol><li> 2 mm의 최종 농도 prewarmed 신선한 문화 매체에 C 12 FDG를 녹입니다. </li><li> 33 μM의 최종 농도에서 세포에 화합물을 추가하고 37 2 시간을 품어 ° C, 5 % CO 2. 비 부착 세포를 사용하는 경우, 4 ° C (속도는 사용되는 세포 유형에 따라 조정해야 할 수도 있습니다)에서 5 분 300 XG에서 세포를 원심 분리, PBS로 2 배 씻는다. 차가운 PBS 500 μL에있는 세포 펠렛을 resuspend. 자기편 세포를 사용하는 경우, 미디어를 제거하고 PBS로 2 배 씻는다. 트립신 용액 원심 분리기를 사용하여 부착 세포를 수확4시 5 분 300 XG에 세포 ° C (속도는 사용되는 세포 유형에 따라 조정해야 할 수도 있습니다). 차가운 PBS 500 μL에있는 세포 펠렛을 resuspend. 두 경우 모두, 세포 농도는 6 10 세포 / ㎖이어야한다. </li><li> 또한 노화 세포의 인구의 특성을 공동 immunolabeling의를 수행합니다. </li></ol> 5. 유동 세포 계측법 분석 <ol><li> 흐름 cytometer에의 제조업체에서 권장하는 제어 구슬을 사용하여 흐름 cytometer에를 보정합니다. 모든 단계는 적어도 10 4 이벤트가 기록되어야한다. </li><li> 레이블이없는 세포를 포함하는 첫 번째 샘플을 실행합니다. 두 매개 변수가 그래픽 표시 앞으로 분산 형 채널 (FSC) 대 사이드 분산 형 채널 (SSC)를 준비합니다. 광전 튜브 (PMTS) 설정 및 샘플 준비 중에 등장했을 수 있습니다 죽은 세포와 세포 파편을 제외하고 관심 영역을 정의합니다. 로그 스케일에 FL1를 표시 한 매개 변수 히스토그램 게이트 관심이 지역을x 축과 y 축에서 이벤트의 수. 이벤트의 총 수는 분석 세포의 수를 나타냅니다. 레이블이 지정되지 않은 세포가 X 축 로그 규모에 첫 10 년에 나타나도록 PMT를 설정합니다. 필요한 경우 세포의자가 형광을 결정합니다. </li><li> 치료 세포를 포함하는 두 번째 샘플을 실행합니다. FL1을 (C 12 FDG의 형광)를 표시 한 매개 변수 히스토그램에서 희미하게 표시 인구 후 커서를 놓습니다. 이 임계 값은 비 - 노화 세포 (희미하게 표시 셀) 및 노화 세포 (밝게 표시된 셀) 사이의 차별을 허용합니다. </li><li> 치료 세포를 포함하는 세 번째 샘플을 실행합니다. 밝게 셀 라벨, 즉 노화 세포는 이전 단계에서 결정된 임계 값 이상으로 나타납니다. 이벤트의 총 수 당 밝은 이벤트의 수를 나누어 노화 세포의 비율을 평가한다. 필요한 경우, β-갈 락토시다 제의 활성은 평균 형광을 사용하여 추정 할 수있다강도. </li></ol>

