MB는 MRC의 교제에서 보조금에 의해 지원됩니다. KP와 AHM은 웨 섹스 의학 연구 및 암 연구 UK / RCS (영국) (C28503/A10013)에서 보조금에 의해 지원된다. 우리는 사우 샘프 턴 조직 화학 연구 단위의 대학의 지원에 대한 감사합니다.

대장의 Explants에서 주요 섬유 아세포 문화 1. 설립 <ol><li> 직접 수술 수술실에서 정상적인 인간의 대장 점막 조직의 샘플을 확보, 7 ~ 10에서 중단 PBS의 ML 100 단위 / ML 페니실린, 100 ㎍ / ml의 스트렙토 마이신, 및 / ㎖ Fungizone 0.25 μg로 보충. </li><li> 실험실에서, 10-cm 조직 배양 접시의 중앙에 시료를 배치하고 PBS / 펜 연쇄상 / Fungizone로 3 회 세척 하였다. 최종 세척 후 흡인하지 마십시오. </li><li> 멸균 집게와 메스로 (약 2 mm) 작은 조각으로 조직을 잘라 12 - 웰 플레이트에 메스 블레이드와 그린 크로스의 중심에 각 조각을 배치합니다. 이 준수 할 수 있도록 조직에 부드럽게 메스 록. </li><li> 농경 750 μL의 시료는 일차 섬유 아세포 성장 배지 subme한다 (DMEM은 20 % 송아지 태아 혈청 (FCS), 100 U / ㎖ 페니실린, 100 ㎍ / ㎖의 스트렙토 마이신 및 292 ㎍ / ml의 L-글루타민)플로트를 사용하지 않고 조직을 RGE. 2 일마다 재 공급을 제외하고 향후 5 일 동안 플레이트를 방해하지 마십시오. </li><li> 향후 5 일 동안 섬유 아 세포는 천천히 조직의 가장자리에서 밖으로 성장하기 시작합니다. 대략 7 일째에, 이것은 더 볼 수 있습니다. 밖으로 성장 충분한 섬유 아 세포가 있는지 후 약 3 ~ 4 주와, 각 웰에 750 ㎕의 트립신을 추가합니다. 세포는 T-25 조직 배양 플라스크에, 수영장 및 전송 (5 ~ 10 분의 시간이 걸릴 수 있음) 분리 한 후. </li></ol> 2.의 Organotypic 준비 섬유 아세포 임신의 Organotypic 겔의 제조 : <ol><li> 10 % FCS 및 292 ㎍ / ml의 L-글루타민이 보충 된 DMEM에서의 Organotypic 당 5 × 105 세포를 포함하는 섬유 아세포의 세포 현탁액을 준비한다. </li><li> 다음과 같은 비율로, 얼음의 Organotypic 젤 메이크업 : <ul><li> 쥐 꼬리 콜라겐의 7 권 : 리겔의 혼합 (1:1) </li><li> 필터링 10 배 DMEM의 1 볼륨 </li><li> FCS의 1 볼륨 </li><li> 5 × 5 섬유 아세포를 함유하는 섬유 아세포의 세포 현탁액 1 부피 9 젤 (젤 당 1 ㎖)의 경우 10 ㎖를 준비하고 거품을 피하기 위해 조심스럽게 섞는다. </li></ul></li><li> 24 웰 플레이트의 각 웰에 1 mL를 넣고 37 ℃에서 1 시간 동안 배양 젤이 설정할 수 있도록 가습 분위기에서 C를 °. 1 시간 후 젤 위에 1 ML DMEM (10 % FCS) / 과잉 추가 하룻밤 인큐베이터로 돌아갑니다. </li><li> 다음 날, 젤 및 5 × 5 CRC 상피 세포를 포함하는 미디어의 판 1 ㎖의 상단에서 대기음 매체. </li></ol> 겔 코팅 된 나일론 시트의 제조 : <ol start="5"><li> 1.5 cm 크기의 멸균 나일론 시트는 사전에, 1.5 cm를 멸균 x 다음 준비합니다. </li><li> 무균 핀셋을 사용하여, 10cm 배양 접시 (통상 4 나일론 시트 인 접시 당 수용 할 수있다)에 나일론 시트의 필요 개수를 배치. </li><li> 다음과 같은 순서로 얼음 젤 메이크업 (250 μL의 총볼륨은) 나일론 장에 필요합니다 : <ul><li> 쥐 꼬리 콜라겐의 7 권 </li><li> 필터링 10 배 DMEM의 1 볼륨 </li><li> FCS의 1 볼륨 </li><li> 1 DMEM의 볼륨 10 % FCS / 공급 과잉 </li></ul></li><li> 액이 황색 인 경우,이 솔루션은 분홍색으로 변할 때까지 50 μL 씩 분주 0.1 M NAOH를 추가하여 중화. </li><li> 각 나일론 시트에이 솔루션을 250 μl를 추가하고 37 품어 ° C 젤이 설정할 수 있도록 30 분 동안 5 % CO 2의 가습 된 분위기에서. </li><li> PBS에 1 % 글 루타 알데하이드 (glutaraldehyde) 솔루션을 구성한다. 