这项研究的经费来自国家癌症研究所 (R01CA166376 和 R01CA191489 D.X.) 和国防部 (W81XWH-16-1-0227 D.X. 阮) 的赠款。

所有实验都是按照耶鲁大学机构动物护理和使用委员会 (IACUC) 批准的协议进行的。
1. 试剂的设置/准备。
<ol><li>
博莱 霉 素 注意: 根据全球统一的化学品分类和标签制度, 博莱霉素被归类为 GHS08 的健康危害。<ol>
<li>用化学罩准备博莱霉素。并用重悬 15 U 入5毫升无菌磷酸盐缓冲盐水 (PBS)。</li>
		<li>整除100-200 µL 的溶液成玻璃瓶, 并冻结在-20 摄氏度, 以供将来使用。用泥沙的日期正确地标注管子。从该日期起6月内使用。 注意: 每个鼠标的应变对博莱霉素26有不同的敏感性。建议对不同剂量的博莱霉素进行小鼠菌株的检测。表 1列出了在代表性实验中使用的鼠株和博莱霉素的相应剂量。</li>
	</ol>
</li>
	<li>
小 鼠
	<ol>
<li>购买实验所需的小鼠, 并允许老鼠在注射前7天适应。在生物安全罩中进行输液。在博莱霉素输液前使用标准的机构程序, 将动物安置在 non-BSL2 兼容的房间里, BSL2 房间。在6-8 周的年龄注射老鼠。 警告: 在危险剂、生物安全等级 2 (BSL2) 和 IACUC 批准的机构指南下注射老鼠。 注: 为代表性实验而购买的鼠标列在材料表中。只有雄性老鼠被使用。</li>
	</ol>
</li>
	<li>
肺癌细胞系
	<ol>
<li>在各自的媒体中培养所需的细胞线。注射前, 进行短串联重复 DNA 分析或基因检测, 以确保正确的细胞线识别。为了促进体内和体外成像, 感染细胞系的慢病毒北疆表达胸苷激酶, 绿色荧光蛋白 (GFP) 和荧光素酶融合记者27, 并排序报告阳性细胞的荧光植入前的活化细胞分类。每6月支原体试验线。<ol>
<li>培养 H2030 人类癌细胞系的建议, 制造商使用罗斯威尔公园纪念研究所培养基 (RPMI) 与10% 胎牛血清 (血清), 青霉素-链霉素, 和两性霉素 B。</li>
			<li>培养368T1 小鼠细胞系 (从KrasG12D;p 53-/ 老鼠) 使用 Dulbecco 的改良鹰的培养基 (DMEM) 与10% 的血清, 青霉素-链霉素, 和两性霉素 B。</li>
			<li>PC9 人类癌细胞系的 RPMI, 采用10% 血清、青霉素-链霉素和两性霉素 B。</li>
		</ol>
</li>
	</ol>
</li>
</ol>2. 博莱霉素治疗
<ol><li>计划在肿瘤细胞植入前14天注射博莱霉素 intratracheally 小鼠。</li>
	<li>在注射前将博莱霉素在冰上解冻2小时。</li>
	<li>解冻后, 将博莱霉素稀释至所需的工作浓度 (0.02 U/50 µL 或 0.005 U/50 µL), 并将其放在冰上。 注: 博莱霉素浓度取决于实验用的小鼠应变 (表 1)。应进行小鼠博莱霉素滴定, 以确定最佳剂量。</li>
	<li>用1毫升注射器和27克针, 通过将氯胺酮和甲苯噻嗪稀释到最终浓度10毫克/毫升和1毫克/毫升, 通过注射腹腔氯胺酮/甲苯噻嗪溶液在100/10 毫克/千克上麻醉每只老鼠, 以制备麻醉药。分别。</li>
	<li>监测老鼠的呼吸, 并使用脚趾捏。确认正确的麻醉时, 鼠标不响应脚趾捏。在麻醉时, 应将兽医软膏涂抹于眼部, 以防干燥。</li>
	<li>放置1鼠标在一个插管平台上, 通过挂在它的前牙与它的背部对平台。</li>
	<li>用光纤照明灯照亮上胸部, 帮助可视化气管。张开鼠标的嘴, 用无菌的扁平钳轻轻地拉出舌头。</li>
	<li>使用静脉导管没有针头, 以避免阻塞呼吸。将导管放置在从气管开口处发出的白色光线上。</li>
	<li>将导管插入气管, 直到导管顶部到达前牙。通过在口腔内打开导管, 通过可视化白光照射, 确认导管在气管内的正确放置。</li>
	<li>使用吸管, 50 µL 的博莱霉素或车辆 (PBS) 直接进入导管, 以确保整个体积被吸入。在无菌条件下执行此步骤。</li>
	<li>如果导管正确插入, 鼠标将立即吸入导管的内容。等待几秒钟, 直到整个音量沿着导管移动。然后从气管取出导管, 在10% 漂白剂溶液中处理。 注: 每个步骤 2.7-2.11 的鼠标平均时间约为5分钟。</li>
	<li>如果鼠标不吸入液体, 仔细监测呼吸并调整导管位置。如果鼠标停止呼吸, 立即取出导管, 并允许鼠标在重新插入导管前正常恢复呼吸。</li>
	<li>将注入的鼠标放在 IACUC 上的已批准的加热垫上, 然后放在平坦的表面上进行恢复。整个过程将采取每只鼠标10分钟。在完全恢复之前, 不要将已经注射到其他动物公司的动物退回。</li>
	<li>将 "危险化学品使用" 卡放在笼子里24小时。</li>
