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.

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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 흉내 낸 산발적 인 인간 종양 개발에 특히 췌 장 β 세포의 premalignant 병 변으로 체세포 유전자 변화 소개의 이점이 있다. 이 방법은 기존의 유전자 변형 모델 개발 중 관심사의 유전자의 소성 식 때문에 종종 관찰 되는 정상적인 조직 형성의 어떤 잠재적인 섭 동을 방지 합니다. 또한, 그것은 관심을 생성 하는 유전자 변형 쥐 보다 유전자를 운반 하는 조류 retroviral 벡터를 생성 하는 훨씬 더 빨리입니다. RCASBP 파생 조류 retroviral 벡터에 tva 표현 세포 생체 외에서 그리고 vivo에서cDNAs (≤2.5 kb), shRNAs, miRNAs, 및 다른 noncoding RNAs를 제공할 수 있습니다. 감염 (그리고 여러 감염)의 효율 대상 셀의 확산 속도 셀의 액세스 가능성에 따라 달라 집니다. titer &#62; ml 당 1 x 108 전염 성 단위는 vivo에서 감염에 대 한 필요. 더 효율적인 바이러스 배달 달성 생체 외에서 유도 된 세포 증식과 바이러스를 모든 셀의 반복 노출의 가능성이 될 수 있습니다.
정확한 intracardiac 주입 기술은 생쥐의 생존 능력에 대 한 중요 하다. 첫째, 바이러스 성 현 탁 액을 그리기 전에 인슐린 주사기에서 공기 공간의 50 µ l 심장 펄스를 보고 결정적 이다. 둘째, 그것은 혈액의 빨간색 펄스 나타나면 주사기의 위치를 수정 하 고 주사기에 표시 혈액의 밝은 빨간 펄스를 볼 때마다 천천히 10-20 µ l 바이러스 성 현 탁 액을 제공 하는 것이 중요. 혈액의 밝은 빨간색 펄스 10-20 µ l 바이러스 성 현 탁 액의 납품 후에 중지, 약간 바늘 위치 도움이 됩니다. 셋째, 마지막 밀어 바이러스 성 현 탁 액을 제공 하는 플런저와 혈액 펄스를 보고 하기 전에, 후 바늘 흉에서 신속 하 게 제거 되 고 부드러운 압력 사출 사이트에 적용 된 내부 출혈을 줄이기 위해 도움이 될 것입니다. 마지막으로, 다시 인슐린 주사기 다른 마우스에 사용 하지 마십시오.
미래 응용 프로그램,이 RIP-태그; RIP-tva 마우스 모델 다른 유전자 변형, 노크, 및 녹아웃 마우스 모델 결합 될 수 있다. 또한, 우리는이 RIP-태그; 결합 구상 RIP-tva 단일 지점 돌연변이, 삭제, 게놈 재배열 뒤집어 및 translocations 22등을 생성 하 게놈 편집 도구 CRISPR Cas9 마우스 모델.

