이 연구의 자금 출처 : 텔 아비브 대학의 기술 이전 회사 라모트와 시바 - 메디컬 센터 (M. CA 및 SI.) ICRF의 공동 기금 - 이스라엘 암 연구 재단 (M. CA.)와 이스라엘 과학 재단 ( SI).

1. 세포 배양 준비 MDA-MB-231 세포는 ATCC (미국 유형 문화 수집)에서 구입하여 액체 질소에 보관 하였다. <ol><li> Dulbeco 수정 이글 중간 (DMEM), 10 % 말 혈청, 1 % L-글루타민, 1 % Penstrep-Amphotericine B를 포함하는 10 ML 완전 배지 92 mm 직경 페트리 접시에 씨앗 10 6 MDA-MB-231 세포는 세포를 허용 80 ~ 100 % 정도의 합류로 증식한다. </li><li> 요리에서 배지를 제거하고 폐기합니다. </li><li> 0.25 % 혈청의 모든 흔적을 제거하는 (W / V) 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) 솔루션을 짧게 세포 층을 씻으십시오. </li><li> 세포 층 (보통 5 - 15 분 이내) 분산 될 때까지 거꾸로 현미경으로 세포를 접시 관찰 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) 솔루션의 2.0 ML를 추가합니다. </li><li> 18 ML 전체 성장 매체를 추가하고 부드럽게 피펫으로 세포를 대기음. 튜브에 전송할 수 있습니다. </li><li> 1,200 rpm으로 세포 현탁액을 원심 분리기. </li><li> CU의 24 ML에 세포 펠렛을 다시 일시 중지매체를 lture. </li><li> 35mm 유리 바닥 요리 (25 % 합류에 대한) 및 인큐베이터의 장소 (5 % CO 2, 37 ° C)에 세포 현탁액 2 ㎖를 추가합니다. </li><li> 솔루션 : <ol><li> 세포 증식을위한 완벽한 매체 : 10 % FBS와 DMEM, 1 % 항생제 (100 단위 / ML 페니실린 G, 100 ㎍ / ㎖ 스트렙토 마이신, 펜 연쇄상-Ampho 솔루션), 2 mM의 L-글루타민. </li><li> 0.25 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)를 포함하는 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) 솔루션. </li></ol></li><li> 요리 : 92mm 직경 페트리 접시. 35mm 직경 폴리 - D - 라이신 코팅 유리 바닥 문화 요리. </li></ol> 2. 라이브 공 촛점 이미징 세포의 준비 <ol><li> 시드 2 × 10 제 1 항에서 언급 한 2 ㎖ 완전 배지에서 유리 바닥 문화 요리 5 MDA-MB-231 세포. 세포 배양은 60 % ~ 70 %의 합류 (접시 당 3-4 × 10 5 세포에 대한)에 도달하면, 형질 진행합니다. </li><li> 두 개의 플라스미드는 엔코딩으로 세포를 형질제조 프로토콜에 따라 리포솜의 transfection 시약 제트-PI를 사용하여 융합 단백질 γ-tubulin의-GFP (중심체의 형광 검출)와 히스톤-RED (H2B-RED, 염색체의 형광 검출을) ING. 간단히, 100 ㎕의 염화나트륨 (150 ㎜)과 관의 각 플라스미드의 2 μg을 혼합한다. 두 번째 튜브에 100 ㎕의 염화나트륨 (150 밀리미터)로 형질 시약 (100 μL)를 혼합하고 실온 (RT)에서 5 분 알을 품다. 다음 두 가지 솔루션, 혼합 (약한 소용돌이를 사용하여) 결합 스핀 다운. RT에서 30 분 알을 품다. </li><li> 형질 혼합물의 배양 기간 동안 PBS로 두 번 세포를 씻으없이 보충 따뜻한 DMEM 2 ㎖ (37 ° C)로 세포 매체를 교체하십시오. </li><li> 부드럽게 DMEM에 세포의 transfection 혼합물을 추가하고 8 시간 동안 (37 ° C, 5 % CO 2) 배양기에 세포를 반환합니다. </li><li> 24 시간 동안 배양 8 시간 후 2 ㎖ 완전 배지와 DMEM를 교체하고 인큐베이터에서 세포를 배양시간. </li><li> 24 시간 형질 전환 한 후 2 ㎖ 완전 배지는 20 μM PJ-34를 포함하여 세포의 매체를 교체하십시오. </li><li> 추가 18 시간 (37 ° C, 5 % CO 2)에 대한 세포를 품어. </li><li> 5 % CO 2, 37 ℃에서 세포를 유지 이미징 챔버에 적어도 16 시간 동안 공 촛점 이미징을 살 수있는 대상 세포 </li><li> 병렬로, 다음과 같이 형광 현미경을 사용하여 형질 효능 36 시간 게시물 형질을 검사 : <ol><li> 시드 2 × 10 1 coverslip에 잘 당 2 ㎖ 완전한 매체를 포함하는 6 - 잘 접시에 5 MDA-MB-231 세포. </li><li> 섹션 2.2-2.5에서 언급 한 세포를 형질. </li><li> 36 시간 포스트 transfection에 차가운 메탄올에 부화하여 coverslip을에 장착 된 형질 전환 세포 수정 : 아세톤 (1:1) 용액, 7 분, -20 ° C. </li><li> 고정 솔루션을 기음과 화학 후드에서 건조 장착 세포와 coverslip을 보자. </li><li> DAPI와 골드 antifade 시약을 연장하고 일을하게 적용전자 coverslip에 6 시간 동안 어둠 속에서 건조. </li><li> 형광 현미경 슬라이드를 검사하고 세포의 전체 인구 (DAPI에 의한 DNA의 얼룩)에서 형질 세포 (적색 및 녹색 신호)의 비율을 계산합니다. 