</ol>3. 在插管后监测小鼠
<ol><li>在插管后立即监测小鼠呼吸窘迫, 然后每15分钟, 直到老鼠从麻醉中醒来。不要让老鼠无人看管, 直到他们恢复足够的意识来维持胸骨卧床。</li>
	<li>注射酮酮 (5 毫克/千克) 腹腔减轻疼痛。</li>
	<li>检查小鼠 24 h 后插管和每周3次的任何发病率的迹象。</li>
	<li>记录程序的日期、动物身份、程序类型、麻醉类型和术后监测观察时间。</li>
	<li>监测小鼠的体重减轻, 呼吸窘迫, 行为异常, 身体状况评分 < 2 (椎柱明显/背骨盆骨分割) 双周后注射博莱霉素。</li>
	<li>弄死小鼠在呼吸窘迫或小鼠经历了15% 的身体质量损失由 CO2或氯胺酮/甲苯噻嗪后, 颈椎脱位。通过对呼吸活动进行触诊确认安乐死。</li>
</ol>4. 植入肺腺癌细胞系。
注: 在注射博莱霉素14天后进行细胞植入 (步骤 2.1)。
<ol><li>允许细胞生长在75厘米2瓶不受干扰的3天前的注射与相应的媒体, 如步骤1.3 所述。 注: 在注射的当天, 它们应该是80% 汇合的 (在每个烧瓶中对应于 2-3 x 106 , 这取决于细胞线)。</li>
	<li>用5毫升的 PBS 在室温下冲洗75厘米2烧瓶上生长的细胞。</li>
	<li>吸入 PBS 和添加1.5 毫升胰蛋白酶到细胞和孵化细胞在37°c, 直到细胞分离 (通常2-5 分钟)。 注: 我们建议每隔2分钟检查从塑料的细胞脱离, 通过简单的可视化烧瓶底部或在光显微镜下。不要超过5分钟的孵化与胰蛋白酶。</li>
	<li>加入4毫升含有10% 种血清的培养基 (与步骤4.1 相同的培养基) 中和胰蛋白酶。将细胞收集成15毫升管, 在室温下将其离心在 200 x g 处3分钟。</li>
	<li>在5毫升的 PBS 中吸入培养基并并用重悬细胞颗粒以洗涤细胞。在室温下将细胞以 200 x g 为3分钟, 在颗粒细胞中进行离心。重复这1次。</li>
	<li>吸入 pbs 和并用重悬1.5 毫升的 pbs 每瓶。</li>
	<li>将50µL 的细胞混合物与50µL 的台盼蓝混合, 用细胞计数器/Hemocytometer 室28计算细胞数。</li>
	<li>通过稀释 PBS 中的细胞来制备细胞悬浮液, 在每只老鼠的50µL 中注射 1 x 105细胞 (或其他指示量)。在注射前把细胞放在冰上。 注: 准备至少两次更多的细胞注射, 以避免耗尽细胞悬浮。</li>
	<li>用1毫升注射器和27克针, 通过将氯胺酮和甲苯噻嗪稀释到最终浓度10毫克/毫升和1毫克/毫升, 通过注射腹腔氯胺酮/甲苯噻嗪溶液在100/10 毫克/千克上麻醉每只老鼠, 以制备麻醉药。分别。</li>
	<li>监测老鼠的呼吸, 并使用脚趾捏。确认正确的麻醉时, 鼠标不响应脚趾捏。在麻醉时, 应将兽医软膏涂抹于眼部, 以防干燥。</li>
	<li>放置1鼠标在一个插管平台上, 通过挂在它的前牙与它的背部对平台。</li>
	<li>用光纤照明灯照亮上胸部, 帮助可视化气管。</li>
	<li>用 p200 吸管轻轻地并用重悬细胞, 以确保它们不形成团簇。张开鼠标的嘴, 用无菌的扁平钳轻轻地拉出舌头。</li>
	<li>使用静脉导管与针移除, 以避免阻塞呼吸。将导管放置在从气管开口处发出的白色光线上。</li>
	<li>将导管插入气管, 直到导管顶部到达前牙。通过在口腔内打开导管, 通过可视化白光照射, 确认导管在气管内的正确放置。</li>
	<li>使用吸管, 将50µL 的细胞直接放入导管中, 以确保整个体积被吸入。在无菌条件下执行此步骤。 注: 如果导管正确插入, 鼠标将立即吸入导管的内容。</li>
	<li>等待几秒钟, 直到整个音量沿着导管移动, 然后从气管中取出导管, 并在10% 漂白剂溶液中处理。</li>
	<li>如果鼠标不吸入液体, 仔细监测呼吸并调整导管位置。如果鼠标停止呼吸, 立即取出导管, 并允许鼠标在重新插入导管前正常恢复呼吸。 注: 步骤 4.11-4. 18 的每个鼠标的平均时间约为5分钟。</li>
	<li>将注入的鼠标放在 IACUC 上的已批准的加热垫上, 然后放在平坦的表面上进行恢复。 注: 整个过程将采取10分钟每鼠标。在完全恢复之前, 不要将已经注射到其他动物公司的动物退回。</li>
	<li>在成像前用电动剃须刀剃掉整个肋区的腹部和背的 B6129SF1/J 和其他小鼠的黑头发。</li>
	<li>2-3 分钟后细胞注射, 注射100µL 荧光素复古轨道 (15 毫克/毫升) 使用胰岛素针。在每次成像会话中交替使用用于注射的眼睛。</li>
	<li>在至少2分钟后, 在一个动物生物发光成像仪中放置多达5只老鼠, 在背卧床位置, 并获得一个侧面图片使用发光设置29。</li>
