Name: in vivo imaging and therapeutic treatments in an orthotopic mouse model of ovarian cancer
Uploaded: 2010-08-17T17:00:00.000+00:00
Duration: 7 min 46 s
Description: Watch this Scientific Journal Video about In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer at JoVE.com

我们感谢他的专家技术支持，哈维亨斯利博士。这项工作得益于以下的福克斯蔡斯癌症中心（FCCC）设施：实验室动物基金和小动物成像设施的使用。这项工作是支持的，一部分由“气候变化框架公约”/宾夕法尼亚大学（P50 CA083638），“气候变化框架公约”的核心授予（P30 CA006927）在卵巢癌的孢子，和卵巢癌研究基金。

一，卵巢肿瘤细胞的制备<ol><li>在文化表达荧光素酶的成长卵巢癌的细胞株。荧光素酶的来源可以是萤火虫，海肾，或其他物种。也可用于荧光蛋白的表达。对于本演示中，我们使用的卵巢癌的细胞株，OVCAR5，表达萤火虫荧光素酶。 </li><li>收获细胞，用常规的细胞培养技术。 </li><li>保持细胞悬液（10,000个细胞/μL）的磷酸盐缓冲液（PBS）冰直到注射时间。 </li></ol>二。 Intrabursal注射液这个过程需要从第二个人的援助。所有的外科手术是在无菌条件下进行。这包括穿着手术的装束和使用无菌手术器械，注射器和针头。 <ol><li>准备3毫升（100毫克/毫升）的盐酸氯胺酮，甲苯噻嗪盐酸1.6毫升（100毫克/毫升），1.5毫升（10毫克/毫升）acepromazine，20毫升0.9％氯化钠混合麻醉的解决方案。 </li><li>检查动物的识别号码和观察到的健康。麻醉与氯胺酮 - 甲苯噻嗪- acepromazine准备麻醉（IP）经腹腔注射给药量为8〜9毫升/公斤体重（BW）的动物。 </li><li>通过与动物的后爪钳或手指脚趾捏，确认该动物是在接受麻醉平原。如果有脚蹬反射，等待麻醉进行更深层次的平原，直到动物是这个程序没有响应。 </li><li>动物背侧上对着它的头和它的尾巴向你面临的无菌纱布垫。切口点位于左边或右边卵巢和中线以上。刮胡子或湿用70％酒精在切口点皮毛。 </li><li>抬起湿润的皮肤使用镊子，做一个小切口用剪刀在背内侧的位置，并直接卵巢脂肪垫以上。卵巢脂肪垫下方的腹腔壁的表面应该是可见的。脂肪垫很容易识别的白色在它周围的暗粉色组织。 </li><li>轻轻抬起腹腔壁衬，如上所述，做一个小切口（5）。 </li><li>中线切口旁放置一个无菌浸泡盐水纱布垫。找到卵巢脂肪垫，轻轻将其拉出上的纱布和休息。卵巢稳定夹紧牛头犬剪辑的脂肪垫。卵巢在解剖显微镜下，位置，让针插入输卵管小管弯曲，导致滑囊（规格30，G）的。当针插入到适当的位置，应该滑囊下可见。 </li><li>轻轻推注射器推杆注入5μL的细胞悬液之间的布尔萨和卵巢，而注射器注射部位定位。这一步需要两个人。一人推柱塞，而其他人保持针的定位。迅速取出针头穿刺部位的密封，但轻轻足够不要撕破滑囊和肾小管。滑囊应该会出现适当注射要稍微扩张。 </li><li>推出猛犬剪辑的脂肪垫，轻轻地取代生殖道进入腹腔的脂肪垫回。轻轻合上腹膜衬拉衬在较低的体壁。关闭手术订书钉或伤口剪辑皮肤。 </li><li>将复苏的动物在笼子里，并提供一个安全的热源，以避免低温和加快恢复。监测呼吸频率和易用性，肌张力的回报，并自愿移动的能力。这是走向复苏的进展的良好指标。订书钉或伤口剪辑可以去除手术后7天或以上。 </li></ol>三。口服灌胃给药<ol><li> 18〜20 G灌胃针或鼻饲管使用一个圆形的提示。灌胃针应不再比从动物的头尖的最后一根肋骨的距离。 </li><li>检查动物的识别号码和观察到的健康。轻轻地用拇指和手指的肩膀皮肤抓住颈背的动物。克制应该只是公司中脱颖而出四肢延伸到每一个侧面和出路。动物不应该能够把握针。 </li><li>动物的头，用食指轻轻拉了回来，形成了直线通过颈部和食管。支持你的拇指内的动物的回。 </li><li>轻轻的和没有力量的，插入口两侧和舌头在灌胃针。在一个平滑的运动，应通过对针口的屋顶上下食道。重力应引导针没有会议抵抗。如果有任何resistanCE，取出针，并再次启动。动物的插科打诨和吞咽反射可能会触发插入时。但是，应无阻力或动物喘气。重要的是要确保动物呼吸时，针在被检查的鼻孔和胸部的运动。 </li><li>慢慢推注射器的柱塞免除剂量体积。 </li><li>灌胃针头轻轻地取出它插入食道的方式在同样的角度和途径。 </li><li> 5〜10分钟，动物笼子里，并监测呼吸和行为。 </li></ol>四， 在活体成像我们用卡尺生命科学监察注入intrabursal腔细胞的行为。使用此系统的实验通常有4〜16周的时间内从肿瘤植入时间。 <ol><li>准备的D -荧光素（萤火虫荧光素酶的底物）的解决方案，通过溶解在1 mL PBS 5〜20毫克，并通过杀菌为0.22μm滤膜过滤。 </li><li>充电打开的氧气和异氟醚气体感应室。接通到感应室的气体流量。异氟醚的流量维持在低水平，直到动物准备进行成像。 </li><li>检查动物的识别号码和观察到的健康。放置在被控异氟醚气体感应室的动物。 </li><li>从室中取出的动物，当动物出现被麻醉。交付200μL准备通过IP使用30克针注射荧光素的解决方案。重要的是要记录注射时间，以保持一致的时间之间的荧光素注射和整个研究的成像性能。 </li><li>放置到感应室的荧光素注射的动物的回。 </li><li>成像系统设置相应的设置。要确保系统初始化之前获得的第一个动物形象。 </li><li>关掉煤气流感应室和成像室的气体流量。 </li><li>麻醉动物的背侧或腹侧了。请务必放置在鼻锥动物的鼻子，以保持适当的麻醉诱导。确保绿色照明“兴趣点网格”内的动物。 </li><li>关闭室的大门，并在适当的时候获得的图像。时间可以由动力学。 </li><li>从成像室取出的动物放到它的笼子。监视“Intrabursal注射液”中描述的动物的恢复。 </li></ol>五，代表性的成果<img src="/files/ftp_upload/2125/2125fig1.jpg" alt="图1" /> 图1。卵巢肿瘤细胞原位小鼠模型的体内成像。 intrabursally OVCAR5细胞表达荧光素酶注射到右侧卵巢，并随着时间的推移成像。图片13（左），17（中），和22（右）天后注射使用IVIS光谱成像系统。在上游面板背侧和腹侧在较低的面板。请注意，腹膜肿瘤细胞的扩散，注射后22天。 福克斯蔡斯癌症中心的机构动物护理和使用委员会规定的准则和法规的规定进行动物实验。