젤이 설정되면 각 10cm 배양 접시에 10 mL를 넣고 1 시간 동안 4 ° C에서 알을 품다. </li><li> 세척 나일론 시트는 PBS와 DMEM에 1X (10 % FCS) / 글루의 3 배, 4 ℃에서 DMEM / 글루에서 하룻밤 배양 </li></ol> 침공 강철 격자에의 Organotypic 젤을 올리기 : <ol start="12"><li> 삼각대 형성에 스테인리스 강판을 접어 발판 그리드를 미리 준비합니다. 사용 전에 압력솥.</li><li> 여섯 웰 플레이트의 각 웰에 철강 격자를 배치 에탄올 소독 집게를 사용합니다. </li><li> 각 그리드에, 최상위 콜라겐면 젤 코팅 된 나일론 시트를 놓습니다. </li><li> 조심스럽게 에탄올 소독 주걱을 사용하여 제기 나일론 시트를 24 웰 플레이트에서의 Organotypic 젤을 전송합니다. </li><li> 나일론 시트 매체와 접촉하지만 침수되지 때까지 10 % FCS와 보충 DMEM 10 % FCS / 공급 과잉으로 잘 입력합니다. 매체의 Organotypic 젤을 만지지 않도록하십시오. </li><li> 2 일마다 미디어를 교체, 5 % CO 2의 습한 분위기에서 37 ° C에서 14 일 동안 배양한다. </li></ol> 3.의 Organotypic 고정 <ol><li> 우물에서 나일론 시트를 포함한 전체의 Organotypic를 제거하고 집착 필름에 배치합니다. </li><li> 의 Organotypic 깨끗한 일회용 메스를 사용하여 나일론 시트를 양분하고 실온에서 24 시간 동안 포름 알데히드의 두 반쪽을 고정합니다. </li><li> 70 %의 에탄과 포름 알데히드를 교체올 24 시간 후 전에, 파라핀에 삽입 구획 및 염색에 하룻밤 둡니다. </li></ol> 침략적인 여백의 4. 레이저 미세 절제 <ol><li> 막 상에 10 ㎛ 두께로 섹션 슬라이드를 장착. </li><li> 1 분 동안 크실렌을 적용하여 Deparaffinize 섹션은​​ 다음 추가로 1 분 동안 크실렌을 제거하고 75 %의 에탄올에 고정합니다. </li><li> 1 분 0.125 % 크레 실 바이올렛 용액을 에탄올과 얼룩을 제거합니다. 크레 실 바이올렛 상피 세포를 강조하고 기질에서 쉽게 차별을 허용합니다. </li><li> 크레 실 바이올렛을 제거하고 100 % 에탄올로 세척하기 전에 추가로 1 분 동안 100 % 에탄올을 적용합니다. 30 분 동안 공기 건조에 슬라이드를 허용합니다. </li><li> 선택 플랫폼 (예를 들어, 라이카 AS 시스템)를 사용하여 레이저 미세 절제를 실시한다. </li><li> 현미경 무대에 얼굴을 아래로 microdissected 할 스테인드 섹션 유리 슬라이드를 놓습니다. </li><li> 컬렉션 카세트에 0.5 ㎖ microcentrifuge 관을 장착하고 50 μL의 추가microdissected 조직을 수집 할에 대한 상한에 세포 용해 버퍼 (예 : DNA, RNA 또는 단백질의 용해 버퍼). </li><li> 직시 하에서, 그 조직을 식별하기 위해 조이스틱을 사용하고, 소프트웨어 인터페이스를 사용하여 종양 침윤 전면 microdissected 할 셀을 강조 더러워 유기 부분을 주석. </li><li> 강조 표시된 부분을 잘라해야 모두 발사 레이저를 지시하고 microcentrifuge 관의 뚜껑에 microdissected 조직을 추진. </li><li> 일단 충분한 자료를 수집 한, 컬렉션 카세트를 꺼내 microcentrifuge 관을 닫고 튜브의 바닥에 용해 버퍼를 그리는 부드럽게 회전. </li><li> 미세 절제가 완료 될 때까지 얼음에 샘플을 놓습니다. 적절한 방법으로 처리 샘플은 관심의 분석을 추출 할 수 있습니다. </li></ol>

