The authors would like to thank Drs. Helene Salmon and Miriam Merad from Icahn School of Medicine at Mount Sinai for providing the B16-F10-YFP cells and for their expert advice on melanoma mouse models. The authors further thank the Animal Imaging Core Facility, the Radiochemistry and Molecular Imaging Probes Core Facility, and the Molecular Cytology Core Facility at Memorial Sloan Kettering Cancer Center (MSK) for their support. This work was supported by National Institutes of Health grants NIH 1 R01 HL125703 (W.J.M.M.), R01CA155432 (W.J.M.M.), K25 EB016673 (T.R.) and P30 CA008748 (MSK Center Grant). The authors also thank the Center for Molecular Imaging and Nanotechnology (CMINT) at MSK for their financial support (T.R.).

该过程包括纳米颗粒的双放射性和荧光标记， 体内 PET-CT成像， 离体生物分布的测量，和离体免疫染色和流式细胞仪分析。所有的动物实验是由纪念斯隆 - 凯特琳癌症中心的机构动物护理和使用委员会批准。 1.双标记的脂质体的制备注:同基因的B16黑色素瘤也可以从吸入含-isoflurane-2％的氧气注入300000 B16-F10细胞到C57BL / 6小鼠的背部侧面麻醉下进行诱导。在手术过程中使用无菌的试剂和工具，在无菌的工作区。手术后，仔细观察动物，直到从麻醉其恢复。把动物放回笼子时，才重新获得全意识和流动性。他们的房子在动物移植瘤无菌室等。具有大小肿瘤300毫米3是理想的纳米颗粒的表征，由于其显着增强的渗透性和滞留效应（EPR），这可以增加纳米颗粒累积。下文提供的详细协议。 <ol><li>在一个圆底烧瓶中，溶解20毫克脂质的混合物在2毫升含氯仿1,2- dipalmitoyl- SN -glycero -3- phophocholine（DPPC），胆固醇，1,2- disearoyl- SN -glycero-的3-磷酸乙醇胺-聚（乙二醇）2000（DSPE-PEG2000），DSPE-去铁胺（DSPE-DFO）8，和1,1- diododecyl-3,3,3,3-tetramethylindodicarbocyanine -5,5-二磺酸（ DiIC12 [5] -DS）以分别61.3％，33.4％，5，0.2％和0.1％，摩尔比。 注意:此脂质组合物非常类似于目前在临床2中使用阿霉素的脂质体制剂。从我们的程序所得到的脂质体将密切模仿临床nanomedic 的体内性能国家统计局。 </li><li>使用旋转蒸发器除去有机溶剂，并形成在室温下的薄的脂质膜。加入20毫升的PBS并超声处理30分钟，通过使用了3.8毫米超声尖端和30瓦的幅度，以在冰上充分冷却，以产生脂质体纳米颗粒。 </li><li>离心机在4000 xg离心脂质体溶液10分钟以除去聚集物和洗涤用PBS离心过滤（分子量截止（MWCO纳米颗粒:100 kDa的）以除去游离脂质和残余有机溶剂的浓缩脂质体，这将留在顶室，然后将它们转移到新管中。脂质体可以在随后的步骤之前被存储在PBS中的冰箱长达1周。 </li><li>对于质量控制，通过动态光散射（DLS）表征脂质体。具体地，混合脂质体的50微升用PBS 950微升，然后将该混合物转移到上浆杯中。放置反应杯在分析仪和测量颗粒尺寸和它们的尺寸分布。的粒径和其粒度分布（多分散性指数（PDI））预计将90 - 分别为0.2， - 120纳米和0.1。 </li><li>对于放射性标记，在6.9和7.1之间的pH用1毫居里89 Zr的草酸，在37℃在1.5-mL管中混合纯化的脂质体，相当于2毫克脂质，在PBS中2小时，（总体积:〜 100μL）。通过离心过滤（MWCO = 100 kDa的），类似于步骤1.3除去游离的，未反应的89锆。用无菌PBS洗涤滞留并稀释至所需的体积。放射化学产率应是80 - 95％。 警告！放射性材料的工作是高度管制。机构必须得到认证，并提供必要的设施，人才培养，使用放射性严格监控。研究人员需要接受适当的培训和处理放射性物质时，佩戴个人防护装备和剂量环/徽章。 </li><li&gt;通过尺寸排阻放射性HPLC，其通常应在98％以上10确定放射性标记的脂质体的放射化学纯度。放射化学纯度低于95％都在1.5步洗指示不足。 </li><li>放射性标记后，确定通过动态光散射纳米颗粒的大小。的尺寸和PDI预计是相同的范围内，在步骤1.4 10测量范围内。 注意:如果放射性样品没有在流式细胞仪或荧光显微镜允许的，非放射性的NAT Zr的草酸盐可以用于在步骤1.5以标记的脂质体。这些非放射性脂质体具有其放射性类似物相同的特性。这个过程的描述在图2中提供。 </li></ol> 2. 在双标记的脂质体的体内 PET-CT成像和生物分布<ol><li>注入大约100微升的双标记的脂质体W为第i约300微居里的放射性成抑制黑素瘤小鼠（C57BL / 6，雌性，10 - 26周龄）通过其尾静脉，在约10毫居里89的Zr / kg体重。 </li><li>为了确定半衰期，从尾静脉从麻醉动物抽血下，使用28-G的胰岛素注射器含有2％异氟醚的氧气。收集血液 - 在预称重管（10 20微升）在2分钟，15分钟，1小时，4小时，8小时，24小时，并在注射后48小时。由差权衡血，用γ计数器测定放射性含量，并计算放射性浓度为每克注射剂量（％ID / g）的百分比。 注:在配备有麻醉系统，回收笼，加热，和生物危害罩的房间执行的过程。确保动物完全恢复，并从回收笼转移他们拿着笼子前，手术后获得正常流动。楼荷瘤动物的房间指定用于动物管理放射性材料。 </li><li> 24或48小时的后注射 脂质体，图像小鼠在小动物MicroPET-CT扫描仪10。水平放置在它的肚子麻醉动物，并保持它麻醉。辖2％异氟醚的氧气通过鼻锥。扫描以防止其干燥前申请眼药膏它的眼睛。 </li><li>执行全身PET静态扫描记录不少于5000万元重合的事件，用15分钟的大致时间。在350-700千电子伏和6纳秒分别设置能源和巧合的时间窗口。之后，执行一个5分钟计算机断层扫描（CT）扫描。在X射线管设置为80千伏和500微安的电流的电压;在CT扫描使用总共220度120旋转的步骤，每帧10大约145毫秒。 </li><li>在PET-CT扫描后，立即通过牺牲二氧化碳窒息的老鼠，然后按确认pinchi死亡纳克动物的爪子。一旦确认，进行颈椎脱位。 </li><li>通过首先切开右心房，然后注入20毫升的PBS到左心室除去在小鼠组织中的血液，并随后收集相关组织如肿瘤，肝，脾，肺，肾，肌肉和其他16。 </li><li>称量组织，测量通过γ计数8,9其放射性含量，并计算组织放射性蓄积为每克注射剂量（％ID / g）的百分比。 </li></ol> 3. 离体流式细胞仪和免疫注意:在剂量为0.5毫克每公斤体重染料注入大约100微升荧光脂质体的进黑素瘤小鼠通过其尾静脉。如果放射性样品没有在流式细胞仪，荧光显微镜允许的，必须使用非放射性的脂质体。 <ol><li>牺牲小鼠24小时后注射，如在步骤2.5，并收集肿瘤，它必须存储在冷PBS中。 </li><li>流式细胞仪，请按照下列步骤17: <ol><li>准备在流由纯化的胶原酶I和II（4 U / mL）中，透明质酸酶（60单位/毫升），和DNA酶I（60单位/毫升）的混合物中的流式细胞仪缓冲液（PBS中的0.5％BSA和1的酶鸡尾酒1mM EDTA）中。 </li><li>混合100毫克肿瘤组织与酶鸡尾酒缓冲的200微升中的1.5ml管和切块的组织小片用细剪刀。添加另外1.3毫升酶鸡尾酒的进入管及其内容物转移到6孔板中。 </li><li>摇上放置一个37℃的烘箱内60分钟以60rpm的水平振荡器6孔板中。 </li><li>与流洗组织匀浆仪缓冲器3次，以获得单细胞悬浮液。 </li><li>与抗体的鸡尾酒染色细胞的特异性生物标记物，包括CD45，CD11b的，的Ly6C，CD11c的，CD64，CD3，MHCII，和CD31（1:200稀释的所有抗体;克隆信息是在材料的电子表格提供）。确定相关的细胞类型，并确定DiIC12其平均荧光强度（MFI）（5）在每个细胞群体-DS用流式细胞仪分析软件的适当流动。 </li></ol></li><li>对于免疫染色，请按照下列步骤操作: <ol><li>将肿瘤放入盛有最佳切割介质（OCT）磁带，并冻结它的干冰。 </li><li>从肿瘤组织做6微米的冰冻切片，并将其放置在组织学载玻片;的部分应涵盖肿瘤的最大横截面，其提供了关于组织最有代表性的信息。 </li><li>染色生物标记（ 例如，CD31对内皮细胞）的与相应的抗体（ 例如，抗CD31，克隆MEC13.3）的兴趣。修正了多聚甲醛的部分;血清匹配二级抗体的物种起源阻止他们; incub用12小时的初级抗体吃在4℃，然后用2小时的第二抗体;最后，用PBS洗和盖玻片覆盖。图片的染色切片用Leica共聚焦直立显微镜SP5 13，16。 </li></ol></li></ol>

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The authors have no disclosure to make.

该议定书中的关键步骤: 双标记的脂质体的高品质的关键是产生在一段长期一致的结果。自由荧光染料或89 Zr离子可以产生完全不同的定位方式，而且必须在纯化步骤中被完全去除。另外，如果免疫系统显著影响实验癌症纳米性能，使用免疫小鼠模型的应该是优选的，如在C57BL / 6小鼠的B16-F10黑素瘤模型中，这是在本协议中使用。如果肿瘤微环境或遗传突变的特定组合是一个主要因素，患者来源的肿瘤异种移植物模型将是理想的选择。 修改和故障排除: 我们的过程提供了一个简单的和可靠的方法来评估纳米医学的体内性能荷瘤的imals。此过程具有作为主干构建具体实验来表征癌症纳米医学不同的应用。例如，研究人员能够确定何时PET-CT扫描在同一动物17多个时间点进行的纳米材料的定量在体内药动学;他们可以通过分析其他组织，如大肠，小肠，胰腺，脑，皮肤，胸腺，胃或唾液腺收集纳米材料的生物分布信息，如图其他出版物18，19，20，21;或者它们可以通过流式细胞术应用此流量和免疫染色协议评价单电池和在比肿瘤其他相关组织中纳米医学的亚细胞特异性。 该技术的局限性: 该协议řequires一些专门的研究基础设施，包括放射性标记，小动物成像和免疫放射化学和成像设备和技术专长。因此，这将是最好的研究人员在相关领域的专业知识的多学科团队执行。我们自己的经验表明，这种程序的最佳执行需要仔细规划和高效的协作。 值得注意的是，在本手稿的具体协议，并提出剂最适合于放射性标记基于脂质的纳米颗粒。然而，双重标记的纳米颗粒的概念可以用于表征被，例如，基于金的纳米晶体，聚合物，或甚至病毒颗粒的其他纳米颗粒制剂。在这些情况下，不同的标记策略是必要的。必须强调的各种各样的生物医学成像中使用的放射性同位素的是重要的，其中包括89个的Zr，18 </suP&gt;楼64的Cu，68 Ga和123 I，仅举几7。放射性同位素的选择必须基于所研究的纳米材料的特定性能进行，主要是它的血液循环半衰期，以允许在稍后的时间点全面表征。然而，其他因素，如化学标记或同位素的可用性，可能会影响最终的选择。在这项研究中，89锆被选中，是因为其长期的物理半衰期（T 1/2 = 78.4 h）为媲美脂质体的血液半衰期。值得注意的是，该协议也可以与其他同位素进行，如99m锝，111 In或125 I，是可商购的并且适合于与单光子发射计算机断层摄影（SPECT）成像。同样，DILC是由于其疏水性高，并在脂质体中，因此高负荷效率选择。其他的荧光染料不利的理化propertiES可直接缀合到磷脂17。 该技术的重要意义: 这个过程呈现适于评估在临床研究纳米医学当高平移潜力。 4键技术 - 即，PET-CT成像，放射性测量，流式细胞术，免疫组织化学和-被常规地用于在患者肿瘤的诊断。因此，这种临床前程序可以容易地转换在临床阶段来评估纳米医学的体内性能。此外，人体器官相比，小鼠的大尺寸允许通过PET-CT显像22，可替代纳米积累的基于微创活检放射性测量更准确的分析。在这种背景下，非侵入性，成像制导方案可以开发将患者并帮助均质患者预后10。德韦跳跃等做作的放射性纳米材料仍然需要大量的基础设施投资。可替代地，基于磁共振成像（MRI）非放射性成像技术已迅速在过去的十年中发展。例如，磁性粒子成像（MPI）使用小氧化铁纳米颗粒（10至20纳米），并可以提供更定量体内与氧化铁纳米颗粒标记的比目前的MRI技术23纳米医学评估。 MPI临床翻译将允许更多的研究人员使用健壮的体内成像技术来评估癌症纳米医学的体内性能。 未来的应用: 一旦研究人员掌握这些协议，它们可应用的程序来表征最荧光和放射性双标记，新材料，包括肽，蛋白质，抗体，抗体片段，等等。这些程序可以帮助科学家们彻底评估新型生物医学材料在体内的性能，并找出那些具有巨大的潜力转化。 总之，我们的目标是全面引入纳米表征方法，可以提供系统性，组织，单细胞和亚细胞水平纳米积累的信息。目前，大多数癌症纳米医学仍然在I期临床试验失败归因于他们的次优的药代动力学，生物分布和毒性反应。另一方面，治疗响应于纳米的极大异质性责成用于选择患者谁可能尤其从这些实验的纳米医学受益播成像策略。我们认为，通过这个程序可以帮助社区找出具有高潜力转化看好纳米医学在临床前的动物模型，并用适当的修饰，还可以帮助临床研究人员确定患者服从nanotherapy。

纳米医学正在改变癌症治疗的发展1的范例。由以前的癌症纳米医学，如脂质体和基于白蛋白nanotherapies 2,3的巨大临床影响的启发，许多新的配方已在过去十年中产生的。然而，最近这些癌症纳米医学的临床转化成功的分析表明，只有少数已被批准用于临床4,5。以新的癌症纳米医学临床翻译的一个主要障碍是有限的治疗指数的提高与自由治疗化合物6直接管理比较。如在临床前动物模型中在全身性的，组织纳米医学，和细胞水平的体内性能等，准确的评价是IDENTIF必不可少Ÿ那些对未来临床翻译最佳的治疗指数。 纳米材料可以是放射性标记的用于在活的动物与正电子发射断层扫描（PET）成像，其具有所有的临床成像方式7之间极好的灵敏度和重现性定量表征。例如，89 Zr的标记的长循环纳米医学已经表征在小鼠模型中的癌症8，9，10，以及在其它疾病模型11。此外，该纳米医学的血半衰期和生物分布可广泛用在个人组织8利用体外放射性测量评估。因此，単允许纳米医学在全身和组织水平的定量评价。 重要的是，radiolabeleð纳米医学通常不能在单细胞或亚细胞水平进行分析，由于放射性信号的空间分辨率有限。因此，荧光标记证明是纳米颗粒的光学成像技术，如流式细胞仪和荧光显微镜12中的评价的互补方式。为此，标记放射性同位素和荧光标签纳米粒子可以定量在体内通过核成像评估和体外通过放射性计数，而且它们也可广泛表征在光学成像的细胞水平。 以前，我们已经开发了模块式程序，以放射性和荧光标记纳入各种纳米粒子，包括高密度脂蛋白（HDL）11，脂质体9，10，聚合物纳米颗粒，抗体片段，和nanoemulsions 10,13。这些标记纳米粒子已经允许在不同层次的相关动物模型，引导这些纳米材料的优化其具体应用定量表征。在目前的研究中，目的是利用纳米脂质体，最成熟的纳米平台14 -作为一个例子来证明综合程序生成双标记纳米颗粒，并把它在一个典型的同系黑色素瘤B16-F10小鼠模型15彻底表征。从结果来看，我们有信心这种纳米粒子的表征方法可以适用于评估相关的小鼠模型其它癌症纳米医学。

<table border="0" cellpadding="0" cellspacing="0" width="638">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DPPC</td>
			<td style="width:136px;">Avantilipids</td>
			<td align="right" style="width:119px;">850355</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cholesterol</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">C8667</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DSPE-PEG2000</td>
			<td style="width:136px;">Avantilipids</td>
			<td style="width:119px;">880120P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DSPE-DFO</td>
			<td style="width:136px;">Home made</td>
			<td align="right" style="width:119px;">110634</td>
			<td>Perez-Medina et al, JNM, 2014</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DiIC12[5]-DS</td>
			<td style="width:136px;">AAT Bioquest</td>
			<td align="right" style="width:119px;">22051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifugal filter</td>
			<td style="width:136px;">Vivaproducts</td>
			<td style="width:119px;">VS2061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rotary evaporator</td>
			<td style="width:136px;">Buchi</td>
			<td style="width:119px;">R-100</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Radio-HPLC</td>
			<td style="width:136px;">Shimadzu HPLC with 2 LC-10AT pumps</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">89Zr-oxalate</td>
			<td style="width:136px;">MSKCC</td>
			<td style="width:119px;">Synthesized in house</td>
			<td>TR19/9 variable beam cyclotron (Ebco Industries Inc)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micro PET-CT</td>
			<td style="width:136px;">Siemens</td>
			<td style="width:119px;">Inveon Micro-PET/CT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gamma counter</td>
			<td style="width:136px;">PerkinElmer</td>
			<td style="width:119px;">2470-0150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flow cytometry</td>
			<td style="width:136px;">BD Biosciences</td>
			<td style="width:119px;">Fortessa</td>
			<td>Any multi-parametric flow cytometry analyzers would suffice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">C57BL/6 mice</td>
			<td style="width:136px;">Jackson Laboratories</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B16-YFP melanoma cells</td>
			<td style="width:136px;">Home made</td>
			<td style="width:119px;">N/A</td>
			<td>Salmon et al, Immunity, 2016</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ly6C (clone HK1.4)--APC-Cy7</td>
			<td align="right" style="width:136px;">128025</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MHCII (M5/114/152)--APC</td>
			<td align="right" style="width:136px;">107613</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD45 (30-F11)--BV510</td>
			<td align="right" style="width:136px;">103137</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD64 (X54-5/7.1)--PE-Cy7</td>
			<td align="right" style="width:136px;">139313</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD11b (M1/70)--BV605</td>
			<td align="right" style="width:136px;">101237</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD3 (17A2)--BV711</td>
			<td align="right" style="width:136px;">100241</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD31 (13.3)--PE</td>
			<td align="right" style="width:136px;">561073</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD11c (M418)--PerCP-Cy5.5</td>
			<td align="right" style="width:136px;">117327</td>
			<td style="width:119px;">BD Biosciences</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD31 (13.3) no fluorophore</td>
			<td align="right" style="width:136px;">550274</td>
			<td style="width:119px;">BD Biosciences</td>
			<td></td>
		</tr>
	</tbody>
</table>

a comprehensive procedure to evaluate the in vivo performance of cancer nanomedicines

在诊所以前的癌症纳米医学的成功的启发，研究人员已经在过去的十年中产生了大量新的配方。然而，只有纳米医学少数已被批准用于临床使用，而根据临床开发的大多数纳米医学的已产生令人失望的结果。到的新的癌症纳米医学成功临床翻译的一个主要障碍是缺乏其在体内表现的准确理解。本文设有严格的程序通过正电子发射断层扫描，计算机断层扫描（PET-CT），放射性量化方法的整合来表征在全身性的，组织，单细胞，和亚细胞水平荷瘤小鼠纳米医学的体内行为，流式细胞仪，荧光显微镜。使用这种方法，研究人员可以准确地评价着性相关的小鼠模型的新纳米制剂河这些协议可以具有高平移电位，以确定最有前途的癌症纳米医学或癌症纳米医学的供将来翻译优化以帮助的能力。

图1示出了该过程的概述。 图2表示在步骤1 10中所述的双重标记的脂质体的概略合成方法。 图3显示一个代表的PET-CT图像（ 图3a），从PET成像（ 图3b），血中半衰期（ 图3c），和放射性纳米颗粒的生物分布（ 图3d）放射性量化，如在步骤2中所述在图3a中 ，脂质体的高度积聚在肿瘤，这是由在图3b的成像结果的定量确认。此外，脂质体显示在图3c约10小时的血液半衰期。 体外生物分布证实了脂质体的高肿瘤积累。最后， 图4提供了一个典型的浇注公关ocedure识别相关细胞在肿瘤对纳米颗粒累积的单细胞水平分析（ 图4a和b），其中显示在肿瘤相关巨噬细胞（TAM）的纳米颗粒的优先积累。代表性共聚焦显微镜图像还示出了纳米颗粒和内皮细胞（ 图4c）之间的重叠。 <img alt="图1" src="/files/ftp_upload/55271/55271fig1.jpg" /> 图1. 在体内的纳米医学表征方法的示意图。此过程包括与两个放射性和荧光标签纳米颗粒的标记;与PET-CT成像，血液半衰期的测量，和生物分布评价放射性标记的纳米粒子的表征;和单细胞并通过流式细胞术和我荧光纳米粒子的亚细胞分析mmunostaining分别。的技术中，从左至右，提供增加的体内蓄积纳米颗粒的空间分辨率。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55271/55271fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55271/55271fig2.jpg" /> 图2. 双标记的脂质体的合成过程。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55271/55271fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55271/55271fig3.jpg" /> 图3. 放射性标记纳米微粒在体内表征代表性的成果。 （a）一种致瘤的PET-CT图像的r-轴承小鼠纳米颗粒注射和（b）的PET衍生的摄取值在多种组织后（N = 3）。 （三）血液放射性的放射性标记的纳米粒子的半衰期，以及（d）在24小时后注射其生物分布（N = 3）。误差棒为标准偏差。从参考8修改许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55271/55271fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55271/55271fig4.jpg" /> 图4. 荧光纳米颗粒在体内的表征代表性的成果。 （a）在典型的流式细胞仪门控程序，以确定肿瘤相关的免疫细胞，肿瘤细胞，和内皮细胞（EC），以及（二）nanoparti的量化CLE积累在这些细胞中8。 3生物重复形象代表。 （三）代表免疫图像显示的RGD-结合纳米乳的特异性靶向内皮细胞肿瘤13细胞。从参考8和13修改许可。肿瘤相关巨噬细胞（TAM）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55271/55271fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines at JoVE.com

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

综合程序，以评估<em&gt;在体内</em&gt;癌症纳米医学的性能

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

a-comprehensive-procedure-to-evaluate-vivo-performance-cancer

Nanyang Technological University

Research

JoVE Journal

Cancer Research

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

8.8K 次观看.  Memorial Sloan Kettering Cancer Center. The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

视频: A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio)

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression. Traditional in vitro models of cancer cell invasion of endothelia typically lack the fluid dynamics that invading cells are otherwise exposed to in vivo. However, in vivo systems such as mouse models, though more physiologically relevant, require longer experimental timescales and present unique challenges associated with monitoring and data analysis. Here we describe a zebrafish assay that seeks to bridge this technical gap by allowing for the rapid assessment of cancer cell vascular invasion and extravasation. The approach involves injecting fluorescent cancer cells into the precardiac sinus of transparent 2-day old zebrafish embryos whose vasculature is marked by a contrasting fluorescent reporter. Following injection, the cancer cells must survive in circulation and subsequently extravasate from vessels into tissues in the caudal region of the embryo. Extravasated cancer cells are efficiently identified and scored in live embryos via fluorescence imaging at a fixed timepoint. This technique can be modified to study intravasation and/or competition amongst a heterogeneous mixture of cancer cells by changing the injection site to the yolk sac. Together, these methods can evaluate a hallmark behavior of cancer cells and help uncover mechanisms indicative of malignant progression to the metastatic phenotype.

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio

This method utilizes zebrafish embryos to efficiently test the vascular invasive ability of cancer cells. Fluorescent cancer cells are injected into the precardiac sinus or yolk sac of developing embryos. Cancer cell vascular invasion and extravasation is assessed via fluorescence microscopy of the tail region 24 to 96 hr later.

测试癌细胞的斑马鱼的血管侵袭能力（<em&gt;斑马鱼</em&gt;）

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression.  Traditional in vitro models of cancer cell invasion of ...

科研

癌症研究

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

用球形发芽法评价 Hemangioblastoma 新生血管的体外性能的综合程序

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

磷酸浓缩结合无标签定量质谱法研究前列腺癌的 Phosphoproteome

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

This protocol describes xenograft and orthotopic mouse models of human thyroid tumorigenesis as a platform to test microRNA-based inhibitor treatments. This approach is ideal to study the function of non-coding RNAs and their potential as new therapeutic targets.

在微RNA的Vivo抑制下，降低小鼠的肿瘤生长

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

体外测定研究肿瘤-巨噬菌体相互作用

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

研究肿瘤分泌性副体结扎对宏噬细胞活化的影响，使用可渗透膜支架共培养

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

肿瘤细胞外侧尾静脉注射后肺转移的病理分析

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

通过颅内注射和磁共振成像对大脑转移进行建模

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

聚酐IL-1α纳米颗粒 体内抗肿瘤 活性评价

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

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此视频中的章节

测试癌细胞的斑马鱼的血管侵袭能力（

体内在肿瘤微环境中 pH 值、 pO₂、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

用球形发芽法评价 Hemangioblastoma 新生血管的体外性能的综合程序

磷酸浓缩结合无标签定量质谱法研究前列腺癌的 Phosphoproteome

在微RNA的Vivo抑制下，降低小鼠的肿瘤生长

体外测定研究肿瘤-巨噬菌体相互作用

研究肿瘤分泌性副体结扎对宏噬细胞活化的影响，使用可渗透膜支架共培养

肿瘤细胞外侧尾静脉注射后肺转移的病理分析

通过颅内注射和磁共振成像对大脑转移进行建模

聚酐IL-1α纳米颗粒体内抗肿瘤活性评价

综合程序，以评估

摘要

探索更多视频

此视频中的章节

测试癌细胞的斑马鱼的血管侵袭能力（

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

用球形发芽法评价 Hemangioblastoma 新生血管的体外性能的综合程序

磷酸浓缩结合无标签定量质谱法研究前列腺癌的 Phosphoproteome

在微RNA的Vivo抑制下，降低小鼠的肿瘤生长

体外测定研究肿瘤-巨噬菌体相互作用

研究肿瘤分泌性副体结扎对宏噬细胞活化的影响，使用可渗透膜支架共培养

肿瘤细胞外侧尾静脉注射后肺转移的病理分析

通过颅内注射和磁共振成像对大脑转移进行建模

聚酐IL-1α纳米颗粒 体内抗肿瘤 活性评价

体内在肿瘤微环境中 pH 值、 pO₂、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

聚酐IL-1α纳米颗粒体内抗肿瘤活性评价