우리는 해 롤 드 Varmus, 브라이언 C. 루이스, 더글러스 Hanahan, 대 니 황, 샤론 Pang, 메 간 웡, 그리고 Manasi M. Godbole 감사합니다. Y.C.N.D.는 국방부 그랜트 W81XWH-16-1-0619와 NIH 그랜트 1R01CA204916에 의해 지원 됩니다.

<ol>
	<li>Vogelstein, B., Kinzler, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multistep+nature+of+cancer.">The multistep nature of cancer.</a> Trends Genet. 9 (4), 138-141 (1993).</li><li>Walrath, J. C., Hawes, J. J., Van Dyke, T., Reilly, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+engineered+mouse+models+in+cancer+research.">Genetically engineered mouse models in cancer research.</a> Adv Cancer Res. 106, 113-164 (2010).</li><li>Khanna, C., Hunter, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+metastasis+in+vivo.">Modeling metastasis in vivo.</a> Carcinogenesis. 26 (3), 513-523 (2005).</li><li>Du, Y. C., Lewis, B. C., Hanahan, D., Varmus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+tumor+progression+factors+by+somatic+gene+transfer+into+a+mouse+model:+Bcl-xL+promotes+islet+tumor+cell+invasion.">Assessing tumor progression factors by somatic gene transfer into a mouse model: Bcl-xL promotes islet tumor cell invasion.</a> PLoS biology. 5 (10), e276 (2007).</li><li>Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+formation+of+pancreatic+beta-cell+tumours+in+transgenic+mice+expressing+recombinant+insulin/simian+virus+40+oncogenes.">Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes.</a> Nature. 315 (6015), 115-122 (1985).</li><li>Fisher, G. H., 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=Development+of+a+flexible+and+specific+gene+delivery+system+for+production+of+murine+tumor+models.">Development of a flexible and specific gene delivery system for production of murine tumor models.</a> Oncogene. 18 (38), 5253-5260 (1999).</li><li>Orsulic, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RCAS-TVA-based+approach+to+designer+mouse+models.">An RCAS-TVA-based approach to designer mouse models.</a> Mamm Genome. 13 (10), 543-547 (2002).</li><li>Tuveson, D., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+medicine:+Cancer+lessons+from+mice+to+humans.">Translational medicine: Cancer lessons from mice to humans.</a> Nature. 471 (7338), 316-317 (2011).</li><li>Choi, S., 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=Bcl-xL+promotes+metastasis+independent+of+its+anti-apoptotic+activity.">Bcl-xL promotes metastasis independent of its anti-apoptotic activity.</a> Nat Commun. 7, 10384 (2016).</li><li>Naik, P., Karrim, J., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rise+and+fall+of+apoptosis+during+multistage+tumorigenesis:+down-modulation+contributes+to+tumor+progression+from+angiogenic+progenitors.">The rise and fall of apoptosis during multistage tumorigenesis: down-modulation contributes to tumor progression from angiogenic progenitors.</a> Genes Dev. 10 (17), 2105-2116 (1996).</li><li>Hughes, S. H., Greenhouse, J. J., Petropoulos, C. J., Sutrave, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptor+plasmids+simplify+the+insertion+of+foreign+DNA+into+helper-independent+retroviral+vectors.">Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors.</a> J Virol. 61 (10), 3004-3012 (1987).