<ol>
	<li>Nystrom, M. L., Thomas, G. J., Stone, M., Mackenzie, I. C., Hart, I. R., Marshall, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+quantitative+method+to+analyse+tumour+cell+invasion+in+organotypic+culture.">Development of a quantitative method to analyse tumour cell invasion in organotypic culture.</a> J Pathol. 205 (4), 468-475 (2005).</li><li>Zhang, 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=miR-153+supports+colorectal+cancer+progression+via+pleiotropic+effects+that+enhance+invasion+and+chemotherapeutic+resistance.">miR-153 supports colorectal cancer progression via pleiotropic effects that enhance invasion and chemotherapeutic resistance.</a> Cancer Res. , (2013).</li><li>Bullock, M. D., 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=Pleiotropic+actions+of+miR-21+highlight+the+critical+role+of+deregulated+stromal+microRNAs+during+colorectal+cancer+progression.">Pleiotropic actions of miR-21 highlight the critical role of deregulated stromal microRNAs during colorectal cancer progression.</a> Cell Death Dis. 20 (4), (2013).</li><li>Jenei, V., Nystrom, M. L., Thomas, 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=Measuring+invasion+in+an+organotypic+model.">Measuring invasion in an organotypic model.</a> Methods Mol Biol. 769, 223-232 (2011).</li><li>De Wever, O., Demetter, P., Mareel, M., Bracke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+myofibroblasts+are+drivers+of+invasive+cancer+growth.">Stromal myofibroblasts are drivers of invasive cancer growth.</a> Int J Cancer. 123 (10), 2229-2238 (2008).</li><li>Fries, K. M., 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=Evidence+of+fibroblast+heterogeneity+and+the+role+of+fibroblast+subpopulations+in+fibrosis.">Evidence of fibroblast heterogeneity and the role of fibroblast subpopulations in fibrosis.</a> Clin Immunol Immunopathol. 72 (3), 283-292 (1994).</li><li>Moutasim, K. A., 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=Betel-derived+alkaloid+up-regulates+keratinocyte+alphavbeta6+integrin+expression+and+promotes+oral+submucous+fibrosis.">Betel-derived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis.</a> J Pathol. 223 (3), 366-377 (2011).</li><li>Winter, J., Jung, S., Keller, S., Gregory, R. I., Diederichs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Many+roads+to+maturity:+microRNA+biogenesis+pathways+and+their+regulation.">Many roads to maturity: microRNA biogenesis pathways and their regulation.</a> Nat Cell Biol. 11 (3), 228-234 (2009).</li><li>Li, 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=Comparison+of+miRNA+expression+patterns+using+total+RNA+extracted+from+matched+samples+of+formalin-fixed+paraffin-embedded+(FFPE)+cells+and+snap+frozen+cells.">Comparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed paraffin-embedded (FFPE) cells and snap frozen cells.</a> BMC Biotechnol. 7 (36), (2007).</li><li>Szafranska, A. E., 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=Accurate+molecular+characterization+of+formalin-fixed,+paraffin-embedded+tissues+by+microRNA+expression+profiling.">Accurate molecular characterization of formalin-fixed, paraffin-embedded tissues by microRNA expression profiling.</a> J Mol Diagn. 10 (5), 415-423 (2008).</li></ol>

저자는 그 잠재적 인 충돌을 공개하지 않습니다.