	<li>在控制面板下调整分辨率和灵敏度设置, 以测量给定细胞线的发光。对于大多数应用程序, 从3分钟 (或 "自动" 如果饱和) 开始, binning = 中等 (4), F/Stop=1, 视野 = D, 主题高度 = 1.50。 注: 如果注射成功, 在上胸区, 在一个肺或两个肺部检测到发光信号。</li>
	<li>翻转小鼠到胸骨卧床位置, 并获得一个背部图片如上所述。 注: 如果细胞注射未正确执行, 细胞可能会聚集在气管入口或颈部。</li>
	<li>注射酮酮5毫克/千克腹腔减轻疼痛。</li>
	<li>把老鼠搬回笼子里, 在笼子里放一张 "BSL-2 剂"。</li>
	<li>在插管后立即监测小鼠呼吸窘迫, 然后每隔15分钟, 直到老鼠从麻醉中醒来。不要让老鼠无人看管, 直到他们恢复足够的意识来维持胸骨卧床。</li>
	<li>检查小鼠 24 h 后插管和每周三次的任何发病率的迹象。</li>
	<li>记录在程序日期, 动物身份, 程序类型, 麻醉类型和术后监测观察和时间的日志。</li>
	<li>监测小鼠的体重减轻, 呼吸窘迫, 行为异常, 身体状况评分 < 2 (脊柱明显/背骨盆骨分割可见) 两周后接种的博莱霉素。 注: 肺肿瘤小鼠可能表现为呼吸刺激、体重减轻、恶病质和/或死亡。</li>
	<li>弄死小鼠在呼吸窘迫或小鼠经历了15% 的身体质量损失由 CO2或氯胺酮/甲苯噻嗪后, 颈部脱位, 如果收集组织。通过对呼吸活动进行触诊确认安乐死。</li>
</ol>5. 生物发光成像对肿瘤生长的监测
<ol><li>重复成像的老鼠在3天后植入和每周。</li>
	<li>麻醉小鼠注射氯胺酮/甲苯噻嗪 (如步骤 2.4-2.5 中所述) 或吸入异氟醚 (2%)。监测小鼠的呼吸, 并使用脚趾捏来确认正确的麻醉。在麻醉时, 应将兽医软膏涂抹于眼部, 以防干燥。</li>
	<li>在成像前用电动剃须刀剃掉整个肋区的腹部和背的 B6129SF1/J 和其他小鼠的黑头发。</li>
	<li>一旦老鼠被麻醉, 注射100µL 的荧光素轨道 (15 毫克/毫升) 使用胰岛素针。等待2分钟交替的眼睛用于注射每一个成像会话。</li>
	<li>将鼠标放在成像仪中, 并获得4.22-4 步24中所述的背侧和腹侧图像。</li>
	<li>在成像后将老鼠放回笼子里, 在他们的背部在平坦的表面恢复。 注: 整个手术过程每组5只老鼠需要5分钟。不要让老鼠无人看管, 直到他们恢复足够的意识来维持胸骨卧床。</li>
	<li>利用成像/生物发光分析软件对图像进行分析。<ol>
<li>选择适当的图像并将它们作为一个组加载到生物发光分析软件中。</li>
		<li>单击roi 工具 | 正方形以添加感兴趣的正方形区域 (roi)。将 ROI 放在上胸部区域, 覆盖整个肺部图像。对每个鼠标图像重复此操作。</li>
		<li>右键单击并选择 "复制所有 roi函数" 将相同的 roi 应用于组中的所有图像, 并将它们放置在鼠标的上胸部, 无论是腹侧视图还是背景色。</li>
		<li>在图像窗口的左上角, 选择光子函数。单击测量控制面板函数以测量 ROIs。将包含总通量 (光子/s) 的输出表复制并粘贴到一个可供选择的软件中, 以便进一步分析数据。</li>
		<li>将每个鼠标的每日 roi 值正常化为其在0天测量的 roi 值。</li>
	</ol>
</li>
</ol>6. 组织分离和加工。
<ol><li>麻醉小鼠注射氯胺酮/甲苯噻嗪 (如步骤 2.4-2.5 或吸入异氟醚 (2%) 所述。监测老鼠的呼吸, 并使用脚趾捏。确认正确的麻醉时, 鼠标不响应脚趾捏。在麻醉时, 应将兽医软膏涂抹于眼部, 以防干燥。</li>
	<li>用胰岛素针注射100µL 荧光素复古轨道 (15 毫克/毫升)。等待2分钟。</li>
	<li>图象老鼠跟随步骤 4.22-4. 24 从这个协议。</li>
	<li>注射到另一只眼睛从步骤6.2 另外100µL 的荧光素复古轨道使用胰岛素针之前, 安乐死。</li>
	<li>弄死小鼠腹腔注射氯胺酮/甲苯噻嗪 (见步骤2.4 的细节), 其次是颈椎脱位, 如果小鼠在深度麻醉下, 根据 IACUC 提供的指导方针。通过对呼吸活动进行触诊确认安乐死。</li>
	<li>使用无菌手术剪刀在胸骨下面做一个皮肤切口。用镊子和手术剪刀在隔膜上打开肌肉层, 通过在两侧两侧的肋骨笼上切开胸腔, 避免胸腔内动脉。</li>
	<li>灌注通过在心脏右心房做一个小切口, 并通过左心室注入5毫升 PBS。如果血液灌注得当, 肺组织的颜色会从红到浅粉色白色。</li>
	<li>用镊子抓住食道或心脏, 仔细地将肺部从胸腔中切除。轻轻拉组织, 并使用手术剪刀仔细削减连接组织避免穿刺的肺组织。在以后的一步中尽量保持气管的膨胀。</li>
	<li>通过浸没和旋转组织3倍的6井板填充3毫升的 PBS, 以清除多余的血液, 洗肺。</li>