<ol>
	<li>Hayflick, L., Moorhead, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+serial+cultivation+of+human+diploid+cell+strains.">The serial cultivation of human diploid cell strains.</a> Experimental Cell Research. 25, 585-621 (1961).</li><li>Campisi, J., d'Adda di Fagagna, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+senescence:+when+bad+things+happen+to+good+cells.">Cellular senescence: when bad things happen to good cells.</a> Nature Review Molecular Cell Biology. 8 (9), 729-740 (2007).</li><li>Shay, J. W., Roninson, I. B. <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+senescence+in+carcinogenesis+and+cancer+therapy.">Hallmarks of senescence in carcinogenesis and cancer therapy.</a> Oncogene. 23 (16), 2919-2933 (2004).</li><li>Bartkova, J., Rezaei, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogene-induced+senescence+is+part+of+the+tumorigenesis+barrier+imposed+by+DNA+damage+checkpoints.">Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints.</a> Nature. 444 (7119), 633-637 (2006).</li><li>Takacova, S., Slany, R., Bartkova, J., Stranecky, V., Dolezel, P., Luzna, 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=DNA+damage+response+and+inflammatory+signaling+limit+the+MLL-ENL-induced+leukemogenesis+in+vivo.">DNA damage response and inflammatory signaling limit the MLL-ENL-induced leukemogenesis in vivo.</a> Cancer Cell. 21, 517-531 (2012).</li><li>Krtolica, A., Parrinello, S., Lockett, S., Desprez, P. Y., Campisi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescent+fibroblasts+promote+epithelial+cell+growth+and+tumorigenesis:+a+link+between+cancer+and+aging.">Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging.</a> Proceedings of the National Academy of Sciences. 98, 12072-12077 (2001).</li><li>Coppe, J. P., Patil, C. K., Rodier, F., Sun, Y., Munoz, D. P., Goldstein, 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=Senescence-associated+secretory+phenotypes+reveal+cell-nonautonomous+functions+of+oncogenic+RAS+and+the+p53+tumor+suppressor.">Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor.</a> PLoS Biology. 6, 2853-2868 (2008).</li><li>Roninson, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+senescence+in+cancer+treatment.">Tumor cell senescence in cancer treatment.</a> Cancer Research. 63 (11), 2705-2715 (2003).</li><li>Dimri, G. P., Lee, X., 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+biomarker+that+identifies+senescent+human+cells+in+culture+and+in+aging+skin+in+vivo.">A biomarker that identifies senescent human cells in culture and in aging skin in vivo.</a> Proceedings of the National Academy of Sciences. 92 (20), 9363-937 (1995).</li><li>Kurz, D. J., Decary, S., Hong, Y., Erusalimsky, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescence-associated+(beta)-galactosidase+reflects+an+increase+in+lysosomal+mass+during+replicative+ageing+of+human+endothelial+cells.">Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells.</a> Journal of Cell Science. 113, 3613-3622 (2000).</li><li>Debacq-Chainiaux, F., Erusalimsky, J. D., Campisi, J., Toussaint, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+to+detect+senescence-associated+beta-galactosidase+(SA-betagal)+activity,+a+biomarker+of+senescent+cells+in+culture+and+in+vivo.">Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo.</a> Nature Protocols. 4 (12), 1798-1806 (2009).</li><li>Cahu, J., Bustany, S., Sola, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescence-associated+secretory+phenotype+favors+the+emergence+of+cancer+stem-like+cells.">Senescence-associated secretory phenotype favors the emergence of cancer stem-like cells.</a> Cell Death and Disease. Dec 20, 3-e446 (2012).</li></ol>

관심 없음 충돌 선언하지 않습니다.

우리는 여기에서 살아있는 암세포의 세포 노화를 정량화하는 유동 세포 계측법 기반 방법을 제시 하였다. 이 방법은 막 투과 화합물 C 12 FDG의 사용을 기반으로하며, 화합물은 세포에 의해 촬영 그러므로 어떤 종류의 세포와 장기 등 다양한 치료 후 사용할 수 있습니다. 세포 흡수시, C 12 FDG는 노화 세포의 리소좀에 풍부한 β-갈 락토시다 아제에 의해 가수 분해됩니다. 레이저 여기 ...

