Name: orthotopic implantation and peripheral immune cell monitoring in the ii-45 syngeneic rat mesothelioma model
Uploaded: 2015-10-02T14:00:00.000+00:00
Duration: 9 min 31 s
Description: Watch this Scientific Journal Video about Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model at JoVE.com

The syngeneic rat mesothelioma II-45 cell line was a kind gift from A/Prof. Emanuela Felley-Bosco, Zurich University.

所有涉及动物的程序进行，根据实践中的动物的护理和使用用于科学目的的澳大利亚准则的建议。该协议为这项研究是经皇家北岸医院的动物护理和伦理委员会。女菲舍尔344大鼠（F344，150-200克）保持在卡恩斯设施，Kolling研究所标准条件（12小时亮/暗周期和自由获得食物和水）下。 注意:一个流程图，用于所有实验程序是在图1。 1.准备细胞植入<ol><li>培养大鼠间皮瘤Ⅱ-45细胞系（也称为IL-45;由腹膜引入青石棉的衍生）的RPMI 1640（RPMI）培养基补充有10％胎牛血清（FBS）和生长在标准条件（37° Ç湿润培养箱5％的CO 2）。 Mainta在通过传代和传代培养在约1:50每周两次在75厘米2烧瓶中。 </li><li>制备试剂的细胞培养和温暖的等分试样在37℃。所需试剂包括无血清RPMI培养基（SFM），RPMI有10％FBS，磷酸盐缓冲盐水（PBS）和0.5％胰蛋白酶-EDTA。 </li><li>培养的细胞植入到约70-80％汇合。这保证它们在线性增长阶段。 </li><li>收获细胞通过丢弃媒体，用5ml无菌PBS洗涤一次，然后加入3毫升0.5％胰蛋白酶-EDTA。 </li><li>返回烧瓶培养箱中约23分钟，直到所有的细胞变成非粘附。 </li><li>一旦细胞是非粘附的，加入3 ml的的RPMI有10％FBS以灭活胰蛋白酶。收集和离心细胞，在300×g离心3分钟。 </li><li>在300×g离心再次在10ml SFM和离心机的洗涤细胞沉淀3分钟。 </li><li>洗涤细胞沉淀再次用10ml的SFM的离心如上。 </li><li>悬浮细胞在10ml的SFM，并执行使用血细胞计数器或类似仪器细胞计数。 </li><li>稀释细胞，使100微升含有细胞被植入的量。 注意:肿瘤生长已被证明在一个剂量低至100个细胞在100μl但一个标准剂量是500,000个细胞在100μl。 </li><li>在媒体准备足够细胞被植入大鼠的数量（即，将100μl/鼠），再加上至少0.5 ml的额外以补偿从引发和针的死体积损失。 </li><li>准备足够的SFM（无细胞）为植入对照大鼠（即，将100μl/鼠），再加上至少0.5 ml的额外。 注:细胞和SFM现在准备植入。他们应保持在37℃，并植入2小时内的收获，保持活力。 </li></ol> 2.在体内植入细胞<ol><li>放置F344大鼠（&gt; 13周龄）插入感应腔和麻醉用1.4％异氟烷吸入（或所使用的方法中的设施）。一旦老鼠似乎是睡着了移动它从室到鼻锥（含1.4％异氟醚流），将其放置在其背部与胸部朝上（腹面）。这使内脏解决远离胸腔。请根据机构的协议来保证大鼠reflexes被完全麻醉。 </li><li>剃须正确的区域选择性costalis（胸）区域去除毛发。 </li><li>清洗，用80％V / V乙醇的剃区域。 </li><li>确定注射部位:在右侧，找到第二压盖开始颅。注射部位是0.5厘米近端于此，在从肋笼的尾端的第三和第四肋之间。 （图2A）。 </li><li>轻轻地混合II-45细胞悬浮。