这项工作得到了 Juntendo 大学青年调查员奖 (2013、2014和 2015) 对 Y.O.、联合项目奖 (2013 和 2014) k.m. 的支持, 并资助了教育部、文化部、体育、科学和技术, 日本 (16K15625 张佑启和16K15598 到 M.G.)。我们特别感谢肛肠外科和分子病理学部的所有成员进行有益的讨论和技术支持。我们还感谢博行 Konno 博士 (滨松大学 Schoolof 医学) 和北岛博士 (国际卫生和福利大学) 在对小鼠进行原位植入手术过程中给予慷慨的技术指导。吉孝河马 (千叶癌症中心) 为执行肿瘤 organoid 文化提供技术咨询。

该病人提供书面知情同意, 该项目被 Juntendo 大学医学院研究伦理学委员会批准。老鼠实验也被 Juntendo 医学院动物研究伦理学委员会批准。
1. Immunodeficient 小鼠 CRC PDXs 的建立
在图 1A中概述了建立 CRC PDXs (步骤 1) 的实验程序。
<ol><li>在患者手术切除肿瘤后立即准备主 CRC 组织。</li>
	<li>在50毫升锥形管中, 用30毫升的冰冷无菌磷酸盐缓冲盐水 (PBS) 多次温和搅拌, 将原 CRC 组织洗净, 并将其放在冰上。</li>
	<li>使用无菌镊子将组织放置在10厘米培养皿上, 使用无菌的剪刀尽可能广泛地去除坏死区域, 用无菌刀片将实体肿瘤组织切成小块 (5 x 5 x 5 毫米3 )。 注意: 坏死区通常比周围区域更白、更柔软、更易碎。</li>
	<li>将小块的肿瘤使用无菌镊子在2毫升的冰冷不育 PBS 在6井板上。</li>
	<li>繁殖6周老点头/石-IL2Rγ空 (酒) 高度 immunodeficient 小鼠在无菌和特定病原体的条件下。</li>
	<li>用小动物吸入麻醉装置麻醉小鼠吸入异氟醚, 用70% 乙醇对皮肤进行消毒。确保正常的呼吸速度和深度, 以及没有脚趾捏反射的适当麻醉。在麻醉时, 眼睛的药膏是一种选择, 以防止眼部干燥。</li>
	<li>使用干热灭菌消毒所有外科工具, 然后使用这些工具为所有的程序, 以防止伤口感染的受体小鼠。</li>
	<li>将麻醉鼠标放在实验室工作台的俯卧位置。做一个小切口, 在右侧和左侧面的收件人鼠标, 以产生皮下口袋的组织植入使用无菌剪刀。注意不要刺穿腹膜膜。这个手术不应该流血。</li>
	<li>将上述制备的肿瘤片段轻轻地浸入50µL 的人工细胞外基质中, 然后将其植入右侧和左侧皮下口袋的下部。</li>
	<li>在手术过程中用伤口夹缝合切口。确保植入的肿瘤组织位于皮肤下切口的某个距离。 注: 根据最小化手术方法, 本研究未对术后疼痛和感染进行治疗。</li>
	<li>把鼠标放在温暖的垫子上, 保持胸骨仰卧位置, 直到恢复足够的意识。一旦完全恢复, 并能够坐在所有四条腿, 老鼠可以返回到他们的家庭笼子。为了防止捕食, 不允许在同一个笼子里饲养非操作动物。</li>
	<li>检查缝合渗漏, 确认小鼠第二天在手术治疗组的状态稳定。检查小鼠是否有疾病迹象, 每周使用卡尺测量每只老鼠的肿瘤大小。</li>
</ol>2. 小鼠 CRC PDXs 的解剖
在图 1B中概述了 CRC PDXs (步骤 2) 解剖的实验程序。
<ol><li>允许肿瘤皮下生长到1厘米3的大小, 这通常需要大约1–3月。</li>
	<li>麻醉用小动物吸入麻醉装置和用70% 乙醇消毒皮肤的异氟醚吸入小鼠。</li>
	<li>把鼠标放在实验室长凳上的俯卧位置上。切开皮肤, 暴露肿瘤, 用无菌剪刀仔细地将皮肤与肿瘤分离。切除肿瘤后, 将其放置在10厘米培养皿上, 从肿瘤中清除任何严重坏死的区域, 然后用5毫升的 PBS 冲洗两次。 注意: 坏死区通常比周围区域更白、更柔软、更易碎。</li>
	<li>
将肿瘤放在冰上, 并将其平均分成4部分用于不同的应用, 包括 1) CRC organoid 培养, 2) 移植到次生小鼠, 3) 组织学检查和 4) 肿瘤片段的冷冻和贮存。
	<ol>
<li>对 CRC organoids 的生成, 将固体肿瘤组织放置在含有1毫升不育 PBS 的6厘米培养皿上。将组织放在冰上直到加工 (步骤 3)。</li>
