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>

<ol>
	<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+The+next+generation.">Hallmarks of cancer: The next generation.</a> Cell. 144 (5), 646-674 (2011).</li><li>Karnoub, A. E., 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=Mesenchymal+stem+cells+within+tumour+stroma+promote+breast+cancer+metastasis.">Mesenchymal stem cells within tumour stroma promote breast cancer metastasis.</a> Nature. 449 (7162), 557-563 (2007).</li><li>Jung, Y., 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=Recruitment+of+mesenchymal+stem+cells+into+prostate+tumours+promotes+metastasis.">Recruitment of mesenchymal stem cells into prostate tumours promotes metastasis.</a> Nat Commun. 4, 1795 (2013).</li><li>Shahar, T., 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=Percentage+of+mesenchymal+stem+cells+in+high-grade+glioma+tumor+samples+correlates+with+patient+survival.">Percentage of mesenchymal stem cells in high-grade glioma tumor samples correlates with patient survival.</a> Neuro Oncol. 19 (5), (2016).</li><li>Behnan, J., 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=Recruited+brain+tumor-derived+mesenchymal+stem+cells+contribute+to+brain+tumor+progression.">Recruited brain tumor-derived mesenchymal stem cells contribute to brain tumor progression.</a> Stem Cells. 32 (5), 1110-1123 (2014).</li><li>Giallongo, 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=Granulocyte-like+myeloid+derived+suppressor+cells+(G-MDSC)+are+increased+in+multiple+myeloma+and+are+driven+by+dysfunctional+mesenchymal+stem+cells+(MSC).">Granulocyte-like myeloid derived suppressor cells (G-MDSC) are increased in multiple myeloma and are driven by dysfunctional mesenchymal stem cells (MSC).</a> Oncotarget. 7 (52), 85764-85775 (2016).</li><li>Baglio, S. R., 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=Blocking+tumor-educated+MSC+paracrine+activity+halts+osteosarcoma+progression.">Blocking tumor-educated MSC paracrine activity halts osteosarcoma progression.</a> Clin Cancer Res. 23 (14), 3721-3733 (2017).</li><li>Pittenger, M. F., 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=Multilineage+potential+of+adult+human+mesenchymal+stem+cells.">Multilineage potential of adult human mesenchymal stem cells.</a> Science. 284 (5411), 143-147 (1999).</li><li>Shi, Y., Du, L., Lin, L., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-associated+mesenchymal+stem/stromal+cells:+emerging+therapeutic+targets.">Tumour-associated mesenchymal stem/stromal cells: emerging therapeutic targets.</a> Nat Rev Drug Discov. 16 (1), 35-52 (2017).</li><li>Ridge, S. M., Sullivan, F. J., Glynn, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells:+key+players+in+cancer+progression.">Mesenchymal stem cells: key players in cancer progression.</a> Mol Cancer. 16 (1), 31 (2017).</li><li>Luo, J., 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=Infiltrating+bone+marrow+mesenchymal+stem+cells+increase+prostate+cancer+stem+cell+population+and+metastatic+ability+via+secreting+cytokines+to+suppress+androgen+receptor+signaling.">Infiltrating bone marrow mesenchymal stem cells increase prostate cancer stem cell population and metastatic ability via secreting cytokines to suppress androgen receptor signaling.</a> Oncogene. 33 (21), 2768-2778 (2013).</li><li>Huang, W. -. H., Chang, M. -. C., Tsai, K. -. S., Hung, M. -. C., Chen, H. -. L., Hung, S. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+promote+growth+and+angiogenesis+of+tumors+in+mice.">Mesenchymal stem cells promote growth and angiogenesis of tumors in mice.</a> Oncogene. 32 (37), 4343-4354 (2013).</li><li>Patel, S. A., Meyer, J. R., Greco, S. J., Corcoran, K. E., Bryan, M., Rameshwar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+protect+breast+cancer+cells+through+regulatory+T+cells:+role+of+mesenchymal+stem+cell-derived+TGF-beta.">Mesenchymal stem cells protect breast cancer cells through regulatory T cells: role of mesenchymal stem cell-derived TGF-beta.</a> J Immunol. 184 (10), 5885-5894 (2010).</li><li>Becker, A., Thakur, B. K., Weiss, J. M., Kim, H. S., Peinado, H., Lyden, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+in+cancer:+Cell-to-cell+mediators+of+metastasis.">Extracellular vesicles in cancer: Cell-to-cell mediators of metastasis.</a> Cancer Cell. 30 (6), 836-848 (2016).</li><li>Skog, J., 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=Glioblastoma+microvesicles+transport+RNA+and+protein+that+promote+tumor+growth+and+provide+diagnostic+biomarkers.">Glioblastoma microvesicles transport RNA and protein that promote tumor growth and provide diagnostic biomarkers.</a> Nat Cell Biol. 10 (12), 1470-1476 (2008).</li><li>Peinado, H., 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=Melanoma+exosomes+educate+bone+marrow+progenitor+cells+toward+a+pro-metastatic+phenotype+through+MET.">Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.</a> Nat Med. 18 (6), 883-891 (2012).</li><li>Zomer, A., 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=In+vivo+imaging+reveals+extracellular+vesicle-mediated+phenocopying+of+metastatic+behavior.">In vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior.</a> Cell. 161 (5), 1046-1057 (2015).</li><li>Webber, J., Steadman, R., Mason, M. D., Tabi, Z., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+exosomes+trigger+fibroblast+to+myofibroblast+differentiation.">Cancer exosomes trigger fibroblast to myofibroblast differentiation.</a> Cancer Res. 70 (23), 9621-9630 (2010).</li><li>Webber, J. P., 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=Differentiation+of+tumour-promoting+stromal+myofibroblasts+by+cancer+exosomes.">Differentiation of tumour-promoting stromal myofibroblasts by cancer exosomes.</a> Oncogene. 34 (3), 290-302 (2015).</li><li>Zhou, W., 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=Cancer-secreted+miR-105+destroys+vascular+endothelial+barriers+to+promote+metastasis.">Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis.</a> Cancer Cell. 25 (4), 501-515 (2014).</li><li>Whiteside, T., Anastasopoulou, E., Voutsas, I., Papamichail, M., Perez, S., Nunes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+and+tumor-mediated+immune+suppression.">Exosomes and tumor-mediated immune suppression.</a> Expert Rev Mol Diagn. 15 (10), 1293-1310 (2016).</li><li>Verweij, F. J., Van Eijndhoven, M. A. J., Middeldorp, J., Pegtel, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+viral+microRNA+exchange+via+exosomes+in+vitro+and+in+vivo.">Analysis of viral microRNA exchange via exosomes in vitro and in vivo.</a> Methods Mol Biol. 1024, 53-68 (2013).</li><li>Baglio, S. R., 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=Human+bone+marrow-+and+adipose-mesenchymal+stem+cells+secrete+exosomes+enriched+in+distinctive+miRNA+and+tRNA+species.">Human bone marrow- and adipose-mesenchymal stem cells secrete exosomes enriched in distinctive miRNA and tRNA species.</a> Stem Cell Res Ther. 6 (1), 127 (2015).</li><li>Naaijkens, B. A., 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=Human+platelet+lysate+as+a+fetal+bovine+serum+substitute+improves+human+adipose-derived+stromal+cell+culture+for+future+cardiac+repair+applications.">Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications.</a> Cell Tissue Res. 348 (1), 119-130 (2012).</li><li>Grisendi, G., 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=Adipose-derived+mesenchymal+stem+cells+as+stable+source+of+tumor+necrosis+factor-related+apoptosis-inducing+ligand+delivery+for+cancer+therapy.">Adipose-derived mesenchymal stem cells as stable source of tumor necrosis factor-related apoptosis-inducing ligand delivery for cancer therapy.</a> Cancer Res. 70 (9), 3718-3729 (2010).</li><li>Cosette, J., Abdelwahed, R. B., Donnou-Triffault, S., Saut&#232;s-Fridman, C., Flaud, P., Fisson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence-based+tumor+quantification+method+for+monitoring+tumor+progression+and+treatment+effects+in+mouse+lymphoma+models.">Bioluminescence-based tumor quantification method for monitoring tumor progression and treatment effects in mouse lymphoma models.</a> J Vis Exp. (113), (2016).</li><li>Carbone, L., 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=Assessing+cervical+dislocation+as+a+humane+euthanasia+method+in+mice.">Assessing cervical dislocation as a humane euthanasia method in mice.</a> J Am Assoc Lab Anim Sci. 51 (3), 352-356 (2012).</li><li>Costa-Silva, B., 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=Pancreatic+cancer+exosomes+initiate+pre-metastatic+niche+formation+in+the+liver.">Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver.</a> Nat Cell Biol. 17 (6), 816-826 (2015).</li><li>Hoshino, A., 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=Tumour+exosome+integrins+determine+organotropic+metastasis.">Tumour exosome integrins determine organotropic metastasis.</a> Nature. 527 (7578), 329-335 (2015).</li><li>Clayton, A., Mitchell, J. P., Court, J., Mason, M. D., Tabi, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+tumor-derived+exosomes+selectively+impair+lymphocyte+responses+to+interleukin-2.">Human tumor-derived exosomes selectively impair lymphocyte responses to interleukin-2.</a> Cancer Res. 67 (15), 7458-7466 (2007).</li><li>Wieckowski, E. U., Visus, C., Szajnik, M., Szczepanski, M. J., Storkus, W. J., Whiteside, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-derived+microvesicles+promote+regulatory+t+cell+expansion+and+induce+apoptosis+in+tumor-reactive+activated+cd8++T+lymphocytes.">Tumor-derived microvesicles promote regulatory t cell expansion and induce apoptosis in tumor-reactive activated cd8+ T lymphocytes.</a> J Immunol. 183 (6), 3720-3730 (2009).</li><li>Valenti, R., Huber, V., Iero, M., Filipazzi, P., Parmiani, G., Rivoltini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-released+microvesicles+as+vehicles+of+immunosuppression.">Tumor-released microvesicles as vehicles of immunosuppression.</a> Cancer Res. 67 (7), 2912-2915 (2007).</li><li>Costa-Silva, B., 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=Pancreatic+cancer+exosomes+initiate+pre-metastatic+niche+formation+in+the+liver.">Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver.</a> Nat Cell Biol. 17 (6), 816-826 (2015).</li><li>Lin, L. Y., 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=Tumour+cell-derived+exosomes+endow+mesenchymal+stromal+cells+with+tumour-promotion+capabilities.">Tumour cell-derived exosomes endow mesenchymal stromal cells with tumour-promotion capabilities.</a> Oncogene. 35 (46), 6038-6042 (2016).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Equipment</td>