慢慢绘制细胞悬浮液（或S​​FM为对照大鼠）到1毫升＃160;注射器没有附加针。如果一个针安装为细胞的图纸上存在对细胞沿着针注入线生长的潜力。附加23G x 1.25针。总理针，并删除任何气泡。 </li><li>一旦注射器和针头都准备，放置20毫米长，直径5毫米间隔件在针轴上。这是用来防止针穿透过深地进入胸膜腔的注射期间。大约5毫米-12毫米暴露针足以通过的肋渗透而不损坏任何器官。 </li><li>慢慢地插入肋骨之间的针，抽回注射器，以确保血管尚未刺破（没血了应该出现在注射器），然后注入100μl的细胞或SFM。 （图2B）。 </li><li>卸下针并轻轻地滚动鼠到另一边蔓延细胞的胸腔。 </li><li>将老鼠进笼子，并检查恢复。钍Ë老鼠应该是在1分钟内清醒并开始走动。 </li><li>重复使用新针头每只大鼠。重复使用相同的针将导致细胞生长沿着针的注入线。 </li><li>每天监视福祉的动物。 </li><li>动物实施安乐死在道义上定义端点作为管辖机构动物伦理委员会。对于大鼠在这些实验伦理终点是重量损失大于10％或呼吸困难。 </li></ol> 3.尾静脉采血<ol><li>如果血液要立即采集后的细胞植入，保持麻醉的老鼠。如果采血在其他时间点，麻醉用异氟醚1.4％的老鼠。请根据机构的协议来保证大鼠reflexes完全麻醉。 </li><li>将大鼠倒向一侧，并找到一个侧尾静脉。 </li><li>消毒用80％乙醇的尾巴和标签0.5ml的EDTAÇollection管。 </li><li>为了收集血液，总是从尾巴的尾端（的方式大约有三分之一沿）。这允许进一步尝试更靠近尾部的颅端的情况下的第一次尝试是不成功的。千万不要重新取样尾部，因为这可能会导致血液凝块。 </li><li>定位一个23G x 1.25针平行于侧静脉和它，所以它渗透约10毫米（图3A）滑入静脉以小角度。 </li><li>注意:如果静脉已成功刺破血液将在针（图3B）的连接端可见。 </li><li>一滴血将形成在尾在针穿刺部位。收集用吸管和转移到标记的0.5毫升（或更小）的EDTA收集管这血。对于免疫细胞测定25微升是足够的。应用纱布的压力，穿刺部位，直到出血停止。 </li><li>弗里克管血混合血液和EDTA公关事件凝固。保持采血并与EDTA作短越好，以防止凝血混合之间的时间。 </li><li>当在室温采集血液从多个大鼠店EDTA-血样在机架中直至分析。在2个小时的采集过程中血液。 </li></ol> 4.样品制备免疫细胞剖析使用基于微珠的方法注意:此单一平台方法依赖于使用具有珠粒每个样品已知数市售绝对计数管中。这些管包含冻干丸粒样品制备过程中溶解，释放珠粒。珠荧光标记和通过门控的珠子群，绝对计数可以计算出来。 <ol><li>确保EDTA全血样品是通过将其放置在一个缓慢旋转混合器几分钟充分混合。标签1的绝对计数管每个样品。一份载有珠颗粒应为t下方可见在管的底部他金属珠保持器。 </li><li>转移25微升EDTA全血成标记的绝对计数管。珠颗粒会溶解在加血。 </li><li>向每个试管添加20微升抗大鼠T / B /天然杀伤（NK）细胞的鸡尾酒，将10μl抗大鼠CD8A的PE，10微升抗大鼠CD4（域1）的FITC和10μl抗大鼠的CD45 PE / Cy7的（图4A）。荧光团在表1中所定义。 </li><li>离心反应管短暂地（300×g离心），以确保所述的抗体和细胞在管的底部，而不是粘在管的一侧。涡旋混合并孵育15分钟，在RT。 </li><li>以裂解红血细胞中添加的10毫摩尔Tris 400微升，0.15M氯化铵缓冲液（pH 7.5）和涡旋以混合。裂解完成时，样品出现半透明的，不混浊（ 图4B和C）。未溶解的样品完全会导致增加研发背景和当流动分析仪假性上升计数。 </li></ol>样品5.流式细胞仪处理注意:请在四色流式细胞仪。 <ol><li>打开采集模式和与8描述一个新的模板的软件，如图5所描绘。 </li><li>调整仪器设置为表1中列出并设置栅极R1（FITC [FL - 1] 与 APC [FL - 4]）， 图5AI）计数荧光珠。