		<li>为了建立人类 crc 的 PDX 模型, 将新的 crc PDXs 成二次小鼠。使用无菌剃刀刀片将肿瘤组织切成小块 (5 x 5 x 5 毫米3英寸), 并将这些组织碎片轻轻地蘸到50µL 的人工细胞外基质中。然后, 如步骤1.8–1.11 中所述, 将这些碎片植入酒鼠标右侧和左侧皮下口袋的下部。</li>
		<li>对于组织学分析, 在室温下将5毫升10% 中性缓冲福尔马林的样品固定在15毫升锥形管中, 2 天。</li>
		<li>为冷冻保存, 将肿瘤组织切成小块 (5 x 5 x 5 毫米3英寸), 并轻轻地将样品浸入1–1.5 毫升 cryovial 管中的500µL 细胞冷冻液中。然后, 把管子转到-80 摄氏度的冰箱里。</li>
	</ol>
</li>
</ol>3. 提取 PDXs 为 CRC Organoid 培养细胞悬浮液的研究
在图 1B中概述了 CRC PDXs (步骤 3) 离解的实验程序。
<ol><li>放置上述 (步骤 2.4.1) 准备的肿瘤片段在一个10厘米培养皿。</li>
	<li>用无菌剃刀刀片将碎片切碎, 并将其转化为15毫升圆锥管, 其中含有8毫升10% 胎小牛血清 (FCS)-Dulbecco 的改良鹰培养基 (DMEM), 80 µL 胶原酶 (见材料表)。</li>
	<li>把管子拧紧, 用 parafilm 包住盖子以防漏水。将切碎的肿瘤碎片与细胞悬浮物分离, 在37摄氏度时轻轻搅动2小时。请注意, 在试管中仍会有许多未消化组织的碎片。</li>
	<li>离心管在 300 x g 5 分钟在4°c 和去除上清。用10毫升的 10% DMEM 来解决细胞颗粒悬浮。</li>
	<li>为了消除未消化的组织碎片, 过滤细胞悬浮两次使用40µm 细胞过滤器设置在50毫升圆锥管。然后, 离心机的流动通过在 300 x g 5 分钟在4摄氏度。去掉上清, 把球团放在冰上。</li>
</ol>4. 人工细胞外基质培养的 CRC Organoids 的生成
在图 1B中概述了结肠 PDXs (步骤 4) 的 CRC organoid 文化的实验程序。
<ol><li>建立适合于 PDX 衍生 CRC organoids 的培养基 (见材料表, "CRC organoid 培养基"), 使用先前描述的人类结肠 organoids11的培养基。</li>
	<li>应用150µL 的人工细胞外基质 (见材料表), 每井在12井板上的冰, 然后孵化30-60 分钟在37°c, 5% CO2孵化器固化凝胶。</li>
	<li>用 CRC organoid 培养基 (从步骤 4.1) 到 5% FCS, 从步骤3.5 中暂停细胞颗粒, 以达到 3 x 105细胞/毫升的调节。然后, 种子1毫升的培养基上的人工细胞外基质涂层板制备的步骤4.2。和孵化过夜在37摄氏度以下 5% CO2。用 hemocytometer 计数细胞悬浮液中的肿瘤细胞, 包括单细胞和多细胞簇 (一组黏附细胞)。 注: 5% 的 FCS 用于抑制胶原酶的残留酶活性的研究。</li>
	<li>仔细收集培养基, 包括从人工细胞外基质涂层板分离出来的漂浮细胞, 在第二天进入1.5 毫升离心管, 并将其离心在 1400 x g 5 分钟。然后, 去除上清液, 在冰上70µL 人工细胞外基质中重新悬浮颗粒。</li>
	<li>为了提高 CRC 细胞的生存能力, 将含有肿瘤细胞的人工细胞外基质覆盖在人工细胞外基质涂层板上的肿瘤 organoid 细胞上, 在37摄氏度、5% CO2孵化器中孵育30分钟以固化人工细胞外基质涂层。然后, 孵化它与1毫升的 CRC organoid 细胞培养培养基与 1% FCS 在37°c 下 5% CO2.
</li>
	<li>每隔一天更改一次培养基。当 CRC organoids 填充介质时, 可能会有必要改变日常的媒介。</li>
</ol>5. GFP 慢病毒载体粒子的产生和富集
在图 1C中概述了 GFP 慢病毒载体粒子的生成和富集的实验程序 (步骤 5)。
<ol><li>文化 HEK293T 细胞与罗斯威尔公园纪念学院 (RPMI) 1640 中等包含10% 个 FCS 并且准备六 10 cm 菜 (2 x 106个细胞每盘) 为以下转染做法。</li>
	<li>每道菜的转染, 准备500µL 的混合物, 包括20µL 的转染试剂在 DMEM 与 PRRL GFP 载体 (5 µg)12和两个慢病毒载体包装质粒, 如 pCMV-VSV G (1 µg) 和 pCMV-dR8.2 dvpr (5 µg), 在无菌1.5 毫升微细.将混合物在室温下保持20分钟, 然后将其覆盖在10厘米的菜肴上, 然后孵化18小时。</li>