</li><li>Petropoulos, C. J., Payne, W., Salter, D. W., Hughes, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appropriate+in+vivo+expression+of+a+muscle-specific+promoter+by+using+avian+retroviral+vectors+for+gene+transfer+[corrected].">Appropriate in vivo expression of a muscle-specific promoter by using avian retroviral vectors for gene transfer [corrected].</a> J Virol. 66 (6), 3391-3397 (1992).</li><li>Dunn, K. J., Williams, B. O., Li, Y., Pavan, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest-directed+gene+transfer+demonstrates+Wnt1+role+in+melanocyte+expansion+and+differentiation+during+mouse+development.">Neural crest-directed gene transfer demonstrates Wnt1 role in melanocyte expansion and differentiation during mouse development.</a> Proc Natl Acad Sci USA. 97 (18), 10050-10055 (2000).</li><li>Loftus, S. K., Larson, D. M., Watkins-Chow, D., Church, D. M., Pavan, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+RCAS+vectors+useful+for+functional+genomic+analyses.">Generation of RCAS vectors useful for functional genomic analyses.</a> DNA Res. 8 (5), 221-226 (2001).</li><li>Bromberg-White, J. 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=Delivery+of+short+hairpin+RNA+sequences+by+using+a+replication-competent+avian+retroviral+vector.">Delivery of short hairpin RNA sequences by using a replication-competent avian retroviral vector.</a> J Virol. 78 (9), 4914-4916 (2004).</li><li>Harpavat, S., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RCAS-RNAi:+a+loss-of-function+method+for+the+developing+chick+retina.">RCAS-RNAi: a loss-of-function method for the developing chick retina.</a> BMC Dev Biol. 6 (2), (2006).</li><li>Huse, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PTEN-regulating+microRNA+miR-26a+is+amplified+in+high-grade+glioma+and+facilitates+gliomagenesis+in+vivo.">The PTEN-regulating microRNA miR-26a is amplified in high-grade glioma and facilitates gliomagenesis in vivo.</a> Genes Dev. 23 (11), 1327-1337 (2009).</li><li>Ahronian, L. G., Lewis, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+high-titer+RCAS+virus+from+DF1+chicken+fibroblasts.">Generation of high-titer RCAS virus from DF1 chicken fibroblasts.</a> Cold Spring Harb Protoc. 2014 (11), 1161-1166 (2014).</li><li>Green, M. R., Sambrook, J., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (2012).</li><li>Du, Y. C., Lewis, B. C., Hanahan, D., Varmus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+tumor+progression+factors+by+somatic+gene+transfer+into+a+mouse+model:+Bcl-xL+promotes+islet+tumor+cell+invasion.">Assessing tumor progression factors by somatic gene transfer into a mouse model: Bcl-xL promotes islet tumor cell invasion.</a> PLoS Biol. 5 (10), 2255-2269 (2007).</li><li>Du, Y. C., Chou, C. K., Klimstra, D. S., Varmus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+for+hyaluronan-mediated+motility+isoform+B+promotes+liver+metastasis+in+a+mouse+model+of+multistep+tumorigenesis+and+a+tail+vein+assay+for+metastasis.">Receptor for hyaluronan-mediated motility isoform B promotes liver metastasis in a mouse model of multistep tumorigenesis and a tail vein assay for metastasis.</a> Proc Natl Acad Sci USA. 108 (40), 16753-16758 (2011).</li><li>Guernet, A., Grumolato, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+editing+of+the+genome+for+cancer+modeling.">CRISPR/Cas9 editing of the genome for cancer modeling.</a> Methods. , (2017).</li></ol>