其他的门都没有在此次收购阶段一样重要，但将需要进行分析。在这个协议中使用的绝对计数的珠含有荧光染料，可在任何通道被检测到，虽然是最弱的蓝色通道。 </li><li>使用准备好的控制血液样品，涡旋，然后加载到流式细胞仪和以低速（12微升/分钟）上设置模式运行，以便数据采集门可以调整。 </li><li>将收购聚集在R1珠门10,000个事件。 </li><li>建立一个文件夹来记录数据，并设置文件号和标签示例文件中采集菜单。 </li><li>加载待分析样品到流式细胞仪，设置流速介质（35微升/分钟）。运行在相同的流速各样品中。流速，可能需要改变，以低（12微升/分钟）或高（60微升/分钟），但介质一般是合适的。以这样的速度，大约需要90至120秒，以获得万珠事件每个样品。 </li><li>一旦样品被加载观看散点图以确保事件出现在R 1胎圈栅极。最初可存在于样品压力引起的漂移在散点图有些不稳定。等待这个稳定。 </li><li>一旦稳定下来，点击获取，并允许样品运行。一旦流式细胞仪已完成收购R1万珠事件流式细胞仪将停止采集并保存所有的数据。 </li><li>取下样品和废弃流涂可以。细胞仪现在准备下一个样品。运行所有的样品，然后进行分析模式。 </li></ol> 6.免疫细胞分析注意:门控策略和布尔代数被用来定义每个细胞群。布尔代数是一个基于逻辑分析方法，其允许在一个单一的定义多个操作。流式细胞仪的分析软件（例如，BD CELLQUEST）允许采用布尔代数。该公式用于积极地占显着的负反应性，其有助于限定细胞更具体标识每个细胞群。 &#39;区域&#39;被用来定义一个"门"。区域限定一个2维空间，而栅极可以通过代数运算符连接众多的区域组成（+，* - ， 在表2中定义）。 <ol><li>切换软件来分析模式。分析模板应产生匹配<str翁&gt;图5示出与情节和大门。 </li><li>分析每个单独的文件（ 即，每个样品）分开。设置栅极R1至到R9和然后设置算法对于每个细胞类型如表2中所定义（也示于图5）。 </li><li>使用电池的统计数据计数器来计算通过门和算法（ 表2和图5）中定义的单个细胞群。该算法将在小区统计计数器自动调节细胞的数量。 </li><li>使用下列公式计算细胞亚群: <img alt="式（1）" src="/files/ftp_upload/53019/53019eq1.jpg" /> 注意:所计数的细胞数的事件（例如，CD4 + T细胞的事件）被利用上述方程，得到每微升血液细胞数计数。例子示于图5。 </li></ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+and+tumor-infiltrating+myeloid+cells+predict+survival+in+human+pleural+mesothelioma.">Circulating and tumor-infiltrating myeloid cells predict survival in human pleural mesothelioma.</a> Cancer. 117 (22), 5234-5244 (2011).</li><li>Weir, C., 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=Streptavidin:+a+novel+immunostimulant+for+the+selection+and+delivery+of+autologous+and+syngeneic+tumor+vaccines.">Streptavidin: a novel immunostimulant for the selection and delivery of autologous and syngeneic tumor vaccines.</a> Cancer Immunol Res. 2 (5), 469-479 (2014).</li></ol>