<ol>
	<li>Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.">Cancer statistics.</a> CA Cancer J Clin. 59, 225-249 (2009).</li><li>Lynch, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hereditary+ovarian+carcinoma:+heterogeneity,+molecular+genetics,+pathology,+and+management.">Hereditary ovarian carcinoma: heterogeneity, molecular genetics, pathology, and management.</a> Mol Oncol. 3, 97-137 (2009).</li><li>Yap, T. A., Carden, C. P., Kaye, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+chemotherapy:+targeted+therapies+in+ovarian+cancer.">Beyond chemotherapy: targeted therapies in ovarian cancer.</a> Nat Rev Cancer. 9, 167-181 (2009).</li><li>Killion, J. J., Radinsky, R., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+models+are+necessary+to+predict+therapy+of+transplantable+tumors+in+mice.">Orthotopic models are necessary to predict therapy of transplantable tumors in mice.</a> Cancer Metastasis Rev. 17, 279-284 (1998).</li><li>Bibby, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+models+of+cancer+for+preclinical+drug+evaluation:+advantages+and+disadvantages.">Orthotopic models of cancer for preclinical drug evaluation: advantages and disadvantages.</a> Eur J Cancer. 40, 852-857 (2004).</li><li>Shaw, T. J., Senterman, M. K., Dawson, K., Crane, C. A., Vanderhyden, 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=Characterization+of+intraperitoneal,+orthotopic,+and+metastatic+xenograft+models+of+human+ovarian+cancer.">Characterization of intraperitoneal, orthotopic, and metastatic xenograft models of human ovarian cancer.</a> Mol Ther. 10, 1032-1042 (2004).</li><li>Greenaway, J., Moorehead, R., Shaw, P., Petrik, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-stromal+interaction+increases+cell+proliferation,+survival+and+tumorigenicity+in+a+mouse+model+of+human+epithelial+ovarian+cancer.">Epithelial-stromal interaction increases cell proliferation, survival and tumorigenicity in a mouse model of human epithelial ovarian cancer.</a> Gynecol Oncol. 108, 385-394 (2008).</li><li>Connolly, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ovarian+cancer.">Animal models of ovarian cancer.</a> Cancer Treat Res. 149, 353-391 (2009).</li><li>Sadikot, R. T., Blackwell, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging.">Bioluminescence imaging.</a> Proc Am Thorac Soc. 2, 537-540 (2005).</li><li>Lehmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+and+multimodal+in+vivo+imaging+of+tumor+hypoxia+and+its+downstream+molecular+events.">Longitudinal and multimodal in vivo imaging of tumor hypoxia and its downstream molecular events.</a> Proc Natl Acad Sci U S A. 106, 14004-14009 (2009).</li><li>Connolly, D. C., Hensley, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenograft+and+Transgenic+Mouse+Models+of+Epithelial+Ovarian+Cancer+and+Non-Invasive+Imaging+Modalities+to+Monitor+Ovarian+Tumor+Growth+In+Situ:+Applications+in+Evaluating+Novel+Therapeutic+Agents.">Xenograft and Transgenic Mouse Models of Epithelial Ovarian Cancer and Non-Invasive Imaging Modalities to Monitor Ovarian Tumor Growth In Situ: Applications in Evaluating Novel Therapeutic Agents.</a> Current Protocols in Pharmacoogy. 45, (2009).</li></ol>