	<li>将肺放在6井板的盖子上, 将含有组织的盖子放在发光成像仪中, 并获得发光图像30。 注: 我们建议从 "采集控制面板" "视图 = A" 和 "主题高度 = 0.75 厘米" 中选择以下设置, 然后 "自动曝光" 以避免获得饱和图像。</li>
	<li>消费和图像其他器官, 如步骤 6.9-6.10 中所做的, 其中转移已被发现的发光, 如脑, 肝, 肾上腺, 骨, 淋巴结, 肾脏或脾脏31。</li>
	<li>灌注1毫升4% 多聚甲醛的肺, 将针头插入鼠标的气管内。如果灌注得好, 肺部会膨胀。 警告: 4% 多聚甲醛是一种毒性、健康危害 GHS07 和 GHS08。</li>
	<li>将肺部转移到15毫升圆锥, 5 毫升4% 多聚甲醛。</li>
	<li>修复肺或其他组织通过孵化的组织与4% 多聚甲醛隔夜在4°c 在一个震动装置在 30-50 rpm。</li>
	<li>根据应用32, 在石蜡或最佳切削温度化合物中嵌入肺部 (或其他组织)。 注: 石蜡是保存肺结构和马尾松三色和苏木精-伊红染色的首选方法。</li>
</ol>

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作者声明没有竞争的财政利益。

在肺癌和其他慢性疾病的肺36之间有明显的临床相似之处。尤其是特发性肺纤维化 (IPF) 患者对发展肺癌的偏爱增加, 而这个协会独立于吸烟史37,38。森林小组的特点是逐步破坏肺结构和受损的呼吸功能, 通过沉积 ECM39。同时, 在手术切除后, 早期的无细胞肺癌患者并发森林小组的结局较差40。多数 NSCLC 肿瘤在诊断时含有纤维化成分, 这种间质反应的程度与预后差18有关。纤维化的急性或持续性组织损伤可能以多种方式增加肿瘤发生率。例如, 在损伤后, 上皮再生的过程可能会扩大容易转化的祖细胞池。另外, 各种免疫细胞的流入和功能可能有助于建立免疫抑制微环境, 而肌的活化可能分泌生长因子或沉积肿瘤促进 ECM。因此, 我们用纤维化诱导剂对肺部进行预调节的方法可能特别适合于研究肺癌的发病率 (如纤维化) 和非 NSCLC 亚型的模型, 后者富含细胞外基质, 并且炎症基质41。
总的来说, 我们的方法大大提高了通过气管注入呼吸道的肿瘤细胞的植入。这种观察适用于不同程度活性的小鼠菌株。其他方法的肿瘤细胞注射到肺部包括尾静脉注射或直接注射到肺实质通过胸壁。对于研究肺癌细胞致瘤性的特定目的, 这些方法是次优, 因为前者要求肿瘤细胞在循环和 extravasate 前生存 (一个过程更类似于转移到肺部), 而后者可导致细胞在注射后很快泄漏到胸膜腔内 (未显示数据)。这两种方法都可能混淆肺癌细胞的局部定量化。另外, 我们的协议确保种子肿瘤细胞首先保留在被肺部基质包围的呼吸道中。对本协议的成功至关重要的是, 对小鼠进行适当的插管, 博莱霉素的耐受剂量, 以及与肿瘤细胞注射有关的气道预适应时间。我们还表明, 该协议允许细胞随后传播到其他组织, 包括大脑。尽管与肺肿瘤负担相关的发病率仍可能限制远 macrometastasis 的综合特征, 但这种方法可能有助于定量和研究来自肺部的循环肿瘤细胞。
尽管本文所述方法有其优越性, 但一些技术变量可能会影响植入的最终效率和肿瘤进展的监测。首先, 有充分的证据表明, 纤维化反应对博莱霉素的小鼠是应变依赖性。例如, C57Bl/6 小鼠比 Balb/c 小鼠更容易发生纤维化42。其次, 对博莱霉素的敏感性是性别和年龄依赖性。根据我们的经验, 雌性和小鼠更容易对整个议定书产生不利影响。这可能限制了需要直接比较男女之间的肿瘤发生研究, 或年轻和年老的动物。此外, 7 周以下的小鼠气管灌注技术具有挑战性, 可能需要对导管大小进行调整。第三, 博莱霉素是有毒的, 可能是诱变由于它的能力诱导 DNA 断裂。在进行实验之前, 对每个小鼠的应变剂量进行测试是很重要的。在这项原则研究的证明, 我们提供建议剂量的雄性小鼠横跨不同的菌株。此外, 深色毛皮小鼠由于组织穿透率低和毛皮对发光信号的掩蔽而不太适合生物发光成像。脱毛小鼠可以改善图像测量, 但其他方法也可以使用, 如磁共振成像和荧光成像应用长波长探头, 如 TdTomato 或 Katushka43。最后, 在选择一个特定的小鼠模型的肿瘤检测模式时, 应考虑给定的报告蛋白的潜在免疫原性。
博莱霉素改造远端肺的第一损伤肺泡型 2 (AT2) 细胞, 然后再生, 试图修复肺泡44。由于 AT2 细胞是引起非细胞肺癌的主要细胞类型之一, 因此, 对肺进行预调节, 也可能改变原位从 GEMMs 产生的肿瘤的起始和进展。其他类型的伤害, 包括流感感染13或萘注入14, 已表明增加植入的人和小鼠肺干/祖细胞。值得注意的是, 萘损害 bronchio-肺泡交界处的茎/祖细胞, 并有可能增加 GEMMs45的肿瘤萌生。对嫁接细胞类型的损伤类型和气道生态位进行微调, 可以进一步优化我们研究不同来源的肺癌同种异体移植或异种移植的方法。最后, 我们建议该方法可以实现肺癌 PDXs 的标准生成。biospecimens 小鼠皮下移植建立人 PDXs 肺癌的成功率相对较低 (30-40%)46,47。PDXs 的原位生长不仅可以通过限制样本量 (从人肺中) 增加这种成功率, 而且还能产生更多的生理相关条件来检验肺癌治疗药物的药代动力学和药效学。

肺癌是全球1癌症相关死亡的主要原因。肺癌患者最终从转移到远端器官, 主要是中枢神经系统, 肝脏, 肾上腺, 骨骼2,3,4。胸恶性肿瘤历来被归类为小细胞肺癌或非小细胞肺癌 (NSCLC)5。NSCLC 是最常见的恶性肿瘤, 可分为不同的组织学亚型, 包括肺腺癌 (LUAD) 和肺鳞状细胞癌 (LUSC)6。经手术切除的人原发性肺癌的基因组分析显示, 某一 histotype 内的肿瘤也能表达多种分子扰动, 进一步促进其临床进展和混淆患者预后。肺癌的显著异质性对合理的设计、临床前试验和有效的治疗策略的实施都是一个重大挑战。因此, 有必要扩大可听话的实验性肺癌模型的汇辑, 以研究这种疾病的不同细胞来源、分子亚型和分期。
使用动物模型的各种方法在体内研究肺癌, 每个都有各自的优缺点, 这取决于所关心的生物学问题。基因工程小鼠模型 (GEMMs) 可以针对特定祖细胞类型的特异基因改变, 导致肿瘤在免疫宿主7中取得进展。虽然非常强大和临床相关的, 潜伏期, 变异性和/或肺部肿瘤发病率与 GEMMs 可能会禁止某些定量测量和检测晚期转移在遥远的器官8。一个补充的方法是使用同种异体模型, 藉以肺癌细胞从老鼠肿瘤直接获得或首先被获取作为建立的细胞线在文化中被重新介绍入自体寄主。类似, 肺癌移植是由人体细胞系或病人衍生的肿瘤样本建立的。人细胞系移植或患者衍生的移植 (PDXs) 一般维持在免疫缺陷小鼠, 因此排除完全免疫监测9。尽管有这个缺点, 它们提供了一个途径来传播限制数量的人类 biospecimens 和研究人类癌细胞的基本的体内性质, 这编码为更复杂的基因组畸变比 GEMM 肿瘤。
同种异体移植和异种移植的一个有用的特性是, 他们可以使用传统的限制性细胞稀释方法, 用于量化10恶性细胞中肿瘤起始细胞 (抽搐) 的频率。在这些实验中, 在动物的侧面注入了一定数量的细胞, 并根据肿瘤的发生率来估计抽搐频率。然而皮下肿瘤可以是更低氧11并且可能不模型主要生理制约肺肿瘤微环境。气管内向小鼠肺内输送上皮干细胞或祖细胞是研究肺再生和气道干细胞生物学12的方法。然而, 这种技术的植入率可能相对较低, 除非肺部首先受到生理形式的伤害, 如病毒感染13,14。炎症基质细胞的支持和 (或) 肺基底膜的破坏, 可改善移植细胞在远端气道15的相关干细胞龛位的保留。纤维化诱导剂也可以预条件肺, 以增强植入诱导多能细胞16和间充质干细胞17。类似的气道损伤形式是否会影响植入率, 肿瘤的启动能力和肺癌细胞的产生还有待系统地评估。
本文通过对损伤小鼠肺的预处理, 描述了提高原位肺癌细胞植入效率的方法。LUAD 出现在远端呼吸道与这些癌症的一个重要的子集发展纤维间质基质18 , 往往与不良预后19。博莱霉素是一种天然非核糖体杂交肽-聚酮体, 已广泛用于诱导小鼠肺纤维化20。博莱霉素的气道灌注首先促进肺泡上皮的磨损和炎症细胞的招募, 包括巨噬细胞、中性粒细胞和单核细胞21。其次是在远端呼吸道组织重塑, 基底膜重组22,23和细胞外基质 (ECM) 沉积24。单个博莱霉素注射液的影响是瞬变的, 在大多数研究中30天后纤维化解决25。利用同种异体移植和异种移植模型, 我们对博莱霉素小鼠呼吸道进行预适应试验, 可以显著提高肺 LUAD 细胞的服用率。

<table><tbody><tr><td>Bleomycin</td><td>Sigma</td><td>B5507-15UN</td><td>CAUTION Health hazard GHS08</td></tr><tr><td>Exel Catheter 24G</td><td>Fisher</td><td>1484121</td><td>Remove needle. For intratracheal injection</td></tr><tr><td>Ketamine (Ketaset inl 100 mg/mL C3N 10 mL)</td><td>Butler Schein</td><td>56344</td><td>To anesthetize mice</td></tr><tr><td>Xylazine</td><td>Butler Schein</td><td>33198</td><td>To anesthetize mice</td></tr><tr><td>Ketoprofen, 5,000 mg</td><td>Cayman Chemical</td><td>10006661</td><td>Analgesic</td></tr><tr><td>Puralube Veterinary Ophthalmic Ointment</td><td>BUTLER ANIMAL HEALTH COMPANY LLC</td><td>8897</td><td>To prevent eye dryness while under anesthesia</td></tr><tr><td>D-Luciferin powder</td><td>Perkin Elmer Health Sciences Inc</td><td>122799</td><td>For luminescent imaging. Reconstitute powder with PBS for a working concentration of 15mg/mL. Protect from Light</td></tr><tr><td>Rodent Intubation stand</td><td>Braintree Scientific</td><td>RIS-100</td><td>Recommended stand for intratracheal injection</td></tr><tr><td>MI-150 ILLUMINATOR 150W MI-150</td><td>DOLAN-JENNER INDUSTRIES</td><td>MI-150 / EEG2823M</td><td>To illuminate and visualize trachea</td></tr><tr><td>Graefe Forceps, 2.75 (7 cm) long serrat</td><td>Roboz</td><td>RS-5111</td><td>For intratracheal injection</td></tr><tr><td>Syringe Luer-Lok Sterile 5ml</td><td>BD / Fisher</td><td>309646</td><td></td></tr><tr><td>Satiny Smooth by Conair Dual Foil Wet/Dry Rechargeable Shaver</td><td>Conair</td><td>-</td><td>To shave mice</td></tr><tr><td>Bonn Scissors, 3.5&quot; straight 15 mm sharp/sharp sure cut blades</td><td>Roboz</td><td>RS-5840SC</td><td></td></tr><tr><td>15 mL conical tube</td><td>BD / Fisher</td><td>352097</td><td></td></tr><tr><td>1.5 mL centrifuge tubes</td><td>USA SCIENTIFIC INC</td><td>1615-5500</td><td></td></tr><tr><td>Vial Scintillation 7 mL Borosilicate Glass GPI</td><td>Fisher</td><td>701350</td><td></td></tr><tr><td>Filter pipette tips (200 &amp;mu;L)</td><td>USA SCIENTIFIC INC</td><td>1120-8710</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>DMEM high glucose</td><td>Life Technologies</td><td>11965-092</td><td></td></tr><tr><td>RPMI Medium 1640</td><td>Life Technologies</td><td>11875-093</td><td></td></tr><tr><td>Fetal bovine serum USDA</td><td>Life Technologies</td><td>10437-028</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Amphotericin B</td><td>Sigma</td><td>A2942-20ML</td><td></td></tr><tr><td>Trypan Blue Stain 0.4%</td><td>Life Technologies</td><td>15250-061</td><td></td></tr><tr><td>Countess Automated Cell Counter</td><td>Life Technologies</td><td>AMQAX1000</td><td></td></tr><tr><td>Flask