The authors have nothing to disclose.

本文详述了胸膜间皮瘤大鼠的同源原位模型以及用于通过纵向血液取样监测疾病进展的简单方法的产生的方法。 在II-45模型通过将菲舍尔344大鼠石棉纤维13开发的。虽然这种曝光表示主机石棉免疫系统的相互作用的真实动态间皮瘤发病机制，它具有长的滞后时间（以年来产生），并可能是危险的，研究人员由于可能暴露于石棉纤维。 Ⅱ-45细胞的原位移植进入胸膜动物?...

恶性间皮瘤（MM）是一种侵袭性的恶性肿瘤其来自转化的细胞中的膜（间皮），该系肺和腹腔，心脏和内部繁殖器官和是肺腔或胸膜1,2的最常见的原发性肿瘤。暴露于石棉纤维占80％，所有的MM，虽然对石棉的使用禁令推出几十年前，在大多数西方国家，其社会各界的广泛使用已经留下了一个致命的遗产。据世界卫生组织估计，107,000名员工每年死于与石棉有关的疾病，与死亡率持续增加。一种新的非职业入射波也正在和有何时，在什么水平，这将达到峰值3知之甚少。 大多数人的MM被确诊晚了，当全身化疗代表的唯一可行的选择4之一。最effecti已经化疗（顺铂5培美曲塞在一起）当前的"护理标准"被认定在10年前。但是失败这种治疗是不可避免的，也没有成熟的第二行选项，使患者的一个严峻的预后只有12 个月2，中位生存期。因此，目前迫切未满足的需要更有效的治疗方法。尽管一批临床试验中的新疗法的检查没有导致的变化在实际中。这是部分由于低5％转移的临床前的结果，一般在异种移植小鼠模型中进行，给临床设定6-8。这类模型不忠实概括在非生理位置中发生的肿瘤微环境的复杂的方面，经常在没有免疫系统功能9。 同源原位模型创建比C一显著更现实的肿瘤环境ommonly使用皮下异种移植模型作为出现在正确的生理部位有一个完整的免疫系统10,11的肿瘤。的大鼠的尺寸较大增强其作为一种啮齿动物疾病模型的使用，尤其是在药物研究，其中串行抽血需要来评估治疗反应和毒性12。此外，在模型中，监测疾病进展是困难的，因为在肿瘤（如在胸膜腔）的位置，用在血液循环中发现的因素是非常有吸引力的，监测疾病进展的能力。使用免疫能干大鼠胸膜间​​皮瘤的同基因原位模型的生成进行说明。此外，一种简单的和相对非侵入性的方法，通过测量循环免疫细胞监测胸膜疾病进展还描述。

<table><tbody><tr><td>EDTA Collection tube (0.5 ml)</td><td>Greiner Bio One GmbH</td><td>450480</td><td></td></tr><tr><td>Rat T/B/NK Cell cocktail</td><td>BD Pharmingen</td><td>558509</td><td>anti-Rat CD3&amp;nbsp; APC (IgM clone 1F4), anti-Rat CD45RA Fitc (IgG1 clone OX-33), anti-Rat CD161a PE (IgG1 Clone 10/78)</td></tr><tr><td>anti-RAT CD8a PE&amp;nbsp;</td><td>Biolegend</td><td>200608</td><td>(IgG1vClone G28)</td></tr><tr><td>anti-Rat CD4 FITC (Domain 1)&amp;nbsp;</td><td>Biolegend</td><td>203406</td><td>(IgG&lt;sub&gt;1&lt;/sub&gt; Clone OX-38)</td></tr><tr><td>anti-Rat CD45 PE/Cy7&amp;nbsp;</td><td>Biolegend</td><td>202214</td><td>(IgG&lt;sub&gt;1 &lt;/sub&gt;Clone OX-1)</td></tr><tr><td>TruCount Tubes</td><td>Becton Dickinson</td><td>340334</td><td>Box of 50 absolute counting tubes</td></tr><tr><td>RPMI 1640 media</td><td>Life Technologies</td><td>11875-119</td><td></td></tr><tr><td>foetal bovine serum (FBS)</td><td>Scientifix</td><td>FBS500-S (lot# 010101-1)</td><td></td></tr><tr><td>trypsin-EDTA</td><td>Life Technologies</td><td>15400-054</td><td></td></tr><tr><td>PBS tablets</td><td>Medicago AB</td><td>09-9400-100</td><td></td></tr><tr><td>23Gx1&amp;frac14; Needle</td><td>Becton Dickinson</td><td>302008</td><td></td></tr><tr><td>1 ml Syringe</td><td>Becton Dickinson</td><td>302 100</td><td></td></tr><tr><td>Fischer 344 Rat</td><td>Animal Resources Centre, Perth Australia</td><td>F344</td><td></td></tr><tr><td>I.S.O (Isoflurane USP)</td><td>Veterinary Companys Australia (VCA)&amp;nbsp;</td><td>B7058</td><td></td></tr><tr><td>II-45 Rat Mesothelioma line</td><td>Zurich University</td><td></td><td>Note: The cell line was given as a gift and is not commercially available at the ATCC</td></tr><tr><td>FACSCalibur 4 color</td><td>Becton Dickinson</td><td>342975</td><td></td></tr><tr><td>TRIS-HCL</td><td>SIGMA</td><td>T3253</td><td></td></tr><tr><td>Ammonium Chloride</td><td>SIGMA</td><td>9718</td><td></td></tr><tr><td>Anaesthetic Machine (The stinger)</td><td>Advanced Anaesthesia specialists</td><td>#00449</td><td></td></tr></tbody></table>