여기에 우리가 특별히 분리하고 상피와 기질 세포의 3 차원 공동 문화 구조의 전이성 진행의 초기 단계에서 암 관련 기질을 공격 할 수있는 능력을 획득 한 종양 세포의 특성을하는 방법을 설명합니다. 생체 인간의 대장 섬유 아 세포를 포함하는 합성 기질과 나란히 CRC 상피 세포로 구성된 공동 문화 모델은 3 차원에서 CRC의 침략을 연구하는 데 사용되었습니다. 생?...

의 Organotypic 공동 배양하는 중요한 세포 외 기질 구성 요소 1-3를 포함하는 콜라겐 겔에 악성 상피 세포와 기질 세포를 병치하여 3 차원 생체 내 종양 미세 환경을 재구성에 도움 조직 공학적 모델입니다. 주로 종양의 침략을 측정하는 방법으로 잉태, organotypics 생체 내 동물 모델에 대한 의존도를 줄이고, 이러한 침입 세포가 단 분산 상태 2-4로 인위적으로 강제되는없이 Transwell 분석과 같은 다른 생체 기술의 단점을 피할 수 있습니다. 이러한 섬유 아세포와 같은 이러한 모델에서 기질 세포의 포함은, 악성 침윤과 전이 3-4 조절에 종양 미세 환경의 필수적인 역할을 반영하지만 기질의 표현형 이질성은하다의 Organotypic의 생리 학적 관련성을 최대화하기 위해 모델, 장기 별, 해부학 적으로 정확한 생체가능하면 3,6 섬유 아 세포가 포함되어야한다. Organotypics 종양 및 간질 세포 사이의 상호 작용을 연구 할 수있는 다양한 플랫폼이며, 점점 화학 억제제의 종양 침윤에 미치는 영향 및 표적 유전자 변경 7,8를 검사하는 새로운 방법으로 사용된다. 침략 종양 마진을 공부하는 것은 특히 흥미로운 전망이다. 의 Organotypic 모델 확립 결장암 (CRC) 세포주는 일반적으로 잘 성층 상피 층을 생산하고, 단면에서, 세포 외 기질 (ECM)를 침입 할 수있는 능력을 획득 한 세포가 쉽게 식별 할 수있다. 레이저 미세 절제를 사용하여 분리를 공부하고이 세포를 추출 종양 및 생체 내에서 종양 관련 기질 세포의 설립 마이크로 지형 이성의 빛으로, 대장 암 전이의 기원에 관한 중요한 생물 학적 통찰력을 보여줄 수 있습니다, 어떻게보다 효율적으로 될 수있다 대상. 나는N 다음과 같은 방법론은 조직의 동의서를 작성 샘플을 제공하고, 연구를 기증 한 모든 환자는 지역 연구 윤리위원회 기관에 의해 승인되었습니다.

<table border="0" cellpadding="0" cellspacing="0" width="634">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Colorectal cancer cell lines - example shown SW480</td>
			<td style="width:132px;">ATCC</td>
			<td style="width:119px;">ATCC CCL-228</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagen</td>
			<td style="width:132px;">BD Biosciences</td>
			<td style="width:119px;">354265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Matrigel</td>
			<td style="width:132px;">BD Biosciences</td>
			<td style="width:119px;">354234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nylon membrane</td>
			<td style="width:132px;">Merck Millipore</td>
			<td style="width:119px;">VVLP01300</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Metal grid</td>
			<td style="width:132px;">The Mesh Company</td>
			<td>WSS20-A4</td>
			<td>themeshcompany.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laser microdissection platform</td>
			<td style="width:132px;">Leica Microsystems</td>
			<td style="width:119px;">Leica AS LMD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Membrane mounted slides</td>
			<td style="width:132px;">Molecular devices</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cresyl Violet</td>
			<td style="width:132px;">Merck Millipore</td>
			<td style="width:119px;">1052350025</td>
			<td></td>
		</tr>
	</tbody>
</table>

molecular profiling of the invasive tumor microenvironment in a 3-dimensional model of colorectal cancer cells and ex vivo fibroblasts