	<li>去除培养基, 加入5毫升新鲜的 10% RPMI 1640 培养基到每道菜, 并离开站立24小时。</li>
	<li>收集每道菜的条件介质成一个50毫升离心管, 并存储30毫升的培养基在4°c 过夜。添加5毫升新鲜 RPMI 1640 到每道菜和离开站立24小时。</li>
	<li>收集每道菜的条件介质成一个50毫升离心管, 并保持, 在总的30毫升的介质在冰上。然后, 丢弃这些单元格。</li>
	<li>过滤60毫升的介质 (从步骤 5.4–5.5) 通过一个0.45 µm 过滤器去除细胞, 并将此数量分成十二5毫升聚丙烯离心管离心。</li>
	<li>要集中病毒, 离心机使用摆动斗转子 (见材料表) 在 85327 x g 1.5 小时4摄氏度。</li>
	<li>立即移除介质。要解决浓缩病毒颗粒, 在十二管中放置250µL 的营养火腿的混合物 F-12 (F12)/DMEM 培养基, 并保持在4°c 过夜。请注意, 病毒颗粒通常是不可见的。</li>
	<li>轻轻地吸管培养基收集3毫升的浓缩 5x gfp 慢病毒北疆股票, 总共, 大概包括 GFP 慢病毒载体粒子与 108传感单位每毫升 (涂/毫升)。然后, 存储十二1.5 毫升管内 (包括每管250µL) 在不育条件下, 在-80 摄氏度, 长达一年。准备许多小整除数的原始溶液, 以避免多次冻融循环。</li>
</ol>6. 用人造细胞外基质培养的 GFP 慢病毒载体颗粒对 CRC Organoid 细胞进行标记
图 1C概述了用 GFP 慢病毒北疆 (步骤 6) 标记 CRC organoid 细胞的实验程序。
<ol><li>允许步骤4.6 中准备的 CRC organoids 在不干扰7–10天的情况下生长, 并使用无菌的细胞刮刀机械收割它们, 然后将它们转化为1.5 毫升的微细。 注: CRC organoids 在培养中的生长取决于患者原发肿瘤的性质。避免 CRC organoids 的过度生长, 维持它们在60–70% 汇合之前, 转移到一个新的人工细胞外基质涂层12井板1:2 分比例。</li>
	<li>离心微细3分钟, 在 1400 x g, 去除培养基, 并通过温和攻丝来解决500µL 中的细胞颗粒。</li>
	<li>离心机的微细为3分钟, 在 1400 x g和消除 PBS。</li>
	<li>为了分离黏附的 CRC organoids, 添加500µL 的细胞脱离溶液的蛋白水解和 collagenolytic 酶的细胞颗粒在管和混合它通过温和的攻丝。然后, 让管子在室温下安定10分钟。</li>
	<li>在1.5 毫升微细中, 轻轻地将细胞悬浮液吸进500µL 1% DMEM, 以大致离解细胞。通过苛刻的吹打从 CRC organoids 中生成单细胞, 显著降低细胞活力。因此, 在1.5 毫升的微细中, 很有必要在细胞悬浮中通过非常温和的吹打来保持细胞的质量。</li>
	<li>离心细胞悬浮液3分钟在 1400 x g和去除上清。</li>
	<li>用100µL 5x GFP 慢病毒北疆的混合物 (> 108涂/毫升) 和400µL 的 CRC organoid 培养基在1.5 毫升微细中, 用柔和的攻丝来调节细胞颗粒, 每5个 10 x 5500个分离肿瘤细胞的浓度µL.使用 hemocytometer 计算肿瘤细胞的数量, 包括细胞悬浮中的单个细胞和细胞组, 如上文所述 (步骤 4.3)。</li>
	<li>如步骤4.2 所述, 准备一个人工胞外基质涂层12井板。然后, 放置500µL 的细胞悬浮在步骤6.7 准备在板材和留给它 18 h 在37°c 在 5% CO2之下。</li>
	<li>收集介质与漂浮细胞分离的人工细胞外基质涂层板成1.5 毫升离心管。将其离心在 1400 x g上, 然后取出上清, 再在70µL 的人工细胞外基质上将颗粒重新悬浮在冰上。</li>
	<li>为了提高 CRC 细胞的生存能力, 将肿瘤细胞内含有的人工细胞外基质覆盖到人工细胞外基质涂层 12-organoids 的肿瘤上, 如步骤4.5 所述。接下来, 在37摄氏度、5% co2孵化器中孵化出30分钟的板材, 以固化人工胞外基质涂层, 然后用 CRC organoid 培养基的1毫升将其培养为1% 摄氏度以下 37 CO5%。</li>