T/C 75cm sq canted neck, blue cap</td><td>Fisher / Corning</td><td>353135</td><td></td></tr><tr><td>IVIS Spectrum Xenogen Bioluminiscence</td><td>Perkin Elmer Health Sciences Inc</td><td>124262</td><td>For &lt;em&gt;in vivo&lt;/em&gt; bioluminescence imaging</td></tr><tr><td>Living image software</td><td>Perkin Elmer Health Sciences Inc</td><td>128113</td><td>For &lt;em&gt;in vivo&lt;/em&gt; bioluminescence analysis</td></tr><tr><td>XGI-8 Gas Anesthesia System</td><td>Perkin Elmer Health Sciences Inc</td><td>118918</td><td>For Isoflurane anesthesia</td></tr><tr><td>BD Ultra-Fine II Short Needle Insulin Syringe 1 cc. 31 G x 8 mm (5/16 in)</td><td>BD / Fisher</td><td>BD328418</td><td>For retro-orbital luciferin injection</td></tr><tr><td>Syringe 1ml</td><td>BD / Fisher</td><td>14-823-434</td><td>For intraperitoneal injections</td></tr><tr><td>26 G x 1/2 in. needle</td><td>BD / Fisher</td><td>305111</td><td>For intraperitoneal injections</td></tr><tr><td>4% Paraformaldehyde</td><td>VWR</td><td>43368-9M</td><td>CAUTION Health hazard GHS07, GHS08. For fixing tissue</td></tr><tr><td>Pipet-Lite Pipette, Unv. SL-200XLS+</td><td>METTLER-TOLEDO INTERNATIONAL</td><td>17014411</td><td></td></tr><tr><td>Mayer&#039;s Hematoxylin</td><td>ELECTRON MICROSCOPY SCIENCES</td><td>517-28-2</td><td></td></tr><tr><td>Eosin Y stain 0.25% (w/v) in 57%</td><td>Fisher</td><td>67-63-0</td><td></td></tr><tr><td>Masson Trichrome Stain Kit</td><td>IMEB Inc</td><td>K7228</td><td>For masson trichrome stain to visualize collagen</td></tr><tr><td>Superfrost plus glass slides</td><td>Fisher</td><td>1255015</td><td></td></tr><tr><td>6 well plate</td><td>Corning</td><td>C3516</td><td></td></tr><tr><td>Universal Mycoplasma Detection Kit</td><td>ATCC</td><td>30-1012K</td><td></td></tr><tr><td>OCT Embedding compound</td><td>ELECTRON MICROSCOPY SCIENCES</td><td>62550-12</td><td>For embedding tissue for frozen sections</td></tr><tr><td>Leica CM3050 S Research Cryostat</td><td>Leica</td><td>CM3050 S</td><td>To section tissue for staining analysis</td></tr><tr><td>Keyence All-in One Fluorescence Microscope</td><td>Keyence</td><td>BZ-X700</td><td></td></tr><tr><td>ImageJ</td><td>US National Institutes of Health</td><td>IJ1.46</td><td>http://rsbweb.nih.gov/ij/ download.html</td></tr><tr><td>Prism 7.0 for Mac OS X</td><td>GraphPad Software, Inc.