대장 암에 침입 (CRC) 세포는 자신의 여동생 세포에서 무료로 휴식 기질에 침투하고, 세포 외 기질 (ECM)를 개조 할 수있는 능력을 획득했다. 세포의 표현형이 서로 다른 그룹의 생물학을 특성화하는 것은 실질적으로 전이 놓기 동안 초기 사건에 대한 우리의 이해를 향상시킬 수 있습니다. 종양의 침략 악성 상피 암 관련 기질 사이의 양방향 상호 작용에 의해 촉진 역동적 인 과정이다. 우리는 공동의 문화와 레이저 마이크로 해부 기술의 Organotypic을 결합했다 종양의 기질 인터페이스에서 세포 고유의 반응을 조사하기 위해. 이러한 섬유 아세포와 같은 주요 기질 성분 3 dimentioanally 암 상피 세포와 공동 배양 된의 Organotypic 모델, 가까운 산도에서 공부하는 침략과 암 - 기질 상호 작용을 가능하게 높은 처리 가능한 실험 도구입니다ysiological 조건. 레이저 미세 절제 (LMD)는 미크론 수준의 정밀도를 가진 종양 조직 내에서 다양한 계층의 외과 절개 및 추출을 수반하는 기술입니다. 게놈 transcriptomic과 성적인 프로파일과 이러한 기술을 결합함으로써 우리는 종양 세포를 침입 및 간질 조직을 주변의 분자 특성에 대한 깊은 이해를 개발하고, 그렇게함으로써 잠재적으로 새로운 바이오 마커 및 CRC에있는 약물 개발을위한 기회를 공개하는 것을 목표로하고 있습니다.

우리는 CRC 세포주 및 간질 세포의 여러 조합에 상기 방법을 적용했다. 여기에 제시 한 예는 인간의 기본 생체 대장 섬유 아세포와 상피 CRC SW480 세포주이다. 3 차원의 공동 문화는 침략 종양 전면 세포 사이의 차등 발현의 miRNAs의 비교 현미경과 레이저 캡처 미세 절제 (그림 2)을 실시한다 (그림 1), 건설 및 마이크로 RNA (miRNA의) 프로파일 (그림 3)에 의해 ?...

Watch this Scientific Journal Video about Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts at JoVE.com

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

합성, 조직 공학 대장 암 모델에서 레이저 microdissected 세포 및 기질의 분자 프로파일은 침습적 전방 및 암 관련 기질 세포에서 종양 세포 간의 계면에서 독특한 생물학을 특성화하고 해부 소설과 같은 처리 방식을 나타낸다.

분자 대장 암 세포의 3 차원 모델의 침윤성 종양 미세 환경의 프로파일 및<em&gt; 예 생체 내</em&gt; 섬유 아세포

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

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Research

JoVE Journal

Medicine

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

11.0K 조회수.  University of Southampton School of Medicine.  합성, 조직 공학 대장 암 모델에서 레이저 microdissected 세포 및 기질의 분자 프로파일은 침습적 전방 및 암 관련 기질 세포에서 종양 세포 간의 계면에서 독특한 생물학을 특성화하고 해부 소설과 같은 처리 방식을 나타낸다. 

비디오: Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

모델링 및 3 차원 이미징 단체 셀 침략

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

연구

의학

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Zebrafish Autochthonous 종양 모델을 사용하여 흑종 조절을위한 검사

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

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 Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

소설 고해상도<em&gt; 생체 내</em종양의 성장과 치료학에 두개 내 구조물의 동적 응답을 공부하기&gt; 이미징 기술

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

유방암의 소설 형질 전환 마우스 모델 : 아데노 바이러스 크레와 유관 상피의 대상으로 전이성 유방암의 개시

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

<em&gt; 생체 내</em&gt; 모델은 인간의 유방암에서 호르몬 행동을 연구하기 위해

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

3 차원 (3D) 종양 회전 타원체 침공 분석

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

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 ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

약물 치료를 종양 미세 환경을 공부하고 대상에 대한 뇌종양 / Organotypic 슬라이스 공동 문화 시스템

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...