卵巢癌是所有妇科恶性肿瘤的 1，死亡的首要原因。本病的死亡率高主要是由于其已故的诊断和缺乏可靠的诊断方法 2 。此外，传统的化疗往往遇到耐药和复发癌症的3。因此，新的治疗是必需的，有效地瞄准这一疾病。针对人类卵巢癌的新疗法的发展，关键是建立一个有代表性的动物模型。 原位动物模型已经超过传统的异种移植模型的优点（如皮下或腹腔注射的肿瘤细胞），1）它再现肿瘤形成的主站点，2）它代表转移的常见部位，以及3）它提供了肿瘤细胞与适当的微环境4，5，6，7，8交互。鼠害有一个独特的法氏囊膜周围的卵巢和输卵管连续的。这种啮齿类动物的独特的解剖结构允许的卵巢肿瘤细胞的注射原位。 Intrabursally注入卵巢肿瘤细胞的行为类似于人类疾病，从而增长内intrabursal膜，并扩散到腹腔肿瘤进展。注射细胞稳定表达荧光素酶或荧光蛋白还可以实时跟踪肿瘤细胞的行为。发光法和/或荧光成像技术使人们有可能超过延长的时间和研究肿瘤的生长，分布，以及在非侵入性的方式9，10，11回归内反复形象的肿瘤细胞。 在建立原位模型，关键是迅速取出针后注入滑囊。针密封穿刺部位突然去除和防止注射细胞的泄漏。然而，在同一时间，运动应该要温柔不要撕破滑囊。如果在注射过程中发生泄漏，可能会导致腹部到种子细胞，并有可能混淆的研究，即外卵巢过早蔓延。在这种情况下，特定​​的动物应被记录下来，并为多种肿瘤部位在腹膜前处理，或者从后续分析所淘汰的发展。在执行灌胃之前，关键是检查灌胃管的长度测量，从动物的头尖最后一根肋骨。它是有帮助的，以纪念在动物的鼻管，和过去不通过这个标志管。过去这个标志的管插入可能会导致在胃的穿孔。确定管的长度是特别重要的的与年轻的动物或动物称量下20克。向下食道灌胃针插入后，应无阻力或从动物的斗争。重要的是管理的溶液或悬浮液太快，因为这可能会导致回流，并随后提供不准确的剂量体积，以及增加动物的压力。对于不时媲美的成像效果，这是最好保持注射荧光素酶的底物和成像之间的时间相一致。时间的推移，可以由基板注射后的一系列图像，观察信号的动力学。卵巢癌，口服给药，药物在体内成像原位小鼠模型的发展是必要的技术，为更好地了解卵巢癌的发展和传播，并评估新的治疗方案，最终可能会提高对病人的与这种致命的疾病结果。