			<td ></td>
			<td ></td>
			<td ></td>
			<td ></td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Ultra Centrifuge</td>
			<td >Beckman</td>
			<td >Optima L-90K</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Rotor SW32Ti</td>
			<td >Beckman</td>
			<td >369650</td>
			<td colspan="4">Referred to in the manuscript as ultra-swinging bucket rotor</td>
			<td></td>
		</tr>
		<tr >
			<td >Transmission electron microscope</td>
			<td >Zeiss</td>
			<td >EM109</td>
			<td>Or similar TEM</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Digital camera</td>
			<td >Nikon</td>
			<td >DMX 1200F</td>
			<td>Or similar camera</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Imaging software TEM&#160;</td>
			<td >Nikon</td>
			<td >ACT-1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Fluorescence microscope</td>
			<td >Zeiss</td>
			<td >Imager.D2</td>
			<td colspan="2">Or similar Fluorescence microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Imaging software FM</td>
			<td>Zeiss</td>
			<td >ZEN Blue</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Incubator</td>
			<td >Nuaire</td>
			<td >4750E</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td >Hettick</td>
			<td >ROTANTA 460R</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >-80 Freezer</td>
			<td >Thermo electro corporation</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >FACS</td>
			<td >BD</td>
			<td >BD FACScalibur</td>
			<td>Or similar flow cytometer</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Drill</td>
			<td >Ferm</td>
			<td >FCT-300</td>
			<td>With 0.8 mm drill</td>
			<td></td>
			<td></td>
			<td></td>
			<td></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>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >IVIS camera</td>
			<td >Xenogen</td>
			<td >Ivis Lumina</td>
			<td colspan="5">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 colspan="2">Xenogen is now part of Perkin Elmer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >10 &#181;L Syringe</td>
			<td >Hamilton</td>
			<td >Neuros Model 1701 RN</td>
			<td></td>
			<td></td>
			<td></td>
			<td></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>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Caliper</td>
			<td >Mitutoyo</td>
			<td >G08004463</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Autoclave</td>
			<td >Astell</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Heat Lamp</td>
			<td >Philips</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Culture media</td>
			<td ></td>
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >Hyclone</td>
			<td >RYG35912</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Platelet Lysate</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >IMDM medium</td>
			<td >Lonza</td>
			<td >BE12-722F</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >alpha-MEM medium</td>
			<td >Lonza</td>
			<td >BE02-002F</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >DMEM medium</td>
			<td >Lonza</td>
			<td>BE12-614F</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >pen/strep/glutamine</td>
			<td >GIBCO</td>
			<td >10378-016</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >heparin</td>
			<td >LEO</td>
			<td >012866-08</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin/EDTA (10x)</td>
			<td >GIBCO</td>
			<td >15400-054</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Cells</td>
			<td ></td>
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >adipose deriverd MSCs</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >GFP-positive MSCs</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >human fibroblasts</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >143B cells</td>
			<td >ATCC</td>
			<td >CRL-8303</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >FLUC-143B cells</td>
			<td >ATCC</td>
			<td >CRL-8303</td>
			<td>Transduced</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Disposables</td>
			<td ></td>
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Culture flasks 175 cm2</td>
			<td >CELLSTAR</td>
			<td >660175</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >50 mL tubes</td>
			<td >Greiner bio-one</td>
			<td >210261</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Freeze tubes</td>
			<td >Thermoscientific</td>
			<td >377224</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Ultra-Clear tubes</td>
			<td >Beckman</td>
			<td >344058</td>
			<td colspan="4">Referred to in the manuscript as ultra-centrifuge tubes</td>
			<td></td>
		</tr>
		<tr >
			<td >0,22 &#181;m filter</td>
			<td >Millex</td>
			<td >SLGV033RS</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >200 mesh Formvar-carbon-coated nickel grids</td>
			<td >EMS (Electron Microscopy Sciences)</td>
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >0.5 mL insulin syringes with 29G Needle</td>
			<td >Terumo</td>