100-200 세포가 계산 될 때 원하는 형질의 비율이 20-40 %이다. </li></ol></li></ol> 3. 라이브 공 촛점 이미징 스캐너 설정의 기술적 인 매개 변수 <ol><li> ScanMode XYZT; 구멍 [통풍] 1.00, 줌 3.5, 해상도 8 비트, 레이저 DPSS 561 nm의, 아르곤, 가시 레이저 488 nm의, 레이저 그 / 네 눈에 보이는 633 nm의, 목표 HCX PL APO CS 63X 1.40 OIL UV, 수치 조리개 1.4; 굴절률 1.52, 속도 700 Hz에서 검색합니다. </li><li> 이미지를 3 차원 프레 젠 테이션 IMARIS 이미징 소프트웨어 7.0으로 제조 하였다. </li></ol> 4. 고정 된 세포의 유사 분열의 공 촛점 이미징 <ol><li> 시드 2 × 10 5 MDA-MB-231 유방암 세포 (ATCC), 정상 마우스 배아 섬유 아세포 (MEF) 또는 PARP1 부족 ME2 ㎖ 완전 배지에서 6 - 잘 접시에 유리 coverslips는에 F (박사 프랑소와 Dantzer에 의해 제조 PARP - - /). 의 coverslips는 2 시간 건조하고, 6 - 잘 접시의 각 우물에 배치 멸균 DD 물로 세척하여 다음, 96 % 에탄올로 세척 하였다. </li><li> 매체에 PJ-34 (10-30 μM)를 추가하고 필요한 기간 (일반적으로 최대 96 시간)에 대한 세포를 품어. </li><li> PBS로 한 번 coverslips를 세척 (인산염 완충 식염수), 그리고 얼음처럼 차가운 메탄올에서 배양하여 세포를 고정 : 아세톤 (1:1) 용액, 7 분, -20 ° C. </li><li> 고정 솔루션을 기음과 coverslips를 화학 후드에서 건조하자 (이 단계에서, coverslips를 여러 주 동안 -20 ° C에 보관 될 수 있습니다). </li><li> 세포막을 permeabilize 및 RT에서 1 시간 PBST 10 % NDS (정상 당나귀 혈청) ( &#39;차단 솔루션&#39;)로 세포를 차단하는 PBST (PBS 0.1 % 트윈 - 20과 보충) 한 번 coverslips를 씻으십시오. </li><li> 기본 antibodi와 투과 고정 된 세포를 배양RT에서 2 시간 동안 ES (스핀들 및 중심체 염색 용). 반대로 α 튜 블린 (1:250 희석) 및 안티-γ 튜 블린 (1:200 희석) : 항체는 것과 같이 따른다 차단 용액에 희석됩니다. 일차 항체는 다음과 같이 적용됩니다 : 6 잘 플레이트 커버 (덮개가 거꾸로) 각 coverslip에 대한 솔루션을 차단하는 항체의 혼합물 100 μL를 (드롭) 적용됩니다. 부드럽게 항체 드롭 coverslip을 넣어 시드 셀의 드롭에 직면. 실온에서 2 시간 동안 항체를 직면 coverslips를 품어. </li><li> 우물에 다시 coverslips를 배치하고 PBST로 세포를 3 회 세척한다. 다음 형광 차 항체의 coverslips에서 세포를 라벨링 4.6에서 설명한 동일한 절차를 사용합니다. 어둠 속에서, 1 시간, RT에 대한 보조 항체의 coverslips에서 세포를 품어. 항체는 다음과 같은 차단 솔루션에 희석되어 있고, 알렉사 플 루어 568 (1:1,000 희석 알렉사 플 루어 488 (녹색 1:1,000 희석)빨간색). </li><li> 를 사용하여 coverslips를이 DAPI (염색체의 염색을 위해)와 금 antifade 시약을 연장하고 건조 어둠 속에서 RT에서 밤새 배양 마운트합니다. </li><li> 공 초점 현미경으로 커버 전표를 확인합니다. </li></ol> 5. 세포 생존율은 ATP 생산에 의해 측정 ATP 생산은 발광 ATP 탐지 분석 장비로 측정됩니다. <ol><li> 96 - 웰 플레이트 각 well에 800 ㎕의 중간에있는 약 20,000 세포의 씨앗 세포를. 세 빈 우물 매체의 배경 발광 측정을 위해 사용되어야한다. </li><li> 약 10 μM에서 100 μM의 ATP 표준 희석 시리즈를 준비하고 얼음에 보관하십시오. </li><li> 각 웰에 세제의 50 μl를 추가하고 궤도 셰이커, 700 rpm으로 5 분 동안 접시를 흔들. </li><li> 키트 &#39;기질 버퍼&#39;의 5 mL로 &#39;동결 건조 된 기질&#39;의 재구성 각각의 병. </li><li> 우물에 재구성 기질 용액 50 μl를 추가하고 흔들어궤도 셰이커, 700 RPM에서 5 분 판. </li><li> 10 분 동안 어둠 속에서 판을 보관하십시오. </li><li> ELISA 마이크로 플레이트 리더 서로 잘 luminiscence을 측정합니다. </li></ol>