60 년대 초반과 헤이 플릭 및 무어 헤드의 발견 이후, 노화는 여전히 휴대 호기심 1 남아있다. 인간의 세포는 무한정 노화에 의해, 헤이 플릭 및 무어 노화의 세포 프로세스를 만들어 낸, 확산되지 않습니다. 노화는 세포의 죽음에 반대하는 세포 대사 활성 상태로 유지하는 안정적인 세포주기 정지됩니다. 텔로미어 단축, DNA 손상, 염색질 교란 및 세포 분열 종양 유전자의 이상 발현 2 일에 네 개의 세포 메커니즘은 노화를 유발하는 것으로 알려져있다. 이것은 정상 세포뿐만 아니라 의미뿐만 아니라 암 세포가 노화 3을받을 수 있습니다. 사실, 노화가 진행 암 세포는 더욱 종양의 진행 4-5 장벽을 구성하는 표시했다. 그러나 몇몇 보고서는 이제 특정 상황에서, 세포의 노화는 악성 6-7을 촉진 할 수 있다고 지적했다. 암 CEL의 노화를 연구LS는 현재 화학 요법 약물은 체외에서뿐만 아니라 생체 8뿐만 아니라 암 세포의 노화로 이어질 수 있습니다 사용되는 암 연구 충동이되기에 관하여이다. 노화 세포의 존재 여부를 조사하기 위해 마스터 분석은 산성 pH에서 β-갈 락토시다 분석이다. 이 조직 화학적 분석은 대부분 노화 세포에서 발생하는 리소좀 생합성 증가를 반영합니다. 간단히, 세포에 적용 외생 X-GAL는, 파랑 색 9 상승을주는, 노화 세포의 리소좀에 풍부한 β-갈 락토시다 제에 의해 절단되어있다. 블루 노화 세포는 그 시야 현미경으로 정량화 할 수있다. 매우 인기가 있지만, β-갈 락토시다 분석은 주요 단점을 제공합니다. 첫째,이 분석은 고정 된 세포에서 수행됩니다. 그것은 현미경으로 노화 세포의 상당히 높은 숫자를 계산하여 노화의 정도를 정량화하는 것을 의미하기 때문에 둘째, 시간이 소요됩니다. 셋째,이 분석노화 및 비 - 노화 세포 사이의 차별은 전적으로 관찰자에 의존로 구분하지 않습니다. 우리는 여기에 살고 노화 세포의 존재 여부를 평가하기 위해 개발되지 않은, 빠른, 민감한 세포 계측법 기반의 분석을 설명합니다. 이 분석은 막 투과 분자의 분해, 5 dodecanoylaminofluorescein 디-β-D-갈 락토 피 라노 (C 12 FDG)을 기반으로, β-갈 락토시다 아제에 의해 노화 세포 충실. 가수 분해 및 레이저 여기 후, C 12 FDG 녹색 형광을 방출하기 때문에 유동 세포 계측법 10, 11에 의해 감지 할 수 있습니다. 유동 세포 계측법을 통해 노화 세포의 검출은 신속하고 민감의 큰 장점을 제공합니다. 또한, 유동 세포 계측법에 의한 노화 세포의 검출은 다른 세포 마커의 검출과 결합 할 수 있습니다. 이 분석은 화학 요법으로 치료 암 세포의 인구 내에서 노화 세포를 감지하는 데 사용할 수 있으며, 정기적으로 설정할 수 있습니다최대 조직 절편에서 세포 분리 후, 진료소합니다.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the Reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>5-dodecanoylaminofluorescein di-&beta;-D-galactopyranoside </td>
			<td>Invitrogen</td>
			<td>D-2893</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dimethyl sulfoxide </td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Bafilomycin A1</td>
			<td>Sigma</td>
			<td>B1793</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Doxorubicin</td>
			<td>Sandoz</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypan blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		 </tr>
		 <tr>
			<td>RPMI 1640 cell culture medium</td>
			<td>Lonza</td>
			<td>BE12-702</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal calf serum</td>
			<td>PAA Laboratories GmBH</td>
			<td>A15 101</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Antibiotics</td>
			<td>Gibco</td>
			<td>5290-018</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gallios flow cytometer</td>
			<td>Beckman Coulter</td>
			<td>A94291</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

a sensitive method to quantify senescent cancer cells

인간의 세포는 무한정 증식하지 않는다. 외부 및 / 또는 내재적 단서에 따라, 세포는 죽거나 노화라는 안정적인 세포주기 정지를 입력 할 수 있습니다. 이러한 텔로미어 단축과 세포 분열 종양 유전자의 이상 발현 등 여러 세포 메커니즘은, 노화를 유발하는 것으로 알려져있다. 노화는 정상 세포에 국한되지 않고, 암 세포는 senesce보고되었다. Chemotherapeutical 약물은 암 세포의 노화를 유발하는 것으로 알려져있다. 그러나 노화를 방지하거나 종양을 촉진하는지 논란이 남아있다. 그것은 결국 환자 특정 할 수 있듯이, 암 세포의 노화를 평가하기 위해 신속하고 민감한 방법은 빨리해야합니다. 이를 위해, 표준 β-갈 락토시다 분석, 현재 사용되는 방법은 주요 단점을 제공합니다 : 그것은 시간이 걸리고 문자를 구분하지 않습니다. 우리는 여기에서 리튬의 노화를 연구하는 유동 세포 계측법 기반 분석을 제안한다세포를 쉬게. 이 분석은 민감하고 신속하다는 장점을 제공하며, 다양한 세포 마커의 immunolabeling의에 결합 할 수 있습니다.

