이 연구는 AACR 헨리 쉐퍼 방광 암 연구 그랜트 (09-60-30-LI)에 의해 자금 지원을하고 있습니다.

동물 과목과 관련된 절차는 캘리포니아 샌프란 시스코 대학의 기관 동물 케어 및 사용위원회 (IACUC)에 의해 승인되었습니다. 1. 셀 준비 <ol><li> 인간의 방광 암 세포주 구-7-luc2-GFP는 (캘리퍼스 생명 과학, Hopkinton, MA) 10 % 태아 소 혈청 및 50 μg / ML gentamicin과 보완 RPMI-1640 미디어에서 재배된다. </li><li> 문화는 37에서 유지됩니다 ° C, 매체와 CO 2는 모든 2-3일 변화 5 %. </li><li> 세포는 트립신의 소화에 의해 수확과 hemocytometer를 사용하여 계산되었다. 50 μl 2 X 10 6 세포의 단일 세포 현탁액은 인산염으로 준비되었다 염분 버퍼 얼음에 보관. </li></ol> 2. Orthotopic 방광 종양 모델 <ol><li> 8~10주 옛 여자 누드 (뉴 / 뉴) 생쥐 (시몬 슨 연구소, Gilory, CA)을 사용 하였다. </li><li> 를 제공하기 위해 가열 패드를 통해 무균 드레이프를 배치SA 외과 플랫폼입니다. 멸균 윤활 젤리, 탐침의 바늘, 실버 질산 용액 및 멸균 물 24 게이지 카테터는 준비가되어 있습니다. </li><li> 일반 마취가 흡입된 isoflurane (유도 3 %, 유지 보수 1~2%)과 nosecone 통해 유지와 생쥐에 시달릴 수 있습니다. 멸균 안과 연고는 동물의 시선에 적용되며, 열 패드는 체온을 유지하는 데 사용됩니다. </li><li> 위로 향한 자세로 마우스를 놓으십시오. </li><li> 부드럽게 안정화를위한 집게로 동물의 외음부를 파악. </li><li> 제거 주사 탐침으로 24 게이지 정맥 카테터를 사용하여 마우스 요도가 catheterized됩니다. 충분한 윤활를 사용해야합니다. 마우스 부정사로 요도가 vulvar 주름과 음부의 앞부분에 즉시 후부에 위치하고 있습니다. 45도 각도는 처음에는 치골 아래에 요도를 cannulate하고 전달하기 위해 가져옵니다. 보다 얕은 각도는 다음 방광으로 요도를 통과하기 위해 가져옵니다. 젠틀 골반을 대표해서요도가 자주 도울 수 펴주십시오. 케어는이 요도 또는 방광 천공을 초래할 수 있으므로, 저항에 대해 너무 심하게 밀어 피하기 위해 이동해야합니다. 카테터 내에서 본 소변이 방광 루멘 내에서 위치를 확인합니다. </li><li> 탐침 바늘을 사용하여 카테터와 방광 루멘에서 소변을 기음. 탐침 바늘이 장소에서 카테터를 떠나, 이후의 모든 열망과 주사에 사용됩니다. 이것은 카테터 내에 사망하는 공간을 제거하여 정확한 볼륨의 주입은 가능하며 반복 catheterizations가 필요합니다. </li><li> 방광 urothelium의 glycosaminoglycan 계층에 악영향을하려면, 0.5 M 실버 질산 중 10 μL을 주입하고 10 초 동안 머물러 허용됩니다. 이것은 남아있는 소변과 흰색 침전물을 형성 있습니다. </li><li> 기음과 탐침 바늘을 사용하여 침전물과 방광 내용물을 버리고. </li><li> 100 μL 멸균 물을 주입하여 방광 씻으십시오. 물을 대기음 및 폐기. Repe4 세차장의 총 멸균 물로 세척 장에서 3 회. </li><li> 카테터 제거를위한 준비 요도를 윗니와 아랫니가 맞물리다하는 요도 meatus 주변 4-0 실크 넥타이를 놓으십시오. </li><li> 주사 이전 탐침 바늘을 이용하여 50 μL에 2 × 10 6 구-7-luc2-GFP 세포를 준비 하였다. </li><li> 동시에 실크 넥타이에 의해 occluded 요도를 떠나는, 카테터와 탐침 바늘을 제거합니다. </li><li> 실크 넥타이가 제거되고 동물이 잠에서되기 전에 마우스는 12 시간 동안 일반적인 마취하에 유지됩니다. </li></ol> 3. 초음파 <ol><li> 일반 마취가 증발 isoflurane과 코 콘을 통해 유지로 유도된다. </li><li> VIVO에서 VisualSonics Vevo 770는 고해상도의 마이크로 이미징 시스템은 (VisualSonics INC, 토론토, 온타리오, 캐나다) RMV-704 40 MHz의 탐침으로 사용되었다. </li><li> 초음파 플랫폼에서 위로 향한 자세로 마우스를 놓으십시오. </li><li> 동물의 뒷다리를 확보테이프로 초음파 프로브의 조작 중에 초과 움직임을 피할 수 있습니다. </li><li> 마우스 골반에 초음파 젤을 적용합니다. </li><li> 가로 또는 세로 이미지를 얻기 위해 마우스 골반에 RMV-704 (40 MHz의) 초음파 프로브를 낮춥니다. </li><li> 종양이 발견되면 이미지는 종양 크기 측정을 위해 저장할 수 있습니다. </li></ol> 4. 