			<td >U-100&#160;</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Petri dish</td>
			<td >Sigma - Aldrich</td>
			<td >P7612</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Filter paper&#160;</td>
			<td >Thermo fisher Scientific</td>
			<td >50363215</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Reagents / kits</td>
			<td ></td>
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >paraformaldehyde</td>
			<td >Alfa Aeser</td>
			<td >43368.9M</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >PBS</td>
			<td >Braun</td>
			<td >220/12257974/110</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >glutaraldehyde</td>
			<td >EMS (Electron Microscopy Sciences)</td>
			<td >16300</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >uranyl oxalate</td>
			<td >EMS (Electron Microscopy Sciences)</td>
			<td >22510</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >urany acetate</td>
			<td >EMS (Electron Microscopy Sciences)</td>
			<td >22400</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >methyl cellulose</td>
			<td >EMS (Electron Microscopy Sciences)</td>
			<td >1560</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >PKH67</td>
			<td >Sigma</td>
			<td >mini67-1kt</td>
			<td colspan="2">Referred to in the manuscript as GFLD</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >BSA</td>
			<td >Sigma</td>
			<td >A8412</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >CBA - human inflammatory cytokine kit</td>
			<td >BD</td>
			<td >551811</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Formaldehyde 37%</td>
			<td >VWR</td>
			<td >104003100</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Carbon Steel surgical blades</td>
			<td >Swann-Morton</td>
			<td >206</td>
			<td colspan="3">Referred to in the manuscript as surgical knife</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >anti-human vimentin antibody</td>
			<td >Santa Cruz</td>
			<td >sc-6260</td>
			<td>Clone V9</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Antibody diluent</td>
			<td >DAKO</td>
			<td >S0809</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >HRP-labeled anti mouse IgG antibody</td>
			<td >Life Technologies</td>
			<td >32230</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >DAB-kit</td>
			<td >DAKO</td>
			<td >K500711</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >hematoxyllin</td>
			<td >Sigma</td>
			<td >GHS232</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >EDTA-buffer</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Citrate buffer</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >rabbit polyclonal anti-GFP antibody</td>
			<td >Abcam</td>
			<td >n.a.</td>
			<td>Ab290</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >DAPI&#160;</td>
			<td >Life Technologies</td>
			<td >D1306</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Paracetamol, 120 mg / 5 ml syrup</td>
			<td >Bayer</td>
			<td >n.a.</td>
			<td colspan="2">Sinaspril, paracetamol solution for kids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Isoflurane 1000 mg/g</td>
			<td >Vumc pharmacy</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >buprenofine hydrochloride, 0.3 mg/ml</td>
			<td >Indivior UK Limited</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >lidocaine-HCL 2%</td>
			<td >Vumc pharmacy</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >70% ethanol</td>
			<td >VWR</td>
			<td >93003.1006</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Tissue glue</td>
			<td >Derma+Flex, formulated medical cyanoacrylate</td>
			<td >Vygon</td>
			<td>LB604060</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Eyedrops: Vidisec Carbogel, 2 mg/ml</td>
			<td >Bausch+Lomb</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >D-luciferin, potassium salt</td>
			<td >Gold Biotechnology</td>
			<td >LUCK-1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Glass slides</td>
			<td >Thermo scientific</td>
			<td >630-0954</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Stainless steel loops&#160;</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Mice experiments</td>
			<td ></td>
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Mice, Hsd:Athymic Nude-Foxn1nu,&#160; female, 6 weeks at arrival, bacterial status conform FELASA</td>
			<td >ENVIGO</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Paper-pulp smart home (cage enrichment)</td>
			<td >Bio Services</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Alpha-dri bedding material</td>
			<td >Shepperd Speciality Papers</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse food: Teklad global 18% protein rodent diet</td>
			<td >ENVIGO</td>
			<td >2918-11416M</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Sutures</td>
			<td >Ethicon</td>
			<td >V926H</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Scissors</td>
			<td >Sigma-Aldrich</td>
			<td >S3146-1EA</td>
			<td>(or similar)</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Tweezers</td>
			<td >Sigma-Aldrich</td>
			<td >F4142-1EA</td>
			<td>(or similar)</td>
			<td></td>
			<td></td>
			<td></td>
			<td></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。
<p class="jove_cont...