	<li>观察细胞在3天后, 在荧光显微镜下感染, 以确认近 100% GFP 阳性, 由于使用高滴度的慢病毒载体粒子 (见图 2A)。</li>
	<li>培养 CRC organoids 7–10天的时间, 以扩大细胞生长之前注射入受体小鼠。</li>
</ol>7. 用 GFP 标记的 CRC Organoids 在受体小鼠中产生转移瘤
图 1C概述了使用 GFP 标记的 CRC organoids (步骤 7) 生成转移瘤的实验程序。
<ol><li>要将 GFP 标记的 CRC organoids 与细胞悬浮液分离, 请使用无菌细胞刮刀机械收割, 并将其转化为1.5 毫升微细, 如上文所述 (步骤 6.1)。</li>
	<li>离心微细为3分钟, 在 1400 x g, 去除培养基, 并通过温和攻丝, 解决500µL 中的细胞颗粒。</li>
	<li>离心机的微细为3分钟, 在 1400 x g和消除 PBS。</li>
	<li>覆盖500µL 的细胞脱离溶液到细胞颗粒在管和混合它通过温和的攻丝。然后, 保持在室温下10分钟的混合物酶离解 organoids, 如上文所述 (步骤 6.4)。</li>
	<li>在1.5 毫升微细中, 轻轻地将细胞悬浮液吸进500µL 1% DMEM, 以大致离解细胞, 如上文所述 (步骤 6.5)。通过苛刻的吹打从 CRC organoids 中生成单细胞, 显著降低细胞活力。因此, 在1.5 毫升的微细中, 通过非常温和的吹打, 使细胞在细胞悬浮中能被肉眼检测到。</li>
	<li>离心细胞悬浮液3分钟在 1400 x g 和去除上清。</li>
	<li>
建立 CRC PDXs 自发性转移小鼠模型:
	<ol>
<li>制备细胞悬浮液, 包括 5 x 105细胞在 PBS 50 µL 与50% 人工细胞外基质每只老鼠。使用前要保持冰层的悬浮。如步骤4.3 所示, 使用 hemocytometer 计数癌细胞。</li>
		<li>麻醉用小动物吸入麻醉装置吸入异氟醚, 并用70% 乙醇对皮肤进行消毒, 如步骤1.6 所示。</li>
		<li>将酒的老鼠放在实验室长凳上的仰卧位置。用无菌镊子抓住直肠黏膜, 轻轻将其拉出肛门。然后, 立即注射用注射器 (针大小:22 克) 将步骤7.7.1 中的细胞悬浮液注入小鼠直肠黏膜下层。通常, 每组第4-6 小鼠用于评估结果。</li>
	</ol>
</li>
	<li>
建立 CRC PDXs 的实验性转移模型:
	<ol>
<li>准备一个 CRC organoid 细胞悬浮, 包括 4 x 104细胞在的 PBS 每只老鼠。使用前要保持冰层的悬浮。使用 hemocytometer 计数癌细胞 (步骤 4.3)。</li>
		<li>麻醉用小动物吸入麻醉装置吸入异氟醚, 并用70% 乙醇对皮肤进行消毒, 如步骤1.6 所示。</li>
		<li>将麻醉酒老鼠放在实验室长凳上的俯卧位置。在左侧面的下部做一个小切口, 然后切开腹膜, 用无菌剪刀和镊子小心地暴露脾脏。用无菌镊子抓住脾脏上的脂肪组织, 然后缓慢注射50µL 细胞悬浮液 (准备如上, 步骤 8.1) 进入脾脏。确保细胞悬浮液适当注射, 无外泄, 脾。通常, 每组4只老鼠用于评估结果。</li>
	</ol>
</li>
	<li>在手术后将所有老鼠放在温暖的垫子上, 并保持胸骨卧位, 直到恢复足够的意识, 如步骤1.11 所述。一旦老鼠完全恢复, 把它们送回他们的笼子里。为了防止捕食, 不允许在同一个笼子里饲养非操作动物。</li>
	<li>检查缝合漏, 确认小鼠在手术治疗组第二天还活着。检查老鼠的病情体征, 并测量肿瘤大小, 使用卡尺, 在所有小鼠每周一次。</li>
	<li>观察小鼠在注射后发现自发性转移 (2.5 个月) 和实验性转移 (1 月)。</li>
	<li>通过麻醉和颈椎脱位在指定的日子里牺牲老鼠。解剖原发肿瘤和各种组织, 包括肝脏和肺部。将解剖过的肿瘤和器官放置在10厘米的培养皿上5毫升的冰冷 PBS 中, 并将其储存在冰上。</li>
	<li>为了检测 GFP 阳性的 CRC organoid 细胞, 在立体荧光显微镜下观察解剖组织。利用 CCD 相机获取图像, 并使用标准图像处理软件进行准备。</li>
</ol>

