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, 我们能够证明这种方法允许对 CRC organoid 衍生淋巴结进行高度灵敏的检测, 并在远端器官中自发地和实验性地形成。
该协议中的关键步骤包括在传代系列中轻轻吹打 CRC organoids, 在不诱导人工细胞外基质的脱落凋亡的情况下保持完整的球体形成。离心浓缩的高滴度慢病毒北疆颗粒的制备对实现 CRC organoids 的近 100% GFP 阳性也很重要.此外, 建议将 GFP 标记的 CRC organoids 在10–14天内注入受体小鼠。感染后的 CRC organoids 扩张期过长, 可能有利于选择弱 GFP 表达 organoids。
将 CRC organoids 注入酒小鼠直肠黏膜下层, 并将其转移到肺部, 而不是进入肝脏, 大概是通过下腔静脉。为了自发产生肝转移, 需要将 CRC organoids 注射入结肠剖腹手术13。
请注意, B 细胞淋巴瘤的发病率在酒 immunodeficient 小鼠中有时会增加, 其中包括淋巴细胞, 感染了 eb 病毒-巴尔 oncovirus, 从人类患者14。因此, 有理由建议, 新出现的肿瘤是否来源于植入的人类 crc, 是通过使用人类 crc 细胞标记 (如癌胚抗原和角蛋白 20) 进行免疫组化来确定的。
当我们调查从只有一个病人的 CRC organoids, 更多的样本来自更多的病人将需要检查, 以推广我们的发现在未来。如果植入 PDX 的 CRC organoids 在原位部位的生长极小, 很少在遥远的部位形成转移, 则鼓励研究者考虑从不同的患者衍生的 PDXs 中提取 crc organoids。
用这种方法对 GFP 标记的微转移进行高度灵敏的检测, 鼓励我们不仅进一步研究几个未解决的生物学问题, 关于微转移向 macrometastasis 的转移, 基质的形成以 PDX 动物为临床前模型, 对转移性殖民化和耐药性习得的生态位进行评估, 并对新型抗癌药物的疗效进行评价。
以 GFP 为基础的 organoids 从原生和远点提取的活 CRC 的分类也有可能允许单细胞检查的基因表达, 以及表观和 metabolomic 剖面, 肿瘤。这些发现很可能会导致阐明 organoids 转移性传播的分子机制。