저자는 공개 없다.

이 연구에서 설명 하는 강력한 마우스 모델, RIP-태그; RIP-tva, 식별 및 전이성 요인의 특성에 대 한 조류 레트로 바이러스를 통해 체세포 유전자 납품을 달성 하기 위해. 비록 RIP-태그; RIP-tva 마우스 췌 장 신경 내 분 비 종양, 전이성 요인이 마우스 모델에서 확인 된 또한 다른 암 종류의 전이 홍보 수 있습니다.
우리의 접근 시간 제어 방식으로, 따라서 더 충실 하 게...

대부분의 암은 체세포 돌연변이 1에서 발생 한다. 기존의 유전자 조작된 마우스 모델 (GEMM)는 특정 유전 변경 tumorigenesis 2에 기여에 대 한 중요 한 통찰력을 제공 합니다. 그러나, 그들은 몇 가지 제한이 있다. 이러한 모델의 주요 단점은 인간, 조직 내의 일부 셀만 유전 변경을 획득에 종양 형성의 산발적 인 특성을 복제 하지 않습니다. 유전자 변형 및 녹아웃 쥐에 있는 돌연변이 생식 개발에 영향을 미칠 가능성이 있습니다. 또한, 이러한 마우스 모델 생성 이며 비싼 시간이 걸리는.
전이 암 분야에서 중요 한 문제입니다. 전이 모델링은 GEMM에서 어렵게 되었습니다. 자연 스러운 전이 마우스에서 희소 하다. Penetrance 변수 이며 대기 시간 전이 3의 GEMM에 오래입니다. 그래서 전이성 캐스케이드의 초기 단계는 제거 실험 전이 모델 마우스의 순환으로 세포의 직접 주사를 사용 합니다.
마우스 모델에서 전이성 요인 공부에 상기 제한 중 일부를 해결 하려면 bitransgenic 마우스 모델, RIP-태그; 개발 했습니다. RIP-tva4. 전략은 하위 그룹 A 조류 leukosis 바이러스, tva 6,7에 대 한 높은 동기화 종양 진행 마우스 모델, RIP 태그 5, 그리고 수용 체의 사용을 결합에 기반. 이 RIP-태그; RIP-tva마우스 모델 유전자 somatically 단일 bitransgenic 마우스 스트레인에 소개 될 수 있습니다. Rb, p53 종양 진압 기능 억제 SV40 T 항 원을 가진 쥐 증식, 혈관 신생, adenoma, 침략 암 등 단계와 비슷한 방식으로 인간의 tumorigenesis, 췌 장 신경 내 분 비 종양을 개발 합니다. RIP-태그 모델이 암, 췌 장 신경 내 분 비 종양에 국한 되지 않음의의 우리의 이해를 위해 매우 유익 했습니다. 그것은 또한 전 임상 실험 8에서 사용 되었습니다.
선물이 RIP-태그;에 조류 레트로 바이러스 intracardiacally의 주입을 통해 체세포 유전자 전송 위한 프로토콜 RIP-tva 쥐. RCASBP 파생 조류 레트로 바이러스와 성공적인 감염 대상 셀을 적극적으로 확산이 필요합니다. 따라서, 우리 선택 RIP-태그; RIP-tva 증식 췌 장 독도의 약 50%에서 발전 하는 때 나이의 7 주에 마우스. 왼쪽된 심 실 intracardiac 주입 높은 titer 바이러스의 감염 10-20 4의 효율성을 달성 하기 위해 필요 합니다. 이 전달 방법 바이러스 췌 장 독도 도달 하기 전에 순환 내에서 바이러스 입자의 중요 한 희석을 감소 시킨다.
이 방법을 사용 하 여, 우리는 이전 Bcl xL 안티 apoptotic 함수 4,9의 독립적인 암 전이 촉진 증명 하고있다. Bcl-xL RIP-태그; tumorigenic 가깝다고 걸쳐 모든 췌 장 β 세포에서 transgene를 통해 표현 했다이 안티 apoptotic 독립적인 전이성 기능 관찰 하지 RIP-Bcl-xL 모델 10마우스. 따라서, 우리의 마우스 모델 식별 tumorigenesis의 나중 단계에서 표현 하는 때 유전자의 기능을 특성화 하는 독특한 기회를 제공 합니다. 독도의 2-4% RIP-태그; 종양으로 발전 하기 때문에 RIP-tva bitransgenic 쥐 바이러스 성 감염 및 모든 premalignant 병 변 없이 RCASBP에서 파생 된 조류 레트로 바이러스 감염만 tumorigenesis의 자연적인 과정을 통해 선택적 우위를 부여 하는 요소를 확인할 수 있습니다. 특히, 전이성 요인 가장 쉽게 인식 됩니다이 방법으로 췌 장의 림프절 이나 다른 장기에 전이 RIP-태그;에서 일반적으로 발생 하지 않습니다 때문에 RIP-tva 쥐.