<table><tbody><tr><td>Ketamine Hydrochloride</td><td>Vedco, Inc.</td><td>Not Available</td><td></td></tr><tr><td>Xylazine Hydrochloride</td><td>Lloyd, Inc.</td><td>Not Available</td><td></td></tr><tr><td>Acepromazine</td><td>Vedco, Inc.</td><td>Not Available</td><td></td></tr><tr><td>D-Luciferin Potassium Salt</td><td><a href="http://www.perkinelmer.com/Catalog/Category/ID/Substrates" target="_blank"><img src="/img/new_window.png" /> Caliper Life Sciences</a></td><td>122796</td><td></td></tr></tbody></table>

in vivo imaging and therapeutic treatments in an orthotopic mouse model of ovarian cancer

更好地代表人类癌症和治疗反应的原位动物模型。本文介绍的卵巢癌的原位小鼠模型，治疗癌症的药物，通过口服给药和肿瘤细胞的行为响应实时的治疗药物在体内成像系统中使用的监测发展。在此原位模型，卵巢肿瘤细胞表达荧光素酶外用他们直接注射到老鼠囊中，其中每个卵巢封闭。萤火虫荧光素酶的底物D -荧光素，注射后，荧光素酶表达细胞产生生物发光信号。这个信号是在活体成像系统的检测和监测肿瘤的生长，分布，并在动物个体的回归的一种非侵入性的手段。通过灌胃的药物管理允许的最大加药量为10毫升/公斤体重直接传送到胃和紧密合作，类似的药物在临床治疗提供。因此，技术描述这里，卵巢癌，口服给药，药物在体内成像原位小鼠模型的发展，有助于更好地理解人类卵巢癌和治疗，并改善针对这种疾病。

Watch this Scientific Journal Video about In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer at JoVE.com

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

卵巢癌的原位动物模型复制更好的人类疾病，因此，加强我们了解癌症的发展和肿瘤对治疗的反应。一个小鼠模型接收intrabursal注射荧光素酶表达的卵巢肿瘤细胞。治疗是通过灌胃给药。监测肿瘤生长<em&gt;在体内</em&gt;成像系统。

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

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Research

JoVE Journal

Biology

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

33.2K 次观看.  Women's Cancer Program.  中文 

视频: In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

原发肿瘤发展小鼠实验模型和腹膜转移<em&gt;通过</em&gt;原位接种卵巢癌细胞

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

科研

癌症研究

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

免疫与感染

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

乳腺癌细胞原位注射到小鼠乳房脂肪板

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

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

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

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

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

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

医学

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of &gt;10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

<meta charset="utf8"></head><body>体内成像测量原位注射乳腺肿瘤细胞的自发性肺转移

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

小鼠模型的非侵入性成像检测和监测卵巢癌复发

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

腹膜内卵巢癌转移的定量

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

活体成像和治疗卵巢癌的原位小鼠模型

副本

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此视频中的章节

原发肿瘤发展小鼠实验模型和腹膜转移

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

乳腺癌细胞原位注射到小鼠乳房脂肪板

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

小鼠模型的非侵入性成像检测和监测卵巢癌复发

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

腹膜内卵巢癌转移的定量

活体成像和治疗卵巢癌的原位小鼠模型

副本

摘要

探索更多视频

此视频中的章节

原发肿瘤发展小鼠实验模型和腹膜转移

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

乳腺癌细胞原位注射到小鼠乳房脂肪板

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

小鼠模型的非侵入性成像检测和监测卵巢癌复发

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

腹膜内卵巢癌转移的定量

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移