<ol>
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저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

공 촛점 이미징 유사 분열 동안 라이브 멀티 centrosomal 세포 (그림 3 및 보충 정보)의 PJ-34의 세포 독성 효과를 실시간으로 문서를 제공 살고 있습니다. 이 PJ-34 5-9으로 유사 분열 재해 세포 사멸의 유도를 제안, 여분의 중심체 드 클러스터링 및 세포 죽음에 인간의 암 세포에있는 PJ-34의 세포 독성을 돌리는 최초의 살아있는 문서이다. 대조적으로, 최고 numerary 중심체의 이중 초점 ...

페난 트렌는, PJ-34 등 PARP1 억제제, 파생 스트레스 조건 (뇌졸중이나 심근 경색 1)에 따라 에너지 소비 PARP1 매개 DNA 수선에 의해 유도 된 세포 사멸 세포 사멸에서 정지 세포를 보호하도록 설계되었습니다. 그러나 최근 우리는 PJ-34, 그 유도 PARP1 억제보다 두 배 높은 농도에서 독점적으로 인간의 암 세포 2,3에서 세포 죽음을 일으킬 수 있다는 것을 발견. 세포의 더 급속한 확산이었다, 세포의보다 효과적인 근절했다. PJ-34의 세포 독성 활동은 여분 중심체 유사 분열 2 드 클러스터링에 기인 하였다. 사람의 암세포 항구는 4,5 multicentrosomes. 효율적으로 유사 분열에서 두 개의 중심체를 숨겨 정지 세포 또는 일부 양성 증식 세포를 저해하지 않고 72-96 시간 이내에 세포를 박멸 20 μM PJ-34와 여분 중심체를 항구 인간의 유방암 세포 MDA-MB-231의 배양 <suP&gt; 2,3. 양성 세포는 인간의 흉선에서 준비 MCF-10 인간 유방 상피 세포, 인간 내피 세포 (HUVEC) 및 기본 간엽 세포가 포함되어 있습니다. 이러한 세포는 PJ-34의 세포 독성에 저항했다. PJ-34 96 시간 배양 동안 자신의 세포주기 방해하거나 자신의 중심체와 이중 초점 스핀들 형성 2,3에 영향을 미치지 않았다. 양극성 중심체 어셈블리는 유사 분열 4,5 양극성 스핀들 형성에 매우 중요합니다. 따라서 두 개 이상의 중심체를 가진 세포는 두 개의 기둥 4-9에서 자신의 별도의 중심체를 클러스터링, 거의 이해하지 분자 메커니즘을 개발했습니다. 자신의 중심체의 양극 어셈블리의 실패 체포 G2 / M 정지의 세포주기 및 세포 죽음에 이르게 4,5 유사 분열 장애에 기인하는 다극 왜곡 스핀들 및 탈선 염색체 분리가 발생할 수 있습니다. 여분 중심체 드 클러스터링을 기본 분자 메커니즘을 집중적으로 조사하는 &lt;&gt; 10을 먹다. 건강한 조직 5,10을 살려주는 동안이 죽음의 메커니즘을 이해하는 것은 암 세포의 독점적 인 박멸을 가능하게 할 것이다. 따라서, 유사 분열 재앙 세포 죽음을 활성화 화합물은 공 촛점 이미징 여분 중심체 클러스터링에 영향을 미치는 분자를 식별하는 데 사용할 수있는 것이 좋습니다 인간의 고체 cancers.Our 결과의 넓은 범위에서 효율적일 수 있습니다 선택적 암 치료의 새로운 모드를 제공 유사 분열 2,3, 약물 후보를 대상으로 이들 화합물의 암을 렌더링합니다. 우리는 고정 살아있는 인간의 암 세포 (유사 분열의 여분 중심체의 높은 발생에) 대 정상 세포를 스캔하여 phenanthridine PJ-34의 세포 독성 활성을 기록했다. 단계별로 인간의 암 세포 PJ-34의 세포 독성 활동을 식별하는 데 사용되는 이미징 절차의 단계 설명은 아래에 포함되어 있습니다.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS </td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen (GIBCO)</td>
			<td>41965</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS (Fetal bovine Serum)</td>
			<td>Invitrogen (GIBCO)</td>
			<td>12657</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen-Strep-Ampho solution</td>
			<td>Biological Industries, Israel</td>
			<td>03-033-1B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen (GIBCO)</td>
			<td>25030-024</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.25% Tripsin-EDTA</td>
			<td>Invitrogen (GIBCO)</td>
			<td>25200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>92 mm Petri dishes</td>