<ol>
	<li>Siegel, R., Desantis, C., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorectal+cancer+statistics,+2014.">Colorectal cancer statistics, 2014.</a> CA Cancer J Clin. 64 (2), 104-117 (2014).</li><li>Hidalgo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+xenograft+models:+an+emerging+platform+for+translational+cancer+research.">Patient-derived xenograft models: an emerging platform for translational cancer research.</a> Cancer Discov. 4 (9), 998-1013 (2014).</li><li>Aparicio, S., Hidalgo, M., Kung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+the+utility+of+patient-derived+xenograft+mouse+models.">Examining the utility of patient-derived xenograft mouse models.</a> Nat Rev Cancer. 15 (5), 311-316 (2015).</li><li>Puig, I., 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=A+personalized+preclinical+model+to+evaluate+the+metastatic+potential+of+patient-derived+colon+cancer+initiating+cells.">A personalized preclinical model to evaluate the metastatic potential of patient-derived colon cancer initiating cells.</a> Clin Cancer Res. 19 (24), 6787-6801 (2013).</li><li>van de Wetering, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+derivation+of+a+living+organoid+biobank+of+colorectal+cancer+patients.">Prospective derivation of a living organoid biobank of colorectal cancer patients.</a> Cell. 161 (4), 933-945 (2015).</li><li>Fujii, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Colorectal+tumor+organoid+library+demonstrates+progressive+loss+of+niche+factor+requirements+during+tumorigenesis.">A Colorectal tumor organoid library demonstrates progressive loss of niche factor requirements during tumorigenesis.</a> Cell Stem Cell. 18 (6), 827-838 (2016).</li><li>Fumagalli, 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=Genetic+dissection+of+colorectal+cancer+progression+by+orthotopic+transplantation+of+engineered+cancer+organoids.">Genetic dissection of colorectal cancer progression by orthotopic transplantation of engineered cancer organoids.</a> Proc Natl Acad Sci U S A. 114 (12), E2357-E2364 (2017).</li><li>O'Rourke, K. 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=Transplantation+of+engineered+organoids+enables+rapid+generation+of+metastatic+mouse+models+of+colorectal+cancer.">Transplantation of engineered organoids enables rapid generation of metastatic mouse models of colorectal cancer.</a> Nat Biotechnol. 35 (6), 577-582 (2017).</li><li>Roper, 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=In+vivo+genome+editing+and+organoid+transplantation+models+of+colorectal+cancer+and+metastasis.">In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis.</a> Nat Biotechnol. 35 (6), 569-576 (2017).</li><li>Kuo, T. 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=Liver+colonization+competence+governs+colon+cancer+metastasis.">Liver colonization competence governs colon cancer metastasis.</a> Proc Natl Acad Sci U S A. 92 (26), 12085-12089 (1995).</li><li>Onuma, K., 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=Genetic+reconstitution+of+tumorigenesis+in+primary+intestinal+cells.">Genetic reconstitution of tumorigenesis in primary intestinal cells.</a> Proc Natl Acad Sci U S A. 110 (27), 11127-11132 (2013).</li><li>Onder, T. 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=Loss+of+E-cadherin+promotes+metastasis+via+multiple+downstream+transcriptional+pathways.">Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways.</a> Cancer Res. 68 (10), 3645-3654 (2008).</li><li>Cespedes, M. V., 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=Orthotopic+microinjection+of+human+colon+cancer+cells+in+nude+mice+induces+tumor+foci+in+all+clinically+relevant+metastatic+sites.">Orthotopic microinjection of human colon cancer cells in nude mice induces tumor foci in all clinically relevant metastatic sites.</a> Am J Pathol. 170 (3), 1077-1085 (2007).</li><li>Fujii, 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=Characterization+of+EBV-related+lymphoproliferative+lesions+arising+in+donor+lymphocytes+of+transplanted+human+tumor+tissues+in+the+NOG+mouse.">Characterization of EBV-related lymphoproliferative lesions arising in donor lymphocytes of transplanted human tumor tissues in the NOG mouse.</a> Exp Anim. 63 (3), 289-296 (2014).</li></ol>