다발성 골수종, 혈액 악성 종양은 chemotherapeutical 절차에 의해 유도 된 노화를 평가하기 위해 사용되었다. 상업적으로 이용 가능한 MM 세포 라인 (RPMI 8226) 독소루비신, chemotherapeutical 약 2 일 동안 처리 하였다 이렇게하려면. 세포는 다음 bafilomycin A1 치료를 받게하고 추가 C 12 FDG와 함께 배양 하였다. 세포 유동 세포 계측법에 의해 분석 하였다. 그림 1은 노화 세포의 비율이 C 12...

Watch this Scientific Journal Video about A Sensitive Method to Quantify Senescent Cancer Cells at JoVE.com

A Sensitive Method to Quantify Senescent Cancer Cells

노화가 방지하거나 종양 논란이 계속 추진할지 여부를 지정합니다. chemotherapeutical 마약 senesce하는 암 세포를 유도 할 수 있기 때문에, 노화를 연구하는 새로운 치료법을 제안 필수적이다. 그러나, 표준 및 널리 사용되는 β-갈 락토시다 분석은 주요 단점을 제공합니다. 우리는 여기에서 노화를 정량화 할 수있는 신속하고 민감한 유동 세포 계측법 기반 분석을 제안한다.

노화 암 세포를 정량화하는 민감한 방법

Human cells do not indefinitely proliferate. Upon external and/or intrinsic  cues, cells might die or enter a stable cell cycle arrest called senescence. ...

a-sensitive-method-to-quantify-senescent-cancer-cells

Research

JoVE Journal

Medicine

20.5K 조회수.  Université de Caen Basse-Normandie. 노화가 방지하거나 종양 논란이 계속 추진할지 여부를 지정합니다. chemotherapeutical 마약 senesce하는 암 세포를 유도 할 수 있기 때문에, 노화를 연구하는 새로운 치료법을 제안 필수적이다. 그러나, 표준 및 널리 사용되는 β-갈 락토시다 분석은 주요 단점을 제공합니다. 우리는 여기에서 노화를 정량화 할 수있는 신속하고 민감한 유동 세포 계측법 기반 분석을 ?...

비디오: A Sensitive Method to Quantify Senescent Cancer Cells

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Murine 아일릿 절연 및 Subcapsular 신장 이식을위한 방법

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

연구

의학

Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. Conventional methods assess neurological deficits and electrophysiology and quantitative sensory testing quantifies functional alterations to detect neuropathy. However, the earliest damage appears to be to the small fibres and yet these tests primarily assess large fibre dysfunction and have a limited ability to demonstrate regeneration and repair. The only techniques which allow a direct examination of unmyelinated nerve fibre damage and repair are sural nerve biopsy with electron microscopy and skin-punch biopsy. However, both are invasive procedures and require lengthy laboratory procedures and considerable expertise. Corneal Confocal microscopy is a non-invasive clinical technique which provides in-vivo imaging of corneal nerve fibres. We have demonstrated early nerve damage, which precedes loss of intraepidermal nerve fibres in skin biopsies together with stratification of neuropathic severity and repair following pancreas transplantation in diabetic patients. We have also demonstrated nerve damage in idiopathic small fibre neuropathy and Fabry's disease.

Corneal Confocal microscopy is a non-invasive clinical technique which may be used to quantify C fibre damage to diagnose and stratify patients with increasing neuropathic severity.

각막 공촛점 현미경 : 주변 Neuropathies에 작은 광섬유 병리를 정할 소설 비침습 기술

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. ...

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

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

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

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

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

마우스에 Intracranial 이종 이식 연구에 대한 간단한 안내 스크류 방법

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

세 가지 차원 문화 : 유방 세포의 악성 변형 대 일반 선포 건축을 연구하는 도구

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

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 ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

고체 시편에서 기본 난소 암 세포를 얻는 방법

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

<em&gt; 체외</em&gt; 골수에서 에스트로겐에 민감한 유방암의 휴면 모델 : 분자 메커니즘 연구 및 가설 생성을위한 도구

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

소프트 한천 콜로니 형성 분석의 활용은 유방암 세포에서 종양 유발의 억제를 확인하는

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

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.