			<td>Nunc,Thermo scientific</td>
			<td>150350</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm poly-D-lysine coated glass bottom culture dishes</td>
			<td>MatTek Corporation, USA</td>
			<td>P35GC-0-14-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Luminescent ATP detection assay kit</td>
			<td>Abcam</td>
			<td>ab113849</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NDS (Normal Donkey Serum)</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti &alpha;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T9026</td>
			<td>1:250 dilution (IF)</td>
		</tr>
		<tr>
			<td>Anti &gamma;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T5192</td>
			<td>1:200 dilution (IF)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat Anti-Mouse IgG</td>
			<td>Invitrogen</td>
			<td>A-11017</td>
			<td>1:1,000 dilution (IF)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Donkey Anti-Rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A-10042</td>
			<td>1:1,000 dilution (IF)</td>
		</tr>
		<tr>
			<td>ProLong Gold antifade reagent with DAPI (mounting)</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>JetPEI (liposomal transfection reagent )</td>
			<td>Polyplus</td>
			<td>101-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica (Mannheim, Germany)</td>
			<td>TCS SP5II</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

cell death associated with abnormal mitosis observed by confocal imaging in live cancer cells

강력한 PARP1 억제제 역할을 페난 트렌 유도체 유사 분열의 멀티 centrosomal 인간의 암 세포에있는 여분 중심체의 이중 초점 클러스터링을 막았다. phenanthridine PJ-34은 가장 강력한 분자이다. 여분 중심체의 Declustering 멀티 centrosomal 세포에서 유사 분열 장애 및 세포 죽음을 발생합니다. 대부분의 고체 인간의 암은 여분 중심체의 높은 발생했습니다. PJ-34의 활동은 살아있는 인간의 유방암 중심체에 매우 풍부 형광 γ-tubulin의를 위해 인코딩 벡터에과 형광 히스톤 H2B 선물로 형질 전환 된 MDA-MB-231 세포의 공 촛점 이미징에 의해 실시간으로 기록되었다 염색체. 비정상적인 염색체 배열 및 declustered 중심체를 나타내는 데 클러스터 γ-tubulin의 병소는 PJ-34 치료 후 형질 전환 MDA-MB-231 세포에서 발견되었다. 두 개의 스핀들 극 유엔 클러스터 여분 중심체는 세포의 죽음을 앞에. 이러한 결과는 FO를 연결R 처음 여분 중심체 유사 분열에 드 클러스터링 및 세포 사망에 이르는 세포 분열 장애를 가진 인간의 암 세포에있는 PJ-34의 최근 발견 독점 세포 독성 활동. 두 개의 중심체와 이중 초점 스핀들과 분열을 겪고 정상 세포를 저해하지 않고 고정 된 세포 다 중심체와 PJ-34 전용 근절 암 세포의 공 촛점 이미징에 의해 관찰 이전의 연구 결과에 따르면. PJ-34이 세포 독성 활성이 다른 강력한 PARP1 억제제에 의해 공유되지 않은, 그리고 PARP1 억제의 독립성을 제안, PARP1 부족 MEF 숨겨 extracentrosomes에서 관찰되었다. 공 촛점 이미징을 살고 유사 분열 동안 세포를 근절 새로운 분자를 식별하기위한 유용한 도구를 제공했다.