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

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

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

开发和维护的临床前患者来源的肿瘤异种移植模型为新的抗癌疗法的调查

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

科研

癌症研究

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

结肠-26癌荷瘤小鼠为癌症恶病质的研究模型

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields (TTFields)

Tumor Treating Fields (TTFields) are an effective treatment modality delivered via the continuous, noninvasive application of low-intensity (1-3 V/cm), alternating electric fields in the frequency range of several hundred kHz. The study of TTFields in tissue culture is carried out using the TTFields in vitro application system, which allows for the application of electric fields of varying frequencies and intensities to ceramic Petri dishes with a high dielectric constant (&#400; &#62; 5,000). Cancerous cell lines plated on coverslips at the bottom of the ceramic Petri dishes are subjected to TTFields delivered in two orthogonal directions at various frequencies to facilitate treatment outcome tests, such as cell counts and clonogenic assays. The results presented in this report demonstrate that the optimal frequency of the TTFields with respect to both cell counts and clonogenic assays is 200 kHz for both ovarian and glioma cells.

Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields TTFields

Tumor Treating Fields (TTFields) are an effective anti-tumor treatment modality delivered via the continuous, noninvasive application of low-intensity, intermediate-frequency, alternating electric fields. TTFields application to cell lines using a TTFields in vitro application system allows for the determination of the optimal frequency that leads to the highest reduction in cell counts.

确定癌细胞使用肿瘤治疗领域的最佳抑制频率（TTFields）

Tumor Treating Fields (TTFields) are an effective treatment modality delivered via the continuous, noninvasive application of low-intensity (1-3 V/cm), ...

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

基于细胞外基质提取物 (ECME) 的三维系统中单核细胞与原代乳癌细胞的通讯分析

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

一种确定环境富集对小鼠结肠肿瘤模型结肠菌群生物多样性影响的方法

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

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 ...

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 ...

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample types have been analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to visualize the distribution of proteins, lipids, and metabolites across the biological sample of interest. We have developed a novel sample preparation method that combines the strengths of the three previous applications to address an underexplored approach for identifying chemical communication in cancer, by seeding mammalian cell cultures into agarose in coculture with healthy tissues followed by desiccation of the sample. Mammalian tissue and cells are cocultured in close proximity allowing chemical communication via diffusion between the tissue and cells. At specific time points, the agarose-based sample is dried in the same manner as microbial colonies prepared for IMS analysis. Our method was developed to model the communication between high grade serous ovarian cancer derived from the fallopian tube as it interacts with the ovary during metastasis. Optimization of the sample preparation resulted in the identification of norepinephrine as a key chemical component in the ovarian microenvironment. This newly developed method can be applied to other biological systems that require an understanding of chemical communication between adjacent cells or tissues.

A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry.

利用成像质谱技术捕获组织与细胞之间的小分子通信

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

研究癌症相关成纤维细胞在肿瘤生长中的作用的小鼠模型

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...