발광 생물 이미징 <ol><li> 200 μL (150 밀리그램 / ㎏) luciferin 기판의 intraperitoneal 주사를 수행합니다. </li><li> isoflurane 증발과 함께 일반적인 마취를 유도하고 코 콘을 통해 유지합니다. </li><li> bioluminescence의 영상기 이내에 위로 향한 자세로 마우스를 놓습니다. </li><li> 5 분 luciferin 주입 후 초당 광자의 동물에서 bioluminescence를 측정. </li></ol> 5. saRNA 준비 <ol><li> 50-100 μmol의 규모 oligonucleotides를 RNA 합성. </li><li> 보완 단일 좌초된 oligonucleotides를 어닐링 전아 니 디럭스 saRNAs. </li><li> [1,3]-dioxolane (DLinKC2-DMA), disteroylphosphatidyl 콜린, 콜레스테롤, 그리고 PEG-DMG - 지질 nanoparticle (LNP) saRNA은 ionizable 지질 1,2-dilinoleyl-4-(- dimethylaminoethyl 2)과 함께 준비하고 있습니다 이전에 설명한대로 자발적인 소포 형성 제제 절차를 사용 1. </li></ol> 6. 공식화 saRNA의 Intravesical 관리 <ol><li> orthotopic 방광 종양 모델을 맺은 후, 마우스는 지질 nanoparticles (LNPs) (표 1)에서 공식화 intravesical 작은 활성 RNA (saRNA)로 처리됩니다. </li><li> 일반 마취를 설립, 유지 isoflurane 증발과 함께, 그리고 무균 안과 윤활유가 적용되어 있습니다. </li><li> 부정사 위치에 동물을 배치합니다. </li><li> 이전에 설명한대로 요도 catheterization을 수행합니다. </li><li> 요도 카테터와 주변 4-0 실크 넥타이를 놓으십시오. </li><li> 카테터에서 모든 소변을 기음과빈 주사기 및 탐침 바늘을 사용하여 방광. </li><li> 50 μL (3 밀리그램 / ㎏)의 총 복용량을 위해 intravesically LNP-saRNA을 주사. 동시에 실크 넥타이에 의해 occluded 요도를 떠나는, 카테터와 탐침 바늘을 제거합니다. </li><li> 마우스는 2 시간 동안 occluded 요도와 anesthetized 유지됩니다. </li><li> intravesical LNP-saRNA과 치료는 종양 이식 후 4 일에 시작되었다. 쥐은 14 트리 트먼트 총, 매 3 일간 순차적으로 처리되었다. </li></ol> 7. 대표 결과 구-7-luc2-GFP 세포에서 형태소 분석 방광 종양은 루시페라제 발광 생물 이미징 (그림 1A와 C)과 방광 초음파 (그림 1B와 D)에 의해 모니터할 수 있습니다. 종양 세포 접종 후 3~5일 이내 자주 감지 있습니다. 우리는 생쥐의 90 % 이상의 성공적인 방광 종양 주입을 발견. intravesical dsRNA와 치료 ensues으로 종양의 진행은 WI를 모니터링할 수 있습니다이러한 modalities 일. 동물 희생 후 종양의 성장 또한 GFP 형광을 사용하여 확인할 수 있습니다. 일반적으로, bioluminescence는 같은 인물 1C 및 1D에 시각적으로 볼, 방광 종양에 대한 평균 초음파 차원과 상호 의미. 동물들이 희생되었으며 종양이 GFP가 확정되자 그러나, 우리는 그 bioluminescence은 초음파 측정했던 것보다 가능한 종양의 존재와보다 밀접하게 상호 발견. 처리 생쥐에서의 잔류 반흔 조직은 종종 초음파에 의해 시각했지만 라이브 종양으로부터 구분하지 못했기 때문에이 saRNA (같은 컨트롤의 반대 의미)로 치료 생쥐 중에서 특히 겉보기했습니다. <img src="/files/ftp_upload/4207/4207fig1.jpg" alt="그림 1" /> 그림 1. 방광 종양의 종양 성장 속도론. () Bioluminescence 이미징 자신의 위치와 성장을 확인하는 방광 종양 내에 루시페라제 활동을 측정합니다. (B)의 생체내 방광 ultrasoun에서D 40MHz 프로브를 사용하는 것은 누구의 치수를 정확하게 측정할 수있는 종양 이미지를 생성합니다. (C) 그래프 bioluminescence 및 초음파 차원에서 종양의 진보적인 증가를 설명. 6 동물의 측정 - 값 (표준 편차 + / -) 의미를 나타냅니다. <a href="http://www.jove.com/files/ftp_upload/4207/4207fig1large.jpg" target="_blank">큰 그림을 보려면 여기를 누르십시오</a> .