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&#947; 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 
			2&#215;10mm</td>
			<td>Natsume manufacturing, Japan</td>
			<td>#C-21-S</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>Hamilton syringe 
			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&#956;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&#176;C. 
			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&#215;PBS. Store aliqupts at -20&#176;C for up to 1 year</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovate Cell Technologies</td>
			<td>#5V2623A</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr>
			<td>DMEM/F-12 with GlutaMAX&#8482;</td>
			<td>Gibco</td>
			<td>#10565018</td>
			<td>Store at 4&#176;C. Warm at 37&#176;C before use</td>
		</tr>
		<tr>
			<td>Cell banker 1plus</td>
			<td>ZENOAQ</td>
			<td>#628</td>
			<td>Store at 4&#176;C. Use within 1 month</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Gibco</td>
			<td>#15140122</td>
			<td>Store at 4&#176;C. Use within 1 month</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Gibco</td>
			<td>#15140122</td>
			<td>Store at 4&#176;C. Use within 1 month</td>
		</tr>
		<tr>
			<td>hEGF</td>
			<td>PEPROTECH</td>
			<td>#AF-100-15</td>
			<td>Store at -20&#176;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&#176;C. Add to medium on 
			same day as use</td>
		</tr>
		<tr>
			<td>Culture medium</td>
			<td>Gibco</td>
			<td></td>
			<td>DMEM/F-12 with GlutaMAX&#8482; supplement supplemented with 5% FBS, 100 U/ml penicillin and 100 &#181;g/ml streptomycin. 
			Store at 4&#176;C. Use within 1 month.</td>
		</tr>
		<tr>
			<td>CRC organoid culture medium 
			with 1% or 5% FCS</td>
			<td></td>
			<td></td>
			<td>DMEM/F-12 with GlutaMAX&#8482; supplement (Gibco #10565018) supplemented with 1% or 5% FCS, 100 U/ml penicillin, 100 &#181;g/ml streptomycin, 2 ng/ml hEGF and 10 &#181;M Y27632, a ROCK inhibitor. 
			Store at 4&#176;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 &#181;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 organoids 组织学上和遗传学上反映了5,6患者的原发性肿瘤的性质, 我们试图开发一个小鼠模型, 允许体内检测 CRC organoid 细胞高灵敏度在转移传播期间。为了实现这一目标, CRC organoids 感染了 gfp 慢病毒北疆, 从而导致几乎所有 organoids 显示非常明亮的 GFP (图 2A)。然后在荧光显微镜下检测远处器官中 GFP 阳性细胞之前, 将这些 organoids 注射原位和 intrasplenically 为次生酒小鼠。我们观察到在所有四只被检查的小鼠的原位部位的 GFP 阳性原发肿瘤的形成 (图 2B, 左)。此外, 在原位注射后2.5 月检查的四只小鼠中, 三的肺中发现了显微转移 (图 2B,中)。此外, 肝转移的观察, 以回应脾注射到所有四只小鼠检查在1月后注射 (图 2B, 右)。
结合起来, 这些发现表明, 高分辨率 GFP 荧光在 PDX 衍生 CRC organoid 细胞允许他们在自发淋巴结和那些实验性的, 在遥远的器官开发的, 当引进到接受者老鼠.这种高灵敏度检测的 PDX 衍生 CRC organoids在体内也提供了一个通用的模型, 不仅用于研究肿瘤转移的生物学, 而且也为发展靶向治疗在临床前的设置。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57374/57374fig1.jpg"> 图 1: 在酒小鼠上用 GFP 慢病毒北疆标记的 PDX 衍生 CRC organoids 产生转移的示意图.(A)将 CRC 组织的小块植入酒小鼠 (步骤 1)。从病人手术解剖的 CRC 组织被切成片, 植入酒小鼠。皮下植入术。(B)产生的 CRC organoids 脱离 PDXs (步骤 2–4)。开发的 CRC 移植被切碎 (步骤 2), 并转移到一个15毫升管包含培养基包括胶原酶 (步骤 3)。在缓慢搅拌孵化后, CRC 细胞悬浮液被过滤 (步骤 3)。然后, organoid 细胞悬浮在 CRC organoid 培养基被播种到一个人工细胞外基质涂层板和孵化一夜之间的 CO2孵化器 (步骤 4)。附在人工细胞外基质上的 CRC organoid 细胞也被涂上额外的人工细胞外基质, 并孵化在 CO2孵化器中 (步骤 4)。皮下植入术。(C)在使用注射入受体小鼠之前, 通过 GFP 慢病毒载体粒子生成 CRC organoids 转基因 (步骤 5–7)。GFP 慢病毒载体粒子以高滴度 (步骤 5) 产生。PDX 衍生的 CRC organoids 在人工细胞外基质中生长, 直接用细胞刮刀收割, 转移到微细 (步骤 6)。离心后, 细胞颗粒被重新悬浮在 PBS。细胞悬浮离心, 细胞颗粒分离。然后, 在联合2孵化器 (步骤 6) 过夜的人工细胞外基质涂层板上, organoid 细胞悬浮液在 crc organoid 培养基上与 GFP 病毒库一起孵化。采用人工胞外基质的 CRC organoid 细胞涂敷额外的人工胞外基质, 并在 CO2孵化器中孵化, 以固化人工胞外基质涂层 (步骤 6)。然后在7–10天内培养 CRC organoids, 以扩大细胞生长 (步骤 6)。为建立自发性转移模型, 将 5 x 105 CRC organoid 细胞标记为绿色荧光蛋白悬浮于50µL 的 PBS 中, 将50% 例人工细胞外基质注入原位酒小鼠 (步骤 7)。为了生成一个实验性转移模型, 4 x 104 CRC organoid 细胞标记与 GFP 在50µL 的 PBS 被注入 intrasplenically 酒小鼠 (步骤 7)。o.t.: 原位注射, 行列: 脾注射液。<a href="/files/ftp_upload/57374/57374fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57374/57374fig2.jpg"> 图 2: 在培养的 gfp 标记的 CRC organoids、原发肿瘤和淋巴结中检测到的 gfp 可视化的高分辨率。(A)近100% 的培养的 CRC organoids 是 GFP 阳性。这些图像是用立体荧光显微镜拍摄的。刻度条 = 250 µm (B) GFP-阳性在原发性肿瘤和淋巴结肺和肝脏。将游离 GFP 表达的 CRC organoid 细胞悬浮液注入酒小鼠直肠黏膜下层 (o.t. 注射液)。大多数肿瘤细胞在原发肿瘤中呈绿色荧光蛋白阳性。绿色荧光蛋白阳性淋巴结殖民肺 (由箭头表示) 也显示。此外, 荧光蛋白阳性淋巴结是在肝脏中检测到的 (由箭头表示), 当细胞悬浮被 intrasplenically 注射 (行列注射液) 成鼠。刻度条 = 500 µm.<a href="/files/ftp_upload/57374/57374fig2large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>

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

Nanyang Technological University

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

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

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 ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

来自沙柯玛患者衍生异种移植物的肿瘤启动细胞的分离和表征

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

患者衍生的人类泌尿细胞癌和结肠直肠癌肿瘤生长和自发转移的正交异种移植模型

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

黑色素瘤患者衍生异种移植模型

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

患者源性肿瘤外植体作为预测患者耐药性的"实时"临床前平台

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

A highly accurate in vitro high-throughput assay system was developed to evaluate anticancer drugs using patient-derived tumor organoids (PDOs), similar to cancer tissues but are unsuitable for in vitro high-throughput assay systems with 96-well and 384-well plates.

使用患者源性肿瘤类器官的高通量 体外 检测

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

分析EGFR突变型NSCLC患者衍生类器官靶向治疗的敏感性

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

从患者来源的肿瘤样本中建立三维微球并评估其对药物的敏感性

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...