作者没有潜在的利益冲突披露。

虽然 CRC PDX 模型已被广泛应用于研究原发性肿瘤的生长, 但该模型是否也适用于肿瘤转移的调查尚未充分阐明。自发性转移也几乎无法检测到的肝脏和肺部的各种报告结肠 PDX 模型4,10。为了检测高灵敏度的淋巴结, 我们开发了一种协议, 传感 GFP 慢病毒载体粒子进入 PDX 衍生 CRC organoids 之前, 他们的原位和静脉注射的受体小鼠。值得注意的是, 我们能够证明...

大肠癌 (CRC) 是1全球癌症死亡的第二大原因。对晚期疾病患者常规疗法的反应不足, 表明试图从根本上治疗 CRCs 是无效的。为了制定更有效的治疗方法, 建立了模拟 CRCs 特征的各种癌前小鼠模型。各种 CRC 细胞系因其方便易操作而被广泛用于产生肿瘤移植。然而, 癌症细胞系的长期培养往往会导致在特定的文化条件下, 在特殊的细胞群中有相当大的增殖, 从而导致临床前药物不可靠的结果和关键的限制。发展。
未经体外培养, 患者源性肿瘤移植 (PDXs) 也被植入到人类 CRC 组织的动物模型中, 经手术解剖的患者2,3,4。PDXs 被广泛认为是综述的主要病理学特征和基因改变最初存在于肿瘤的病人, 从他们的来源。此外, 在3D 条件下建立了由肿瘤细胞簇组成的患者源性肿瘤 organoids, 并对原发肿瘤5、6的生物学特性进行了严密的模拟。这些肿瘤 organoids 也被应用于高通量的药物筛选, 从而允许设计5的个性化疗法。然而, 明显的异种 CRC 种群被认为存在于肿瘤肿块中。特殊的 CRC 种群在体内和体外一系列的 PDX 和肿瘤 organoids 中有选择性地增殖和扩张。这也可能使受影响的 CRCs 的整体基因表达谱和免疫/遗传状态发生变化, 从而导致与父母 CRC 的最小相似性。
患者衍生的 CRC organoids 和从癌人结肠工程中提取的致癌突变的组合, 也被用来研究肿瘤侵袭转移的人肿瘤细胞的特征。6,7,8,9。然而, 由于患者源性 CRC 原位植入 immunodeficient 小鼠的自发性转移发生率极低, 因此难以研究侵袭转移级联的多步法过程, 包括局部入侵, intravasation, 运输在血液, 渗出和殖民化遥远的器官4,10。由患者衍生的 CRC organoids 形成的≤2毫米肿瘤细胞沉积所代表的微转移常常被忽略在实验性小鼠模型中受影响的远端器官切片的组织病理学分析中。自发淋巴结的可视化在体内也很少, 因为在注射到受体小鼠之前, 很难有效地将荧光标记引入肿瘤 organoid 细胞。在这项研究中, 我们开发了一个协议, 以有效地传感器 GFP 慢病毒北疆进入 PDX 衍生的 CRC organoid 细胞在3D 的文化注入受体小鼠之前, 并允许高度敏感的检测, 使用立体荧光显微镜, 其不同器官的定植形成淋巴结。