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><tbody><tr><td>RCASBP-Y DV plasmid</td><td>Addgene</td><td>11478</td><td></td></tr><tr><td>RCAS-RNAi plasmid</td><td>Addgene</td><td>15182</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>fetal bovine serum</td><td>Atlanta Biologicals</td><td>25-005-CI</td><td></td></tr><tr><td>L-glutamine, 100x</td><td>Corning</td><td>25-005-CI</td><td></td></tr><tr><td>Penicillin-Streptomycin solution, 100x</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>PBS&lt;sup&gt;-/-&lt;/sup&gt;, 1x</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Superfect</td><td>Qiagen</td><td>301305</td><td></td></tr><tr><td>Polyallomer centrifuge tube</td><td>Beckman Coulter</td><td>326823</td><td></td></tr><tr><td>0.45 mm Nalgene&lt;br/&gt; Syringe Filters with PES Membrane</td><td>Thermo Scientific</td><td>194-2545</td><td></td></tr><tr><td>Insulin Syringes</td><td>BD</td><td>329461</td><td></td></tr><tr><td>synaptophysin</td><td>Vector Laboratories</td><td>VP-S284</td><td></td></tr><tr><td>VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG)</td><td>Vector Laboratories</td><td>PK-6101</td><td></td></tr><tr><td>AmpliTaq DNA Polymerase with Buffer II</td><td>Life Technologies</td><td>N8080153</td><td></td></tr><tr><td>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 마우스에서 종양 세포의 충분 한 금액이입니다. 이 시스템을 사용 하 여, 전이에서 Bcl xL의 새로운 핵 기능 확인 된 9이었다. RIP-태그; RIP-tva RCASBP-Bcl xL 감염 쥐 전시 높은 발생률 침략 carcinomas의 제어 바이러스에 감염 된 생쥐 보다 RCASBP-ALPP 74% (96%). 또한, RCASBP-xL-에 Bcl의47%-감염 RIP-태그; RIP-tva 마우스 개발 때 아무 전이 제어 쥐 20에서 발견 하는 동안 (그림 3A)의 16 주에 안락사 췌 림프절에 전이.
또한, 우리 RIP-태그;에서 암 유전자의 도서관 상영 RIP-tva 마우스, 전이 췌장암의 림프절과 간 21 (그림 3B와 3c)을 추진 하 고 첫 번째 유전자를 확인 하 고. 이 유전자 hyaluronan 중재 운동 B isoform (RHAMMB) 단백질에 대 한 수용 체 인코딩하고 EGFR 21신호를 활성화 합니다. 우리는 그 간 특정 전이 실험 전이 있는 N134 종양 세포는 처음 받는 사람 immunodeficient 마우스 21의 폐 모 세관 침대를 통해 유통의 꼬리 정 맥 분석 결과 지 수 설명 했다.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55890/55890fig1.jpg"> 
그림 1 : RCASBP(A), RCAS-X 및 Y RCAS 구문을의 도식. 복제 사이트, 굵은 글꼴로 표시 됩니다. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55890/55890fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55890/55890fig2.jpg"> 
그림 2 : Intracardiac 주입의 위치. 해부학 적 랜드마크 (화이트에 명시 된) 흉 골에 수평 파선으로 표시 됩니다. 마우스의 피부에 sternal 노치와 xyphoid 프로세스, 랜드마크로 서 역할 하 고 바늘 삽입 중간 흉 골에서 1 m m 이며 약간 (해 부)는 흉 골의 왼쪽. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55890/55890fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55890/55890fig3.jpg"> 
그림 3 : Immunostaining에 의해 검출 전이성 췌 장 β 세포. 사진 표시 대표 synaptophysin 전이성 췌 장 신경 내 분 비 종양 (A, B) 췌 장의 림프 노드 또는 간 (C)의 얼룩. RIP-태그; RIP-tva 쥐 나이의 7 주에 표시 된 RCASBP 레트로 바이러스에 감염 되었고 나이의 16 주에 안락사. 눈금 막대 = 50 µ m. 원래 확대 = 20 X. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55890/55890fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>

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 ...

identification-characterization-metastatic-factors-gene-transfer-into

Nanyang Technological University

Research

JoVE Journal

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

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

방사성 핵 종 형광 취재 원 유전자 설치류 종양 모델에서 종양의 진행을 추적 하기 위해 이미징

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

연구

암 연구학

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 ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

꼬리 정맥 전이 검사의 결합된 사용 및 생체 내 실시간 생체 내 이미징을 결합하여 폐암 전이성 식민지화 및 폐의 부담을 정량화

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

신속하고 정량적인 병아리 Chorioallantoic 멤브레인 모델을 사용하여 전이성 레귤레이터의 발견

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

인간의 전립선 암 전이의 동소 쥐과 모델

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

의학

A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was established using robust, reproducible in vivo models and standardized methods to identify novel players in melanoma metastasis. This approach allows for data inference at specific experimental stages to precisely characterize a gene's role in metastasis. Models are established by introducing genetically modified melanoma cells via intracardiac, intradermal, or subcutaneous injections into mice, followed by monitoring with serial in vivo imaging. Once preestablished endpoints are reached, primary tumors and/or metastases-bearing organs are harvested and processed for various analyses. Tumor cells can be sorted and subjected to any of several 'omics' platforms, including single-cell RNA sequencing. Organs undergo imaging and immunohistopathological analyses to quantify the overall burden of metastases and map their specific anatomic location. This optimized pipeline, including standardized protocols for engraftment, monitoring, tissue harvesting, processing, and analysis, can be adopted for patient-derived, short-term cultures and established human and murine cell lines of various solid cancer types.

This article describes a workflow of techniques employed for testing novel candidate mediators of melanoma metastasis and their mechanism(s) of action.

흑색종 전이의 새로운 중재자 식별을 위한 강력한 발견 플랫폼

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was ...

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 ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

정량 실시간 PCR에 의해 마우스 유방 종양 모델에서 폐 전이의 평가

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

예는 폐 전이 및 그들의 미세 환경의 라이브 영상을 생체 내

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable model for tracing tumor progression and angiogenesis simultaneously in a visual and sensitive manner. Bioluminescence imaging displays its unique superiority in living imaging due to its advantages of high sensitivity, strong specificity, and accurate measurement. Presented here is a protocol to establish a tumor-bearing mouse model by injecting a Renilla luciferase-labeled murine breast cancer cell line 4T1 into the transgenic mouse with angiogenesis-induced Firefly luciferase expression. This mouse model provides a valuable tool to simultaneously monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging in a single mouse. This model may be widely applied in anti-tumor drug screening and oncology research.

This protocol describes the establishment of a tumor-bearing mouse model to monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging.

종양 진행 및 혈관 신생의 이중 생물 발광 이미징

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable ...