纳丹 Baglio 得到了由欧洲联盟出资的 Associazione 意大利每 la Ricerca Cancro (AIRC) 的奖学金。此外, 该项目还从欧洲联盟2020《玛丽·斯卡洛多斯卡·居里-居里赠款协定》660200号 (纳丹 Baglio) 下的研究和创新方案中获得资金。

在机构伦理委员会批准和书面知情同意后, Tergooi 医院 (荷兰 Hilversum) 整形外科部获得了人骨髓间充质干细胞分离的人体脂肪组织。GFP-阳性脂肪间充质干细胞是从医学和外科科学部为儿童和成人 (摩德纳大学和艾米利亚)。
动物实验是按照荷兰动物实验法进行的, 该议定书是荷兰阿姆斯特丹大学医学中心动物实验委员会批准的。
1. 分离肿瘤分泌的胞外囊。
<ol><li>用离心在 7万 x g 夜 (16 小时), 没有刹车, 在4°c 使用一个超摆动的铲斗转子 (预期 1 h 为减速) 制备 EV 耗尽胎牛血清 (血清)。仔细收集上清 (EV 耗尽的血清) 而不干扰颗粒。用0.22 µm 过滤器过滤 EV 耗尽的血清。</li>
	<li>用100毫升青霉素、100µg/毫升链霉素、2毫米谷氨酰胺 (1x P/Iscove/G) 和 5% EV 耗尽的血清补充的改性 Dulbecco 培养基 (IMDM) 制备 EV 耗尽培养基。</li>
	<li>种子 3 x 106 143B 细胞在 175 cm2烧瓶和养殖他们在 EV 被耗尽的文化媒介在一个孵化器在37°c 和 5% CO2。当细胞是 80-90% 汇合 (通常在36小时到48小时之后), 收集从 8x 175 cm2烧瓶 (28 毫升/瓶) 的上清为 EV 隔离。</li>
	<li>根据下面的协议, 预先清除细胞上清液, 通过差分离心 (离心上一次旋转的上清) 去除细胞和细胞碎片:<ol>
<li>将上清液离心两次, 500 x 克, 10 分钟。</li>
		<li>将上清液离心两次, 2000 x 克, 15 分钟。</li>
		<li>将上清液离心两次, 1万 x 克, 30 分钟。</li>
		<li>执行所有 centrifugations 在4°c 与最大加速度和减速速度设置。每一步后, 仔细收集含 EV 的上清, 留下约1毫升。立即进行步骤1.5 或存储预清除条件介质在-80 °c, 直到 EV 隔离。</li>
	</ol>
</li>
	<li>使用超摆动斗式转子, 在超离心管 (最终容积38.5 毫升/管) 中, 用 7万 x g 离心预清除条件介质, 将其隔离为1小时, 慢速刹车, 4 °c。</li>
	<li>小心移除上清, 留约1毫升后, 并用重悬的含 EV 颗粒在剩余的体积, 把它们全部放在一个 ultracentrifuge 管, 并添加磷酸盐缓冲盐水 (PBS) 直到完整的管充填 (最终体积38.5 毫升)。</li>
	<li>离心机再一次在 7万 x g 为1小时在4°c, 没有刹车 (期望 1 h 为减速)。小心取出上清液, 不干扰颗粒, 并留下 100-200 µL。</li>
	<li>并用重悬 ev 颗粒, 并调整最终体积到200µL 与 PBS (标准化为所有 EV 隔离)。立即使用 EV 准备或存储在-80 °c, 直到使用22。 注: 电动车可储存6月至-80 摄氏度。</li>
</ol>2. EV 特性。
纯化电动汽车可以通过透射电镜 (TEM)23进行可视化。
<ol><li>混合 EV 眼影与4% 多聚甲醛在磷酸盐缓冲容量相等。</li>
	<li>涂层200网格 Formvar-碳包覆的镍 TEM 网格, 5 µL 的电动汽车悬浮在室温下为20分钟。</li>
	<li>用1% 戊二醛在0.1 米磷酸酯缓冲液 (ph 7.0) 与草酸铀 (ph 7.0) 进行对比, 将 EV 样品集中在 TEM 网格上, 并将4% 个醋酸盐和2% 甲基纤维素的混合物嵌入到1:9 的冰上。</li>
	<li>用不锈钢环去除网格, 用滤纸涂抹多余的液体, 以确保甲基纤维素膜的适当厚度。</li>
	<li>干燥后, 用透射电子显微镜检查网格, 用数码相机捕捉图像, 并与相应的图像软件相耦合。</li>
</ol>3. 用肿瘤衍生的电动车对 MSCs 进行教育。
<ol><li>用 4% EV 耗尽的人血小板裂解液 (hPL)24, 肝素 (10 U/毫升) 和1x 的 P/秒来补充α最小必需培养基 (alpha), 制备 MSC 培养基。 注: EV 耗尽的 hPL 是按照1.1 节中描述的程序获得的。</li>
	<li>种子 1.4 x 106脂肪派生的 MSCs 每 75 cm2烧瓶和养殖他们在 EV 耗尽的 MSC 培养基在一个孵化器在37°c 和 5% CO2。</li>
	<li>当细胞达到70% 汇合, 添加143B 骨肉瘤-或控制人成纤维细胞 (hf)-电动车, 10 µL EV 准备/cm2培养瓶。孵育24小时, 并添加相同数量的电动车额外的6小时。注: 人成纤维细胞是在 Dulbecco 的改良鹰培养基 (DMEM) 中培养的, 10% 的血清和电动车被隔离, 如第1节所述。</li>
	<li>收集上清液, 离心机1x 在 500 x g 5 分钟4摄氏度 (最大加速度和减速速度设置), 转移到新的管和存储清除清液-80 °c, 以使未来的细胞因子分析 (5 节)。</li>
	<li>对于体内实验 (第6节), 教育 GFP 阳性 MSCs25 , 如3.1 至3.3 节所述, 在 PBS 中收集单元格并将其并用重悬为10 6 细胞每100µL 的最后浓度. 把细胞放在冰上直到注射。</li>
</ol>4. EV 内化试验。
为了可视化 MSCs 的 EV 吸收, 标签电动车与绿色荧光链接器染料 (GFLD) (商业上可用) 后, 制造商的协议:
<ol><li>简要地, 增加180µL 稀释剂到200µL EV 准备。稀释1µL 的 GFLD 染料50µL 稀释剂, 并添加20µL 稀释染料的稀释 EV 准备。</li>
	<li>轻轻地混合吹打和孵化3分钟在室温下在黑暗中。</li>
	<li>在 pbs 中加入5毫升 0.1% BSA, 转移至超离心管, 并将管与 PBS (最终体积38.5 毫升/管) 填满。</li>
	<li>离心机1x 在 7万 x g 1 小时在4°c, 没有刹车 (期望 1 h 为减速)。小心地去除上清和并用重悬标记 EV 颗粒在最后的容量200µL (调整容量与 PBS)。</li>
	<li>将标记的 EVs 添加到 MSC 区域性, 或将其存储在摄氏-80 摄氏度。夜间孵化后, 通过荧光显微镜和流式细胞术7评估囊泡内化。</li>
</ol>5. 细胞因子表达谱的评估。
<ol><li>对于 interleukin-8 (IL-8)、interleukin-1β (IL-1β)、interleukin-6 (IL-6)、interleukin-10 (IL-10)、肿瘤坏死因子 (TNF) 和 interleukin-12p70 (IL-12p70) 蛋白质水平的多项量化的标准条件培养基 (见步骤 3.4), 使用商业可用的细胞珠阵列 (CBA) 的人炎症细胞因子后, 制造商的指示。</li>
	<li>简单地, 混合抗体共轭捕获珠, 并添加到所有化验管。将细胞因子标准稀释或条件培养基样品添加到管中。</li>
	<li>添加检测试剂和孵化3小时在 RT。</li>
	<li>用所提供的洗涤液清洗珠子。使用流式细胞仪测量样品, 并根据制造商的说明分析数据。</li>
</ol>6. 生成骨肉瘤同种异体原位移植小鼠模型。
<ol><li>允许六周大, 雌性, 裸 Nude-Foxn1nu 小鼠在开始实验过程前至少一周适应。</li>
	<li>前一天的手术过程和围手术期镇痛添加扑热息痛的饮用水。稀释 "对孩子的扑热息痛解决方案" (参见材料表) 用水获得最终浓度为2毫克/毫升。根据平均水摄入量150毫升/千克/天, 和平均体重20克, 这一剂量足以达到剂量6毫克/鼠/天 (300 毫克/千克/天)。</li>
	<li>持续止痛治疗, 直到手术后24小时, 或更长的时间, 如果动物显示疼痛的迹象。</li>
	<li>在手术的当天, 收集培养的荧光素阳性 (Fluc) 143B 细胞 (以前由慢病毒载体转导) 由离心在 300 x g 10 分钟, 并用重悬细胞集中2 x 10 5 细胞/µL 在 PBS。保持细胞悬浮在冰上长达6小时。</li>
	<li>高压釜手术设备。准备和清洁工作区域, 并将灭菌的剪刀和镊子放在无菌床单上。手术前二十分钟 (步骤 6.8), 注射以丁丙诺啡 (0.05 毫克/千克, 稀释0.9% 生理盐水) 为镇痛剂的动物, 使用0.5 毫升胰岛素注射器 (29 口径针)。</li>
	<li>就在麻醉每只动物之前, 用10µL 注射器填满至少1µL 的浓缩 (Fluc) 143B 细胞悬浮 (见步骤 6.4)。</li>
	<li>麻醉的动物与异氟醚吸入麻醉 (2-3% 的氧气)。用脚趾捏的动物检查麻醉的水平。当动物对捏不起反应时,即,它被深深地麻醉, 在眼睛上涂抹药膏以防止麻醉时的干燥。</li>
	<li>将动物放在背部, 头部用麻醉面罩和腿部向操作员。弯曲左膝。用70% 乙醇擦拭皮肤, 应用利多卡因 (2%) 与棉花尖3倍作为局部镇痛剂。</li>
	<li>使用无菌手术刀在膝盖下面的皮肤上做一个小切口 (大约5毫米) 暴露胫骨。在胫骨上钻一个针孔, 膝盖下面大约2毫米使用0.8 毫米微麻花钻。小心不要钻两个胫骨皮质。</li>
	<li>使用5口径针将1µL 细胞悬浮 (大约 2 x 105细胞) 缓慢地 (在大约26秒内) 插入孔中。用一滴纸巾将孔闭合, 防止悬浮液回流。</li>
	<li>用单丝缝合和一滴组织胶水关闭皮肤。让动物在炎热的环境中恢复 (使用热灯)。不要让动物无人看管, 直到他们恢复足够的意识来维持胸骨卧床。只有在动物完全从麻醉中恢复后, 才能把它们送回自己的笼子里。</li>
	<li>在肿瘤接种两天后, 注射 EV 教育或天真的 GFP 阳性 MSCs (106细胞在100µL PBS, 看见步 3.5) 静脉注射与0.5 毫升胰岛素注射器在小鼠的尾巴静脉。</li>
	<li>通过生物荧光成像26每周两次肿瘤生长。为此, 用胰岛素注射器注射150µL d-荧光素 (30 毫克/毫升) 的小鼠。注射后十分钟, 将动物放置在生物发光成像系统中, 测量肿瘤细胞产生的光子通量。</li>
	<li>另外, 用卡尺测量原发性骨肿瘤的直径。肿瘤体积 (毫米3) 估计宽度 x 长度2 x 0.5。</li>
	<li>注意: 人性化端点被定义为肿瘤直径 > 15 毫米或体重损失 > 15%。</li>
</ol>7. 肺结核数目的评估
<ol><li>当其中一个动物到达步骤6.14 定义的人文端点 (大约 3-4 周), 终止实验, 收集和分析所有相关组织。</li>
	<li>euthanization 前十分钟, 注射150µL d-荧光素 (30 毫克/毫升) 与0.5 毫升胰岛素注射器。</li>
	<li>麻醉的动物与异氟醚吸入麻醉 (见步骤 6.7)。如果没有老鼠对脚趾捏的反应, 请确认麻醉深度。弄死颈椎脱位27对小鼠进行了实验。</li>
	<li>用消毒过的剪刀和镊子收集肺部、肝脏、脾脏和肾脏。清洗 PBS 中的器官以清除血液痕迹并将其放在培养皿上。</li>
	<li>在生物发光成像系统中放置培养皿与器官。测量器官两侧的生物发光信号。测量后, 立即将4% 福尔马林中的器官浸入 (24-72 小时, RT), 用于未来的组织学分析。</li>
	<li>使用适当的图像软件进行手工阈值处理获得的图片。计算每张图片上的肺结节数。计算肺部两侧的肺结节总数。</li>
</ol>8. 组织学分析
<ol><li>肺组织的组织学分析:<ol>
<li>将福尔马林固定器官嵌入石蜡, 准备6µm 组织切片。</li>
		<li>Deparaffinate 肺组织切片, 并在0.01 米柠檬酸盐缓冲液中煮沸15分钟进行抗原提取 (pH 6)。</li>
		<li>在室温下, 用抗人波形抗体 (200 µg/毫升) 稀释 1:150, 在抗体稀释剂 (商用) 中进行水平孵化, 随后用二次, 人工标记抗体 (0.5 毫克/毫升, 稀释 1:500)。用 3 '-diaminobenzidine (民建) 和苏木素计数器染色染色组织。</li>
		<li>用光学显微镜与相应的相机和图像软件相结合观察染色组织幻灯片。</li>
	</ol>
</li>
	<li>评价 GFP 阳性 MSCs 在小鼠胫骨中的存在:<ol>
<li>除去石灰质胫骨 (EDTA) 0.24 米, pH 7.2-7.4。每隔一天刷新 EDTA 缓冲, 直到骨骼变得灵活 (大约1周)。</li>
		<li>脱水胫骨, 用石蜡渗入。在石蜡块中嵌入胫骨 (包括骨肉瘤组织)。</li>
		<li>使用切片准备6µm 石蜡切片。将石蜡切片放到玻璃滑梯上。干燥后, 贮存在室温下过夜。</li>
		<li>用柠檬酸缓冲剂进行热介导的抗原检索 (见 8.1.2), 在1:900 稀释的抗体稀释剂中用抗 GFP 抗体 (全抗体) 染色组织。Counterstain 组织与 4 ', 6-diamidino 2-苯基吲哚 (DAPI)。</li>
		<li>用荧光显微镜观察组织的幻灯片, 并结合相应的成像软件。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

肿瘤分泌的胞外囊 (EVs) 可以改变局部和远端间充质细胞的生理, 从而产生肿瘤支持的环境。在这里, 我们描述的一代骨肉瘤的前体小鼠模型, 允许解剖的 EV 介导的相互作用的肿瘤细胞和间充质干细胞 (MSCs)在体内。我们表明, 系统注射的人肿瘤 EV 培养的小鼠骨肉瘤移植通过激活 IL-6/STAT3 信号通路7强促进肿瘤的生长和转移形成。
最近的研究表明, 癌症电?...

肿瘤微环境积极参与了肿瘤发生和肿瘤进展的大部分 (如果不是全部) 方面, 包括转移形成和对治疗的抵抗力的发展1。这强调需要对前体肿瘤小鼠模型, 允许解剖复杂的肿瘤基质相互作用发生在肿瘤的利基。
在肿瘤微环境的许多细胞成分中, 间充质干细胞 (MSCs) 在多种癌症类型 (如乳腺癌、前列腺癌、脑肿瘤、多发性骨髓瘤和骨肉瘤) 中有强烈的促进作用2 ,3,4,5,6,7。MSCs 是驻留在各种成人和胎儿组织中的多能干细胞, 包括骨髓、脂肪组织、胎盘、脐血和其他8,9。为应对癌症产生的炎症信号, MSCs 向肿瘤部位迁移, 纳入肿瘤微环境, 最终分化为肿瘤支持细胞10。这些与癌症相关的 MSCs 提供了重要因素 (例如,生长因子、趋化因子、细胞因子和免疫抑制剂), 用于肿瘤细胞和周围基质的作用2,3,11,12,13. 虽然在众多的癌症模型中研究了癌症相关的 mscs 的促肿瘤作用, 但是肿瘤细胞重新编程 mscs 以形成促进癌症的利基的机制却不甚了解。在这里, 我们描述了一个同种异体原位移植模型的生成, 它特别允许通过细胞外囊泡 (EVs) 研究骨癌细胞与骨髓间充质干细胞之间的亲癌变相互作用。
EVs 是肿瘤与基质细胞间通讯的关键介质14。EVs 携带原细胞的功能生物分子, 包括蛋白质、血脂和调控 rna。一旦在细胞外空间释放, 这些囊泡可以被周围的细胞占用, 或者通过血液或淋巴循环携带到远处的部位, 并能深刻地影响靶细胞的行为。15,16, 17例如, 基质成纤维细胞对癌电动车的吸收可能导致上皮细胞分化支持血管生成和加速肿瘤生长体内 18 19, 内皮内部化细胞可以刺激肿瘤血管生成, 增加血管通透性16,20, 与免疫细胞的相互作用可能导致抑制抗肿瘤免疫应答21。
我们最近用一种生物发光的同种异体骨肉瘤模型证明, 肿瘤细胞释放出大量的电动车, 促使 MSCs 获得亲癌变和支持转移的表型。这种影响是由于 msc 细胞因子表达谱 (称为 "理学硕士教育") 的戏剧性变化, 并可通过管理治疗 interleukin-6 受体 (IL-6R) 抗体7来预防。我们的研究表明, 癌症电动车是 MSC 行为的关键调节剂, 从而为以微环境为目标的方法阻止骨肉瘤进展提供了理论依据。在此, 我们描述了一个逐步的协议, 以调查 EV 介导的肿瘤理学硕士互动在体内。该模型的目的是: 1) 明确定义的癌症 EV 诱导改变的肿瘤微环境, 2) 评估这种相互作用是如何促进骨肿瘤的生长和转移形成, 3) 研究是否干预EV 介导的串扰在体内防止癌症进展。

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ultra Centrifuge</td><td>Beckman</td><td>Optima L-90K</td><td></td></tr><tr><td>Rotor SW32Ti</td><td>Beckman</td><td>369650</td><td>Referred to in the manuscript as ultra-swinging bucket rotor</td></tr><tr><td>Transmission electron microscope</td><td>Zeiss</td><td>EM109</td><td>Or similar TEM</td></tr><tr><td>Digital camera</td><td>Nikon</td><td>DMX 1200F</td><td>Or similar camera</td></tr><tr><td>Imaging software TEM&amp;nbsp;</td><td>Nikon</td><td>ACT-1</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Zeiss</td><td>Imager.D2</td><td>Or similar Fluorescence microscope</td></tr><tr><td>Imaging software FM</td><td>Zeiss</td><td>ZEN Blue</td><td></td></tr><tr><td>Incubator</td><td>Nuaire</td><td>4750E</td><td></td></tr><tr><td>Centrifuge</td><td>Hettick</td><td>ROTANTA 460R</td><td></td></tr><tr><td>-80 Freezer</td><td>Thermo electro corporation</td><td>n.a.</td><td></td></tr><tr><td>FACS</td><td>BD</td><td>BD FACScalibur</td><td>Or similar flow cytometer</td></tr><tr><td>Drill</td><td>Ferm</td><td>FCT-300</td><td>With 0.8 mm drill</td></tr><tr><td>HSS micro twist drills, 0.8 mm</td><td>Proxxon</td><td>28 852</td><td>0.8 mm drill</td></tr><tr><td>IVIS camera</td><td>Xenogen</td><td>Ivis Lumina</td><td>Referred to in the manuscript as bioluminescence camera. Xenogen is now part of Perkin Elmer</td></tr><tr><td>Living image software2.60</td><td>Xenogen / Igor Por</td><td>n.a</td><td>Xenogen is now part of Perkin Elmer</td></tr><tr><td>10 &amp;micro;L Syringe</td><td>Hamilton</td><td>Neuros Model 1701 RN</td><td></td></tr><tr><td>Needle: Hamilton RN Needle for Syringe, 26 Gauge, Pointstyle AS, custom length 2 cm</td><td>Hamilton</td><td>n.a.</td><td></td></tr><tr><td>Caliper</td><td>Mitutoyo</td><td>G08004463</td><td></td></tr><tr><td>Autoclave</td><td>Astell</td><td>n.a.</td><td></td></tr><tr><td>Heat Lamp</td><td>Philips</td><td>n.a.</td><td></td></tr><tr><td>&lt;strong&gt;Culture media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>RYG35912</td><td></td></tr><tr><td>Platelet Lysate</td><td>n.a.</td><td>n.a.</td><td></td></tr><tr><td>IMDM medium</td><td>Lonza</td><td>BE12-722F</td><td></td></tr><tr><td>alpha-MEM medium</td><td>Lonza</td><td>BE02-002F</td><td></td></tr><tr><td>DMEM medium</td><td>Lonza</td><td>BE12-614F</td><td></td></tr><tr><td>pen/strep/glutamine</td><td>GIBCO</td><td>10378-016</td><td></td></tr><tr><td>heparin</td><td>LEO</td><td>012866-08</td><td></td></tr><tr><td>Trypsin/EDTA (10x)</td><td>GIBCO</td><td>15400-054</td><td></td></tr><tr><td>&lt;strong&gt;Cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>adipose deriverd MSCs</td><td>n.a.</td><td>n.a.</td><td></td></tr><tr><td>GFP-positive MSCs</td><td>n.a.</td><td>n.a.</td><td></td></tr><tr><td>human fibroblasts</td><td>n.a.</td><td>n.a.</td><td></td></tr><tr><td>143B cells</td><td>ATCC</td><td>CRL-8303</td><td></td></tr><tr><td>FLUC-143B cells</td><td>ATCC</td><td>CRL-8303</td><td>Transduced</td></tr><tr><td>&lt;strong&gt;Disposables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Culture flasks 175 cm2</td><td>CELLSTAR</td><td>660175</td><td></td></tr><tr><td>50 mL tubes</td><td>Greiner bio-one</td><td>210261</td><td></td></tr><tr><td>Freeze tubes</td><td>Thermoscientific</td><td>377224</td><td></td></tr><tr><td>Ultra-Clear tubes</td><td>Beckman</td><td>344058</td><td>Referred to in the manuscript as ultra-centrifuge tubes</td></tr><tr><td>0,22 &amp;micro;m filter</td><td>Millex</td><td>SLGV033RS</td><td></td></tr><tr><td>200 mesh Formvar-carbon-coated nickel grids</td><td>EMS (Electron Microscopy Sciences)</td><td></td><td></td></tr><tr><td>0.5 mL insulin syringes with 29G Needle</td><td>Terumo</td><td>U-100&amp;nbsp;</td><td></td></tr><tr><td>Petri dish</td><td>Sigma - Aldrich</td><td>P7612</td><td></td></tr><tr><td>Filter paper&amp;nbsp;</td><td>Thermo fisher Scientific</td><td>50363215</td><td></td></tr><tr><td>&lt;strong&gt;Reagents / kits&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>paraformaldehyde</td><td>Alfa Aeser</td><td>43368.9M</td><td></td></tr><tr><td>PBS</td><td>Braun</td><td>220/12257974/110</td><td></td></tr><tr><td>glutaraldehyde</td><td>EMS (Electron Microscopy Sciences)</td><td>16300</td><td></td></tr><tr><td>uranyl oxalate</td><td>EMS (Electron Microscopy Sciences)</td><td>22510</td><td></td></tr><tr><td>urany acetate</td><td>EMS (Electron Microscopy Sciences)</td><td>22400</td><td></td></tr><tr><td>methyl cellulose</td><td>EMS (Electron Microscopy Sciences)</td><td>1560</td><td></td></tr><tr><td>PKH67</td><td>Sigma</td><td>mini67-1kt</td><td>Referred to in the manuscript as GFLD</td></tr><tr><td>BSA</td><td>Sigma</td><td>A8412</td><td></td></tr><tr><td>CBA - human inflammatory cytokine kit</td><td>BD</td><td>551811</td><td></td></tr><tr><td>Formaldehyde 37%</td><td>VWR</td><td>104003100</td><td></td></tr><tr><td>Carbon Steel surgical blades</td><td>Swann-Morton</td><td>206</td><td>Referred to in the manuscript as surgical knife</td></tr><tr><td>anti-human vimentin antibody</td><td>Santa Cruz</td><td>sc-6260</td><td>Clone V9</td></tr><tr><td>Antibody diluent</td><td>DAKO</td><td>S0809</td><td></td></tr><tr><td>HRP-labeled anti mouse IgG antibody</td><td>Life Technologies</td><td>32230</td><td></td></tr><tr><td>DAB-kit</td><td>DAKO</td><td>K500711</td><td></td></tr><tr><td>hematoxyllin</td><td>Sigma</td><td>GHS232</td><td></td></tr><tr><td>EDTA-buffer</td><td>n.a.</td><td>n.a.</td><td></td></tr><tr><td>Citrate buffer</td><td>n.a.</td><td>n.a.</td><td></td></tr><tr><td>rabbit polyclonal anti-GFP antibody</td><td>Abcam</td><td>n.a.</td><td>Ab290</td></tr><tr><td>DAPI&amp;nbsp;</td><td>Life Technologies</td><td>D1306</td><td></td></tr><tr><td>Paracetamol, 120 mg / 5 ml syrup</td><td>Bayer</td><td>n.a.</td><td>Sinaspril, paracetamol solution for kids</td></tr><tr><td>Isoflurane 1000 mg/g</td><td>Vumc pharmacy</td><td>n.a.</td><td></td></tr><tr><td>buprenofine hydrochloride, 0.3 mg/ml</td><td>Indivior UK Limited</td><td>n.a.</td><td></td></tr><tr><td>lidocaine-HCL 2%</td><td>Vumc pharmacy</td><td>n.a.</td><td></td></tr><tr><td>70% ethanol</td><td>VWR</td><td>93003.1006</td><td></td></tr><tr><td>Tissue glue</td><td>Derma+Flex, formulated medical cyanoacrylate</td><td>Vygon</td><td>LB604060</td></tr><tr><td>Eyedrops: Vidisec Carbogel, 2 mg/ml</td><td>Bausch+Lomb</td><td>n.a.</td><td></td></tr><tr><td>D-luciferin, potassium salt</td><td>Gold Biotechnology</td><td>LUCK-1</td><td></td></tr><tr><td>Glass slides</td><td>Thermo scientific</td><td>630-0954</td><td></td></tr><tr><td>Stainless steel loops&amp;nbsp;</td><td>n.a.</td><td>n.a.</td><td></td></tr><tr><td>&lt;strong&gt;Mice experiments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mice, Hsd:Athymic Nude-Foxn1nu,&amp;nbsp; female, 6 weeks at arrival, bacterial status conform FELASA</td><td>ENVIGO</td><td>n.a.</td><td></td></tr><tr><td>Paper-pulp smart home (cage enrichment)</td><td>Bio Services</td><td>n.a.</td><td></td></tr><tr><td>Alpha-dri bedding material</td><td>Shepperd Speciality Papers</td><td>n.a.</td><td></td></tr><tr><td>Mouse food: Teklad global 18% protein rodent diet</td><td>ENVIGO</td><td>2918-11416M</td><td></td></tr><tr><td>Sutures</td><td>Ethicon</td><td>V926H</td><td></td></tr><tr><td>Scissors</td><td>Sigma-Aldrich</td><td>S3146-1EA</td><td>(or similar)</td></tr><tr><td>Tweezers</td><td>Sigma-Aldrich</td><td>F4142-1EA</td><td>(or similar)</td></tr></tbody></table>

a preclinical mouse model of osteosarcoma to define the extracellular vesicle-mediated communication between tumor and mesenchymal stem cells

在肿瘤微环境中, 居民或被征募的间充质干细胞 (MSCs) 有助于多种癌症类型的恶性进展。在特定环境信号的影响下, 这些成体干细胞可以释放分泌介质, 从而加速肿瘤的生长和转移。定义肿瘤和骨髓间充质之间的串扰, 对于了解癌症进展的机制和确定治疗干预的新靶点至关重要。
癌细胞产生大量的胞外囊泡 (EVs), 这会深刻地影响肿瘤微环境或远处的靶细胞的行为。肿瘤电动车包围功能生物分子, 包括炎症 rna 和 (onco) 蛋白, 可以教育基质细胞, 以增强癌细胞的转移行为或参与转移前的利基形成。在这篇文章中, 我们描述了一个前肿瘤小鼠模型的发展, 它可以对 EV 介导的肿瘤和间充质干细胞之间的串扰进行具体评估。首先, 我们描述了肿瘤分泌的电动车的纯化和表征, 以及 MSCs 对电动汽车内化的评价。然后利用复用珠基免疫分析法, 对癌症电动车诱导的细胞因子表达谱的改变进行评价。最后, 我们说明了概括骨肉瘤的生物荧光原位异种移植小鼠模型的生成, 并显示了 EV 培养的骨髓间充质干细胞对肿瘤生长和转移形成的贡献。
我们的模型提供了一个机会来定义癌症电动车如何形成肿瘤支持的环境, 并评估是否封锁的 EV 介导的肿瘤和 MSCs 之间的沟通, 以防止癌症进展。

在这项研究中, 我们探讨了骨肉瘤分泌的电动车的能力, 以培养骨髓间充质干细胞向癌变和支持转移表型。我们表明, 骨肉瘤细胞释放外切样的电动车, 由 MSCs 内化。我们测量了癌症电动车诱导的细胞因子表达谱的变化, 并评价了 EV 培养的骨髓间充质干细胞对肿瘤生长和转移形成的影响。研究设计的一般表示形式在图 1中进行了说明。
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Watch this Scientific Journal Video about A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells at JoVE.com

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

骨肉瘤前小鼠模型对肿瘤与间充质干细胞细胞外囊泡介导通信的定义

直接注射癌症衍生细胞外囊 (EVs) 导致骨髓支持肿瘤进展的重新编程;然而, 哪些细胞调解这种影响是不清楚的。在这里, 我们描述了一步一步的协议来调查 ev 介导的肿瘤间充质干细胞 (MSC) 相互作用的在体内, 揭示了一个关键的作用, 电动汽车教育的 MSCs 在转移。

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

a-preclinical-mouse-model-osteosarcoma-to-define-extracellular

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

10.1K 次观看.  VU Medical Center. 直接注射癌症衍生细胞外囊 (EVs) 导致骨髓支持肿瘤进展的重新编程;然而, 哪些细胞调解这种影响是不清楚的。在这里, 我们描述了一步一步的协议来调查 ev 介导的肿瘤间充质干细胞 (MSC) 相互作用的在体内, 揭示了一个关键的作用, 电动汽车教育的 MSCs 在转移。

视频: A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

应用 Fraumeni 综合征诱导多潜能干细胞建模骨肉瘤

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

科研

癌症研究

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

骨肉瘤的三维骨细胞外基质模型

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

肺转移瘤在小鼠体内的组合在单细胞分辨率的可视化改进<em&gt;在原位</em灌注肺组织和X-gal染色<em&gt; lacZ的</em&gt;标记的肿瘤细胞

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

医学

LINE-1 Methylation Analysis in Mesenchymal Stem Cells Treated with Osteosarcoma-Derived Extracellular Vesicles

Methylation-specific probe amplification (MSPA) is a simple and robust technique that can be used to detect relative differences in methylation levels of DNA samples. It is resourceful, requires small amounts of DNA, and takes around 4&#8211;5 h of hands-on work. In the presented technique, DNA samples are first denatured then hybridized to probes that target DNA at either methylated or reference sites as a control. Hybridized DNA is separated into parallel reactions, one undergoing only ligation and the other undergoing ligation followed by HhaI-mediated digestion at unmethylated GCGC sequences. The resultant DNA fragments are amplified by PCR and separated by capillary electrophoresis. Methylated GCGC sites are not digested by HhaI and produce peak signals, while unmethylated GCGC sites are digested and no peak signals are generated. Comparing the control-normalized peaks of digested and undigested versions of each sample provides the methylation dosage ratio of a DNA sample. Here, MSPA is used to detect the effects of osteosarcoma-derived extracellular vesicles (EVs) on the methylation status of long interspersed nuclear element-1 (LINE-1) in mesenchymal stem cells. LINE-1s are repetitive DNA elements that typically undergo hypomethylation in cancer and, in this capacity, may serve as a biomarker. Ultracentrifugation is also used as a cost-effective method to separate extracellular vesicles from biological fluids (i.e., when preparing EV-depleted fetal bovine serum [FBS] and isolating EVs from osteosarcoma conditioned media [differential centrifugation]). For methylation analysis, custom LINE-1 probes are designed to target three methylation sites in the LINE-1 promoter sequence and seven control sites. This protocol demonstrates the use of MSPA for LINE-1 methylation analysis and describes the preparation of EV-depleted FBS by ultracentrifugation.

Described here is the use of a methylation-specific probe amplification method to analyze methylation levels of LINE-1 elements in mesenchymal stem cells treated with osteosarcoma-derived extracellular vesicles. Ultracentrifugation, a popular procedure for separating extracellular vesicles from fetal bovine serum, is also demonstrated.

用骨肉瘤衍生细胞外囊泡处理的美相干细胞中的LINE-1甲基化分析

Methylation-specific probe amplification (MSPA) is a simple and robust technique that can be used to detect relative differences in methylation levels of ...

遗传学

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

通过实体瘤移植植入骨模拟原发性骨肿瘤和骨转移

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

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 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 ...

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

胫骨肉瘤细胞注射生成原位骨肉瘤和肺转移小鼠模型

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...

<meta charset="utf8"></head><body>骨肉瘤 PDX 小鼠模型的方案：研究生长和耐药性

Establishment of Patient-Derived Xenograft Mouse Model with Human Osteosarcoma Tissues

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, the prognosis for osteosarcoma patients has not significantly improved. Therefore, it is crucial to establish a robust preclinical model with high fidelity. The patient-derived xenograft (PDX) model faithfully preserves the genetic, epigenetic, and heterogeneous characteristics of human malignancies for each patient. Consequently, PDX models are considered authentic in vivo models for studying various cancers in transformation studies. This article presents a comprehensive protocol for creating and maintaining a PDX mouse model that accurately mirrors the morphological features of human osteosarcoma. This involves the immediate transplantation of freshly resected human osteosarcoma tissue into immunocompromised mice, followed by successive passaging. The described model serves as a platform for studying the growth, drug resistance, relapse, and metastasis of osteosarcoma. Additionally, it aids in screening the target therapeutics and establishing personalized treatment schemes.

<meta charset="utf8"></head><body>作者聚焦：在免疫缺陷小鼠中复制人骨肉瘤进展用于癌症研究

The present protocol describes the method for establishing a patient-derived xenograft (PDX) mouse model using human osteosarcoma tissue.

人骨肉瘤组织患者来源的异种移植小鼠模型的建立

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, ...

骨肉瘤前小鼠模型对肿瘤与间充质干细胞细胞外囊泡介导通信的定义

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此视频中的章节

应用 Fraumeni 综合征诱导多潜能干细胞建模骨肉瘤

骨肉瘤的三维骨细胞外基质模型

肺转移瘤在小鼠体内的组合在单细胞分辨率的可视化改进

用骨肉瘤衍生细胞外囊泡处理的美相干细胞中的LINE-1甲基化分析

通过实体瘤移植植入骨模拟原发性骨肿瘤和骨转移

同源原位骨肉瘤Sprague Dawley大鼠模型，截肢控制转移率

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

胫骨肉瘤细胞注射生成原位骨肉瘤和肺转移小鼠模型

人骨肉瘤组织患者来源的异种移植小鼠模型的建立

人骨肉瘤组织患者来源的异种移植小鼠模型的建立