<table><tbody><tr><td>NOD/Shi-scid IL2R&amp;gamma; null (NOG) mice</td><td>The Central Institute for Experimental Animals,Kanagawa, Japan</td><td></td><td>Breed 6-week-old male mice under germ-free and specific pathogen-free conditions</td></tr><tr><td>wound clips&lt;br/&gt; 2&amp;times;10mm</td><td>Natsume manufacturing, Japan</td><td>#C-21-S</td><td>Autoclave before use</td></tr><tr><td>Hamilton syringe&lt;br/&gt; needle size:22 gauge</td><td>Tokyo Science, Japan</td><td></td><td>Disinfect with 70% alcohol and sterile PBS.</td></tr><tr><td>6-well plate</td><td>BMBio</td><td>#92006</td><td></td></tr><tr><td>12-well plate</td><td>BMBio</td><td>#92412</td><td></td></tr><tr><td>15ml conical tube</td><td>Sumitono Bakelite</td><td>MS-57150</td><td></td></tr><tr><td>50ml conical tube</td><td>Sumitomo Bakelite</td><td>MS-57500</td><td></td></tr><tr><td>microtube</td><td>Eppendorf</td><td>#0030120086</td><td>Autoclave before use</td></tr><tr><td>Hemocytometer</td><td>Erma</td><td>#03-202-1</td><td></td></tr><tr><td>40&amp;mu;m cell strainer</td><td>Corning</td><td>#352340</td><td></td></tr><tr><td>Matrigel basement membrane matrix</td><td>Corning</td><td>#354234</td><td>Store aliqupts at -20&amp;deg;C.&lt;br/&gt; Place on ice until use</td></tr><tr><td>Collagenase type 1</td><td>Sigma</td><td>#C1030</td><td>150 mg/ml collagenase type1 in 1&amp;times;PBS. Store aliqupts at -20&amp;deg;C for up to 1 year</td></tr><tr><td>Accutase</td><td>Innovate Cell Technologies</td><td>#5V2623A</td><td>Store at 4&amp;deg;C.</td></tr><tr><td>DMEM/F-12 with GlutaMAX&amp;trade;</td><td>Gibco</td><td>#10565018</td><td>Store at 4&amp;deg;C. Warm at 37&amp;deg;C before use</td></tr><tr><td>Cell banker 1plus</td><td>ZENOAQ</td><td>#628</td><td>Store at 4&amp;deg;C. Use within 1 month</td></tr><tr><td>Penicillin</td><td>Gibco</td><td>#15140122</td><td>Store at 4&amp;deg;C. Use within 1 month</td></tr><tr><td>Streptomycin</td><td>Gibco</td><td>#15140122</td><td>Store at 4&amp;deg;C. Use within 1 month</td></tr><tr><td>hEGF</td><td>PEPROTECH</td><td>#AF-100-15</td><td>Store at -20&amp;deg;C. Add to medium on same day as use</td></tr><tr><td>Y27632, a ROCK inhibitor</td><td>Wako</td><td>#253-00591</td><td>Store at -20&amp;deg;C. Add to medium on&lt;br/&gt; same day as use</td></tr><tr><td>Culture medium</td><td>Gibco</td><td></td><td>DMEM/F-12 with GlutaMAX&amp;trade; supplement supplemented with 5% FBS, 100 U/ml penicillin and 100 &amp;micro;g/ml streptomycin.&lt;br/&gt; Store at 4&amp;deg;C. Use within 1 month.</td></tr><tr><td>CRC organoid culture medium&lt;br/&gt; with 1% or 5% FCS</td><td></td><td></td><td>DMEM/F-12 with GlutaMAX&amp;trade; supplement (Gibco #10565018) supplemented with 1% or 5% FCS, 100 U/ml penicillin, 100 &amp;micro;g/ml streptomycin, 2 ng/ml hEGF and 10 &amp;micro;M Y27632, a ROCK inhibitor.&lt;br/&gt; Store at 4&amp;deg;C. Use within 1 month.</td></tr><tr><td>the FuGENE 6 transfection regent</td><td>Roche</td><td>11814 443001</td><td></td></tr><tr><td>Minisart 0.45 &amp;micro;m filter</td><td>Sartorius stedim</td><td>17598-K</td><td></td></tr><tr><td>5 ml polypropylene centrifuge tubes</td><td>Beckman Coulter</td><td>326819</td><td></td></tr><tr><td>PRRL-GFP vector</td><td>Gift from Dr. Robert A. Weinberg</td><td></td><td></td></tr><tr><td>pCMV-VSV-G</td><td>Gift from Dr. Robert A. Weinberg</td><td></td><td></td></tr><tr><td>pCMV-dR8.2 dvpr</td><td>Gift from Dr. Robert A. Weinberg</td><td></td><td></td></tr><tr><td>the SW55Ti swinging bucket rotor</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>a Zeiss Axioplan 2 stereo-fluorescence microscope</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

high-sensitivity detection of micrometastases generated by gfp lentivirus-transduced organoids cultured from a patient-derived colon tumor

尽管目前人类大肠癌 (crc) 治疗的进展, 但很少有根治性疗法对晚期的 CRC 有效。为克服这一临床挑战, 采用长期建立的人肿瘤细胞系和许多转基因小鼠模型, 以肿瘤为基础的模型, 开发了移植瘤小鼠模型。它们部分模仿人类肿瘤的特点, 但往往无法概括人类恶性肿瘤的关键方面, 包括侵袭和转移。因此, 人们长期以来一直在等待替代模式, 更好地代表人类 CRC 的恶性进展。
我们在这里显示由病人衍生的肿瘤移植 (PDXs) 通过皮下植入的小 CRC 片段手术解剖的患者。结肠 PDXs 发育, 病理类似于患者的 CRC。然而, 在 PDX 模型中, 在受影响的远端器官的常规剖面中, 很少有自发淋巴结可检测到。为了便于检测转移到远处的器官, 我们从培养的结肠 PDXs 中提取出肿瘤 organoid 细胞, 并在注射至高度 immunodeficient 点头/石-免疫慢病毒北疆前感染了 GFP(酒) 老鼠。原位注射 PDX 衍生的 CRC organoid 细胞持续形成原发肿瘤阳性的 GFP 在接受小鼠。此外, 在这些小鼠的肺部, 通过荧光显微术, 自发地开发出表达 GFP 的淋巴结微转移菌落。此外, 脾注射液的 CRC organoids 经常产生肝定植。结合起来, 这些发现表明 GFP 标记的 PDX 衍生 CRC organoid 细胞在一个多步骤的过程中被视觉检测, 称为侵袭转移级联。所述的协议包括建立 PDXs 的人 crc 和3D 培养的相应的 CRC organoid 细胞转基因由 GFP 慢病毒载体粒子。

经诊断为中度分化的原发性大肠腺癌经手术切除, 76 岁女性, TNM 分级, 术后化疗后。主要的 CRC 细胞免疫组化染色阳性的癌胚抗原, Ki-67, pan 角蛋白和 e-钙黏素。切除的肿瘤的片断也被皮下植入酒小鼠, 以生成结肠 PDX 模型。然后从这些小鼠皮下发育的结肠 PDX 提取 CRC organoid 细胞进行组织培养。在人工细胞外基质的一系列通道中, CRC organoids 能够持续形成多细胞簇。由于肿瘤 or...