<ol>
	<li>Semple, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+cationic+lipids+for+siRNA+delivery.">Rational design of cationic lipids for siRNA delivery.</a> Nature. 28, 172-176 (2010).</li><li>Li, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+dsRNAs+induce+transcriptional+activation+in+human+cells.">Small dsRNAs induce transcriptional activation in human cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 17337-17342 (2006).</li><li>Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+effect+of+dsRNA-induced+p21(WAF1/CIP1)+gene+activation+in+human+bladder+cancer+cells.">Antitumor effect of dsRNA-induced p21(WAF1/CIP1) gene activation in human bladder cancer cells.</a> Molecular cancer therapeutics. 7, 698-703 (2008).</li><li>Chancellor, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+results+of+myoblast+injection+into+the+urethra+and+bladder+wall:+a+possible+method+for+the+treatment+of+stress+urinary+incontinence+and+impaired+detrusor+contractility.">Preliminary results of myoblast injection into the urethra and bladder wall: a possible method for the treatment of stress urinary incontinence and impaired detrusor contractility.</a> Neurourology and urodynamics. 19, 279-287 (2000).</li><li>Hadaschik, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+validated+mouse+model+for+orthotopic+bladder+cancer+using+transurethral+tumour+inoculation+and+bioluminescence+imaging.">A validated mouse model for orthotopic bladder cancer using transurethral tumour inoculation and bioluminescence imaging.</a> BJU international. 100, 1377-1384 (2007).</li><li>Fu, C., Apelo, C. A., Torres, B., Thai, K. H., Hsieh, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Bladder+Wall+Injection.">Mouse Bladder Wall Injection.</a> J. Vis. Exp. (53), e2523 (2011).</li><li>Dobek, G. L., Godbey, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Orthotopic+Model+of+Murine+Bladder.">An Orthotopic Model of Murine Bladder.</a> Cancer. J. Vis. Exp. (48), 2535 (2011).</li><li>Dinney, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+metastatic+variants+from+human+transitional+cell+carcinoma+passaged+by+orthotopic+implantation+in+athymic+nude+mice.">Isolation and characterization of metastatic variants from human transitional cell carcinoma passaged by orthotopic implantation in athymic nude mice.</a> The Journal of urology. 154, 1532-1538 (1995).</li></ol>

LCL은 캘리포니아 샌프란 시스코 대학에서 제기한 및 Alnylam 제약 회사에 허가되었습니다 saRNA에 관련된 특허 출원이 보류에 관한라는 발명가이다.