PJ-34 phenanthridine 1 용해 안정적인 물 (그림 1)입니다. 우리의 이전 결과 세포의 죽음을 발표하고 PJ-34로 치료했다 고정 멀티 centrosomal 암 세포의 여러 유형의 엑스트라 중심체를 드 클러스터. 대조적으로, 일반적인 증식 세포는 2,3 손상되지 않았다. 중심체는 고정 여분 centrosomal 세포 2 centrine1 및 γ-tubulin에 대한 항체를 가진 두 배 레테르를 붙이기에 의해 확인되?...

Watch this Scientific Journal Video about Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells at JoVE.com

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

유사 분열을 겪고있는 암 세포에있는 phenanthridine PJ-34의 세포 독성 활동은 라이브 공 촛점 이미징 실시간으로 기록되었다. PJ-34 근절 인간의 유방암 MDA-MB-231 세포의 유사 분열에 여분의 중심체를 숨겨 세포. 정상 이중 초점 유사 분열과는 달리, 여분 중심체는 PJ-34의 존재에있는 두 개의 스핀들 극에 클러스터되지 않았습니다.

비정상적인 유사 분열과 관련된 세포의 죽음은 라이브 암 세포의 공 촛점 이미징에 의해 관찰

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

cell-death-associated-with-abnormal-mitosis-observed-confocal-imaging

Research

JoVE Journal

Medicine

14.8K 조회수.  Sheba Medical Center. 유사 분열을 겪고있는 암 세포에있는 phenanthridine PJ-34의 세포 독성 활동은 라이브 공 촛점 이미징 실시간으로 기록되었다. PJ-34 근절 인간의 유방암 MDA-MB-231 세포의 유사 분열에 여분의 중심체를 숨겨 세포. 정상 이중 초점 유사 분열과는 달리, 여분 중심체는 PJ-34의 존재에있는 두 개의 스핀들 극에 클러스터되지 않았습니다.

비디오: Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

재조합 Adeno - 관련 바이러스 혈청형 3 벡터에 의해 간암 세포의 고효율 전달

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

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의학

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

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

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

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

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

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

Compartmentalizing 마이크로 유체 장치와 라이브 세포 이미징을 사용하여 암 줄기 세포 마이그레이션 평가

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

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

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

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

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

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

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Azoxymethane (AOM)과 Dextran의 황산 나트륨 (DSS)와 모델링 대장염 - 관련 암

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

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 ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

유방암의 마우스 모델에서 종양 Microenvironment에 약물 응답의 영상을 라​​이브

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

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

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

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

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

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

Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination of dead cells in stroke by waste-management phagocytes of any cellular lineage. Although numerous cells of different origins that are capable of phagocytosis are present in ischemic brain, only part of them actively engulf and digest cell corpses. The selective visualization, quantification and analysis of such active phagocytic waste-management are helpful in assessing brain response to ischemia. Efficient cell death clearance is important for brain recovery from ischemic injury, as it opens the way for the subsequent regenerative processes. The failure to clean the corpses would result in a toxic reaction caused by non-degraded DNA and proteins. The described procedure uses fluorescent probes selectively ligated by a viral topoisomerase to characteristic DNA breaks produced in all phagocytes during engulfment and digestion of cells irreversibly damaged by ischemia. The method is a new tool for the investigation of brain reaction to ischemic injury.

We present a new fluorescence technique for selective in situ labeling of active phagocytic cells, which clear off cell corpses in stroke. The approach is important for assessing brain reaction to ischemia because only a small proportion of phagocytes present in ischemic brain participate in clearance of cell death.

Ligatable 형광 프로브에 의한 실험 스트로크에서 세포 죽음의 식세포 정리 평가

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

유방암 세포에서 에스트로겐 조절 마이크로 RNA의 프로파일

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...