Watch this Scientific Journal Video about High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor at JoVE.com

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

为了允许高度敏感的检测传播的人大肠癌 (CRC) 细胞的殖民组织, 我们在此显示了一个协议, 以有效地转导绿色荧光蛋白 (GFP) 慢病毒载体粒子到 PDX 衍生 CRC organoid 细胞在注射到受体小鼠之前, 用立体荧光显微镜观察。

淋巴结慢病毒北疆-转基因 Organoids 培养的大鼠结肠肿瘤的高灵敏度检测

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

high-sensitivity-detection-micrometastases-generated-gfp-lentivirus

Research

JoVE Journal

Cancer Research

9.9K 次观看.  Juntendo University Faculty of Medicine. 为了允许高度敏感的检测传播的人大肠癌 (CRC) 细胞的殖民组织, 我们在此显示了一个协议, 以有效地转导绿色荧光蛋白 (GFP) 慢病毒载体粒子到 PDX 衍生 CRC organoid 细胞在注射到受体小鼠之前, 用立体荧光显微镜观察。

视频: High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

可追踪记者乳腺癌患者来源的移植瘤贴标肿瘤生长和转移的研究

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

科研

癌症研究

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

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

研究使用正交乳腺癌模型的三阴性乳腺癌

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Medium-Throughput Drug- and Radiotherapy Screening Assay using Patient-Derived Organoids

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a near-native state. When derived from resected or biopsied tumor tissue, organoids closely recapitulate in vivo tumor morphology and can be used to study therapy response in vitro. Biobanks of tumor organoids reflect the vast variety of clinical tumors and patients and therefore hold great promise for preclinical and clinical applications. This paper presents a method for medium-throughput drug screening using head and neck squamous cell carcinoma and colorectal adenocarcinoma organoids. This approach can easily be adopted for use with any tissue-derived organoid model, both normal and diseased. Methods are described for in vitro exposure of organoids to chemo- and radiotherapy (either as single-treatment modality or in combination). Cell survival after 5 days of drug exposure is assessed by measuring adenosine triphosphate (ATP) levels. Drug sensitivity is measured by the half-maximal inhibitory concentration (IC50), area under the curve (AUC), and growth rate (GR) metrics. These parameters can provide insight into whether an organoid culture is deemed sensitive or resistant to a particular treatment.

We describe detailed protocols to use patient-derived organoids for medium-throughput therapy sensitivity screenings. Therapies tested include chemotherapy, radiotherapy, and chemo-radiotherapy. Adenosine triphosphate levels are used as a functional readout.

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

使用宽场活细胞成像进行批量和单类器官分析的多参数肿瘤类器官药物筛选

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

用于患者来源的肿瘤类器官药物评估的自动动力学成像

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

作者聚焦:为基于类器官的药物反应分析开发多重动力学分析

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

多重活细胞成像在患者来源的癌症类器官模型中用于药物反应

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...

Establishing Experimental Metastases Mouse Model: Implanting GFP Expressing CRC Organoid Cells in PDX Model to Detect Micrometastases

Source: Okazawa, Y. et al.&#160; <a href="https://www.jove.com/v/57374"> High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor.</a> J. Vis. Exp. (2018).
This video describes the protocol for generating metastases by the PDX derived CRC organoids labeled with GFP lentivirus into the spleen of an immunodeficient mouse. Cancer micrometastases colonizing other organs can be detected using a fluorescence microscope to assess the GFP expression.

淋巴结慢病毒北疆-转基因 Organoids 培养的大鼠结肠肿瘤的高灵敏度检测

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此视频中的章节

可追踪记者乳腺癌患者来源的移植瘤贴标肿瘤生长和转移的研究

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

研究使用正交乳腺癌模型的三阴性乳腺癌

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

Medium-Throughput Drug- and Radiotherapy Screening Assay using Patient-Derived Organoids

使用宽场活细胞成像进行批量和单类器官分析的多参数肿瘤类器官药物筛选

多重活细胞成像在患者来源的癌症类器官模型中用于药物反应

多重活细胞成像在患者来源的癌症类器官模型中用于药物反应

多重活细胞成像在患者来源的癌症类器官模型中用于药物反应

建立实验转移小鼠模型:在 PDX 模型中植入表达 GFP 的 CRC 类器官细胞以检测微转移