<table border="0" cellpadding="0" cellspacing="0" width="747">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">RCASBP-Y DV plasmid</td>
			<td style="width:123px;">Addgene</td>
			<td style="width:119px;">11478</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RCAS-RNAi plasmid</td>
			<td style="width:123px;">Addgene</td>
			<td style="width:119px;">15182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:123px;">Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal bovine serum</td>
			<td>Atlanta Biologicals</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine, 100x</td>
			<td style="width:123px;">Corning</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin solution, 100x</td>
			<td style="width:123px;">Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">PBS-/-, 1X</td>
			<td style="width:123px;">Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfect</td>
			<td>Qiagen</td>
			<td>301305</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyallomer centrifuge tube</td>
			<td>Beckman Coulter</td>
			<td>326823</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">0.45 mm Nalgene 
			Syringe Filters with PES Membrane</td>
			<td style="width:123px;">Thermo Scientific</td>
			<td style="width:119px;">194-2545</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Insulin Syringes&#160;</td>
			<td style="width:123px;">BD</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">synaptophysin</td>
			<td style="width:123px;">Vector Laboratories</td>
			<td>VP-S284</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG)&#160;</td>
			<td style="width:123px;">Vector Laboratories</td>
			<td>PK-6101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AmpliTaq DNA Polymerase with Buffer II</td>
			<td>Life Technologies</td>
			<td>N8080153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MyTaq DNA Polymerase</td>
			<td>Bioline</td>
			<td>BIO-21106</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification and characterization of metastatic factors by gene transfer into the novel rip-tag; rip-tva murine model

전이성 암은 단단한 종양을 가진 환자에 있는 죽음의 90%를 차지 한다. 암 전이의 드라이버를 더 잘 이해 하 고 소설 치료 목표를 식별 하는 긴급 한 필요가 있다. 조사를 진행 주 암에서 전이를 분자 이벤트, 우리 bitransgenic 마우스 모델, RIP-태그; 개발 RIP-tva. 이 마우스 모델 쥐 인슐린 발기인 (RIP) 췌 장 β 세포에 SV40 T 항 원 (태그)와 하위 조류 leukosis 바이러스 (tva)에 대 한 수용 체의 식 드라이브. 쥐 증식, 혈관 신생, adenoma, 침략 암 등 단계와 비슷한 인간의 tumorigenesis는 잘 정의 된 단계를 통해 100 %penetrance 췌 장 신경 내 분 비 종양을 개발 합니다. 때문에 RIP-태그; RIP-tva 마우스 전이성 질병을 개발 하지 않습니다, 전이 촉진 유전자 변경 쉽게 확인 될 수 있다. 체세포 유전자 tva 표현으로 전송, 췌 장 β 확산 premalignant 병 변 원하는 유전 변경 은닉 조류 레트로 바이러스의 intracardiac 주사를 통해 달성 된다. titer &#62; ml 당 1 x 108 감염 단위 vivo에서 감염에 대 한 적절 한 것으로 간주 됩니다. 또한, 조류 레트로 바이러스 RIP-태그; 종양에서 파생 된 셀 라인을 감염 시킬 수 있습니다. RIP-tva 고효율 쥐. 셀 라인 또한 전이성 요인 특성을 사용할 수 있습니다. 여기이 마우스 모델을 이용 하 여 세포 선 종양 전이에 후보 유전자의 기능을 평가 하는 방법을 보여 줍니다.

Vivo에서 그리고 생체 외에서 감염 속도 RIP-태그; RIP-tva RCASBP 기반 바이러스에 의해 종양 세포는 20~ 20%와 ~ 80% 각각. RIP 태그; RIP-tva 마우스 모델, 각 마우스에 400 췌 장 독도의 약 4% 종양 20;에 개발 자연스럽 게 따라서 바이러스에 의해 전달 되는 유전자의 잠재적인 효과의 조직학 및 phenotypic 분석에 대 한 각 마우...