orthotopic implantation and peripheral immune cell monitoring in the ii-45 syngeneic rat mesothelioma model

在免疫为基础的治疗的癌症如疫苗和免疫检查点抑制剂，和在治疗响应肿瘤微环境中的作用的进一步了解感兴趣的巨大热潮，共同指向需要免疫能干正位模型的临床前试验这些新的治疗方法。本文演示了如何建立胸膜恶性间皮瘤原位免疫能力的动物模型。在原位模型监测疾病进展是由肿瘤的内部位置混淆。纵向监测疾病的进展及其对循环免疫细胞在这方面和癌症等大鼠模型的效果，单管流式细胞术检测只需要25全血中描述微升。这提供了七个免疫指标准确定量:总淋巴细胞，单核细胞和嗜中性粒细胞，以及T细胞亚群CD4和CD8，B细胞和自然杀伤细胞。不同的潜艇这些参数的ETS在不同的情况和模型有用，与中性粒细胞淋巴细胞比率具有用于监测疾病进程中的间皮瘤模型最大的效用。分析循环使用这种单管方法还可以协助监测响应免疫为基础的治疗和了解，导致治疗成功或失败的根本机制免疫细胞的水平。

在本文用于使用II-45细胞胸膜间皮瘤的原位模型​​的生成的方法，导致动物屈服于间皮瘤可再现的和快速的时间内，没有大鼠垂死由于植入方法。细胞植入的数量的滴定中确定1×10 3个细胞是需要的充分渗透模型（100％的植入）的最小数目。的不同数目的细胞植入到大鼠的改变疾病的时间过程中没有出现影响疾病的严重程度。动物接受1×10 3，1×10 4和5×10 5个细胞40天，30和...

Watch this Scientific Journal Video about Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model at JoVE.com

II-45同基因大鼠间皮瘤模型中的原位植入和外周免疫细胞监测|医学|视频

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

胸膜恶性间皮瘤由II-45间皮瘤细胞进入胸膜免疫活性大鼠腔植入的原位大鼠模型的产生提出。甲流式细胞法分析从25微升血样在这些动物7免疫细胞亚群也被描述。

在II-45同基因大鼠模型间皮瘤原位移植和外周免疫细胞监视

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...

orthotopic-implantation-peripheral-immune-cell-monitoring-ii-45

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9.1K 次观看.  Royal North Shore Hospital, University of Sydney. 胸膜恶性间皮瘤由II-45间皮瘤细胞进入胸膜免疫活性大鼠腔植入的原位大鼠模型的产生提出。甲流式细胞法分析从25微升血样在这些动物7免疫细胞亚群也被描述。 