우리는 transcriptional 활성화를 위해 p21 CIP1/WAF1 (p21)의 발기인을 대상으로 LNP 책정 saRNA와 종양의 intravesical 치료 다음에 마우스 orthotopic 이종 이식의 방광 종양 모델의 구축을위한 프로토콜을 제시한다. 2, 3 결과 종양이 될 수 있습니다 발광 생물 이미징 및 방광 초음파로 확인 및 모니터링. Orthotopic 모델은 소변과 밀접 내생 방광 암 대략 것을 urothelium의 환경을 제공함으로써 intraperito...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 (옵션) </td></tr><tr><td> 구-7 룩-GFP 세포 </td><td> 캘리퍼스 </td><td> 128,091 </td><td></td></tr><tr><td> thymic 누드 마우스 (뉴 / 뉴) </td><td> 시몬 슨 실험실 </td><td> 6~7주 이전 </td><td> 그렇소 : (NCR) 뉴 / 뉴 fisol </td></tr><tr><td> 초음파 장치 </td><td> Visualsonics </td><td> Vevo 770 </td><td> RMV-704 프로브 </td></tr><tr><td> Bioluminescence 단위 </td><td> 캘리퍼스 </td><td> IVIS 스펙트럼 </td><td></td></tr><tr><td> Exel safelet의 카테터 </td><td> 어부 </td><td> 14-841-21 </td><td> 24G X 잡았다 &quot; </td></tr><tr><td> 실버 질산염 </td><td> 시그마 </td><td> S8157 </td><td></td></tr><tr><td> 해밀턴 주사기 </td><td> 해밀턴 공동mpany </td><td> 80,301 </td><td></td></tr><tr><td> LNP-saRNA </td><td> Alnylam 제약 </td><td></td><td></td></tr></tbody></table> 표 1. 특정 시약 및 장비.

an orthotopic bladder tumor model and the evaluation of intravesical sarna treatment

우리는 orthotopic 이종 이식 마우스 방광 종양 모델에서 intravesically 배달 작은 활성 RNA (saRNA)로 방광 암 치료를위한 새로운 방법을 제시. 마우스 모델은 흡입된 일반적인 마취하에 요도 catheterization에 의해 설립되었습니다. 화학 화상 그러면 방광 glycosaminoglycan 계층을 혼란 intravesical 실버 질산염 용액을 사용하여 방광 점막을 소개하고 세포 첨부할 수 있습니다. 멸균 물, 인간의 방광 암 구-7-luc2-GFP 세포와 여러 세차장에 이어 2 시간 머물지 방광에 카테터를 통해 얻은됩니다. 방광 종양의 후속 성장 생체내 방광 초음파와 발광 생물 이미징에 의해 확인하고 감시하고 있습니다. 종양 그러면 saRNA은 지질 nanoparticles (LNPs)에 공식화와 intravesically 처리됩니다. 종양 성장 초음파 및 bioluminescence으로 감시하고 있습니다. 이 절차의 모든 단계가 수반 비디오에 증명하고 있습니다.

Watch this Scientific Journal Video about An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment at JoVE.com

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

초음파와 발광 생물 이미징에 의한 intravesically 전달 saRNA 및 모니터링 종양 성장의 antitumor 효과를 평가하는 orthotopic 방광 종양 모델을 수립.

Orthotopic 방광 종양 모델 및 Intravesical saRNA 트리 트먼트의 평가

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

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JoVE Journal

Medicine

14.7K 조회수.  University of California, San Francisco. 초음파와 발광 생물 이미징에 의한 intravesically 전달 saRNA 및 모니터링 종양 성장의 antitumor 효과를 평가하는 orthotopic 방광 종양 모델을 수립.

비디오: An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

Murine 방광 암의 Orthotopic 모델

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

연구

의학

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

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

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

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

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

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Orthotopic 마우스 뇌종양 모델의 조합 방사선 치료

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

역 형성 갑상선 암의 동소 마우스 모델

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

유전자 전달 연구 동소 방광암 모델

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

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  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

쥐과 동 소성 방광 이종 이식의 최소 침습 설립

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

피하 종양 모델에서 종양 침투 백혈구 하위 집합의 평가

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

효능 평가<em&gt; H. 파이로리</em동소 이식 쥐 모델에서 방광암의 치료를위한 치료 도구로&gt; 단백질 HP-NAP

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

&quot;환자와 같은&quot;동소 이식 동계 마우스 모델 간세포 암의 전이

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...