Watch this Scientific Journal Video about Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model at JoVE.com

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

선물이 RIP-태그;를 활용 하 여 소설 체세포 유전자 전달 시스템을 설명 하는 프로토콜 RIP-tva 마우스 모델 전이에서 유전자의 기능 연구. 조류 레트로 바이러스 intracardiacally 성인 쥐에서 췌 장 β 세포의 사전 악성, 비 침 투 적인 병 변으로 유전자 전송을 보장 하기 위해 전달 됩니다.

식별 및 태그-소설 RIP;으로 유전자 이동에 의해 전이성 요인의 특성 RIP-tva Murine 모델

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis ...

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Cancer Research

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

8.7K 조회수.  Weill Cornell Medicine. 선물이 RIP-태그;를 활용 하 여 소설 체세포 유전자 전달 시스템을 설명 하는 프로토콜 RIP-tva 마우스 모델 전이에서 유전자의 기능 연구. 조류 레트로 바이러스 intracardiacally 성인 쥐에서 췌 장 β 세포의 사전 악성, 비 침 투 적인 병 변으로 유전자 전송을 보장 하기 위해 전달 됩니다.

비디오: Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

고급 동물의 간에서 대장 전이의 모델 : 이미징 기술 및 전이성 클론의 특성

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

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암 연구학

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

전임상 치료의 양적 및 종 방향 평가를위한 동계 마우스 모델 전이성의 신장 세포 암

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

주변 방사선 조사에 의해 유도 된 피로의 마우스 모델

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

혀 기질과 혀 편평 세포 암 종에서 생체 외에서 의 재구성의 제조

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

발광 및 형광 Orthotopic Syngeneic Murine 모델 Androgen 종속 및 거세 내성 전립선 암

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) for prostate cancer are hampered by tumor heterogeneity and its complex microevolution dynamics. Traditional prostate cancer mouse models include, amongst others, germline and conditional knockouts, transgenic expression of oncogenes, and xenograft models. Generation of de novo mutations in these models is complex, time-consuming, and costly. In addition, most of traditional models target the majority of the prostate epithelium, whereas human prostate cancer is well known to evolve as an isolated event in only a small subset of cells. Valuable models need to simulate not only prostate cancer initiation, but also progression to advanced disease.
Here we describe a method to target a few cells in the prostate epithelium by transducing cells by viral particles. The delivery of an engineered virus to the murine prostate allows alteration of gene expression in the prostate epithelia. Virus type and quantity will hereby define the number of targeted cells for gene alteration by transducing a few cells for cancer initiation and many cells for gene therapy. Through surgery-based injection in the anterior lobe, distal from the urinary track, the tumor in this model can expand without impairing the urinary function of the animal. Furthermore, by targeting only a subset of prostate epithelial cells the technique enables clonal expansion of the tumor, and therefore mimics human tumor initiation, progression, as well as invasion through the basal membrane.
This novel technique provides a powerful prostate cancer model with improved physiological relevance. Animal suffering is limited, and since no additional breeding is required, overall animal count is reduced. At the same time, analysis of new candidate genes and pathways is accelerated, which in turn is more cost efficient.

This protocol describes a newly established method for virus delivery to the murine prostate. Using either CRISPR/Cas9 technology, gene overexpression, or Cre recombinase delivery, the technique allows orthotopic alteration of gene expression and implements a novel mouse model for prostate cancer.

CRISPR의 바이러스 배달 Murine 전립선 유전자 변경에 대 한 가이드

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

농축 및 설립된 피하 Murine 종양에서 종양 면역 및 비 면역 Microenvironments의 특성

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

식별, 조직학 특성화 및 체 외 3D 회전 타원 체 문화 모델에 대 한 마우스 전립선 돌출부의 해 부

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

다리에 대 한 3 차원 뼈 세포 외 매트릭스 모델

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

허혈 재관류 손상의 설정에서 전이성 간 종양의 뮤린 모델

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...