视频: Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

生物发光成像评估的免疫反应，植入心脏组织工程（东隧）

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

科研

生物工程

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

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without the ability to target a specific or well-delineated tumor volume. The delivery of radiation was achieved using fixed radiation sources or linear accelerators producing megavoltage (MV) X-rays. These devices are unable to achieve sub-millimeter precision required for small animals. Furthermore, the high doses delivered to healthy surrounding tissue hamper response assessment. To increase the translation between small animal studies and humans, our goal was to mimic the treatment of human glioblastoma in a rat model. To enable a more accurate irradiation in a preclinical setting, recently, precision image-guided small animal radiation research platforms were developed. Similar to human planning systems, treatment planning on these micro-irradiators is based on computed tomography (CT). However, low soft-tissue contrast on CT makes it very challenging to localize targets in certain tissues, such as the brain. Therefore, incorporating magnetic resonance imaging (MRI), which has excellent soft-tissue contrast compared to CT, would enable a more precise delineation of the target for irradiation. In the last decade also biological imaging techniques, such as positron emission tomography (PET) gained interest for radiation therapy treatment guidance. PET enables the visualization of e.g., glucose consumption, amino-acid transport, or hypoxia, present in the tumor. Targeting those highly proliferative or radio-resistant parts of the tumor with a higher dose could give a survival benefit. This hypothesis led to the introduction of the biological tumor volume (BTV), besides the conventional gross target volume (GTV), clinical target volume (CTV), and planned target volume (PTV).
At the preclinical imaging lab of Ghent University, a micro-irradiator, a small animal PET, and a 7 T small animal MRI are available. The goal was to incorporate MRI-guided irradiation and PET-guided sub-volume boosting in a glioblastoma rat model.

In the past, small animal irradiation was usually performed without the ability to target a well-delineated tumor volume. The goal was to mimic the treatment of human glioblastoma in rats. Using a small animal irradiation platform, we performed MRI-guided 3D conformal irradiation with PET-based sub-volume boosting in a preclinical setting.

PET 与 MRI 引导下微器对大鼠胶质瘤模型的照射

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

癌症研究

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Here we describe a minimally invasive syngeneic orthotopic transplantation model of mouse lung adenocarcinoma cells as a time- and cost-reducing model to study non-small cell lung cancer.

合成肺腺癌细胞原位移植研究 pd-l1 表达

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

小鼠鳞状细胞细胞的肿瘤免疫分析及治疗反应评价

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

同源原位骨肉瘤Sprague Dawley大鼠模型，截肢控制转移率

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival ...

An Immunocompetent Murine Model for Laser Interstitial Thermal Therapy of Glioblastoma

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and has no cure. Laser interstitial thermal therapy (LITT) is a Food and Drug Administration (FDA)-approved treatment for GB and is used in patients who may not be candidates for conventional surgical resection. While the clinical efficacy of LITT has been established, research beyond clinical case studies and case series is limited and hindered by the lack of an established animal model. This protocol uses C57BL/6 mice and syngeneic CT2A glioma cancer cell line to closely recapitulate human GB&#160;while also using a 1064 nm Neodymium-doped Yttrium Aluminum Garnet (Nd:YAG) laser, such as is used in one of the two FDA-approved LITT systems, providing excellent pre-clinical relevance. The successful establishment of this LITT murine model will provide a valuable platform for investigating the unique features of LITT ablation and its effects on the tumor microenvironment, potentially leading to improved therapeutic strategies.

Glioblastoma is a devastating form of primary brain cancer, and laser interstitial thermal therapy is emerging as a promising alternative to conventional surgical resection for inoperable glioblastoma. This protocol describes an optimized pre-clinical mouse model that can be used to study treatment effects or adjuvant and combinatorial treatments.

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

体外和活体评估移植受体的 T、B 和髓细胞抑制活性和体液反应

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

大鼠肠系膜血管生成法

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

生物学

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

小鼠中人类胸膜皮质瘤的PET/CT的植入与监测

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...