</td><td>-</td><td></td></tr><tr><td>Athymic (Crl:NU(NCr)-Foxn1nu) mice</td><td>Charles River</td><td>NIH-553</td><td></td></tr><tr><td>NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice</td><td>Jackson Laboratories</td><td>5557</td><td></td></tr><tr><td>B6129SF1/J mice</td><td>Jackson Laboratories</td><td>101043</td><td></td></tr><tr><td>NIH-H2030 cells</td><td>ATCC</td><td>CRL-5914</td><td></td></tr><tr><td>368T1</td><td>generously provided by Monte Winslow (Standford University)</td><td>-</td><td></td></tr><tr><td>PC9 cells</td><td>Nguyen DX et al. Cell. 2009;138:51&amp;ndash;62</td><td>-</td><td></td></tr><tr><td>H2030 BrM3 cells</td><td>Nguyen DX et al. Cell. 2009;138:51&amp;ndash;62</td><td>-</td><td></td></tr></tbody></table>

pre-conditioning the airways of mice with bleomycin increases the efficiency of orthotopic lung cancer cell engraftment

肺癌是一种致命的治疗顽固性疾病, 是生物异构的。为了了解和有效地治疗胸恶性肿瘤的完整临床谱, 需要额外的动物模型来重述不同的人肺癌亚型和分期。同种异体或异种移植模型是多种多样的, 可以利用小鼠或人类的恶性细胞,在体内癌变能力的量化。然而, 以前所描述的肺癌细胞植入的方法已经在非生理部位进行, 如小鼠的侧面, 由于细胞移植到肺部的效率低下。在本研究中, 我们描述了一种通过对小鼠肺纤维化诱发博莱霉素进行预调节来增强原位肺癌细胞植入的方法。作为一种概念验证实验, 我们将这种方法应用于从老鼠或人源获得的肺腺癌亚型的嫁接肿瘤细胞, 并将其转化为不同品系的小鼠。我们证明, 在肿瘤细胞注射前, 与博莱霉素的呼吸道损伤增加了肿瘤细胞从0-17% 到71-100% 的植入。显著地, 该方法通过不同模型和小鼠菌株增强肺肿瘤发生率和后续生长。此外, 嫁接肺癌细胞从肺部传播到相关的远端器官。因此, 我们提供了一个协议, 可用于建立和维持新的肺癌原位模型, 限制数量的细胞或 biospecimen, 并定量评估肺癌细胞在生理相关设置的癌变能力.

为了提高 LUAD 癌细胞植入到小鼠肺中的效率, 我们制定了一项协议, 首先使用博莱霉素的呼吸道, 其次是原位肿瘤细胞注射 (图 1)。我们证实, 即使在免疫裸小鼠, 博莱霉素诱导瞬变纤维化14天, 证明了气道结构的损失和增加胶原沉积 (图 2)。这些小鼠的毛纤维化在博莱霉素注射液后50天解决 (图 2; 右面板) 与以前的研究一致24。
根据这些观察, 我们嫁接的 LUAD 细胞不同的起源到肺部的各种小鼠菌株, 已预先条件与单一剂量的车辆或博莱霉素前14天。例如, 从已建立的人类 LUAD 细胞系 (H2030) 中悬浮 LUAD 细胞, intratracheally 到裸小鼠的肺部。35天后, 用博莱霉素预处理的小鼠有很高的肺肿瘤负担, 如生物发光检测 (图 3A)。相反, 在经过车辆处理的动物中, 没有发现同一时间框架内的肿瘤 (图 3A)。尸检后组织学证实肺肿瘤负担。用车辆预处理的肺部没有肿瘤结节的证据, 而博莱霉素预处理的肺部有大的肿瘤结节 (图 3B)。使用该协议, 我们还增加了另一个人类 LUAD 细胞线植入裸小鼠 (PC9;图 3C)和一个小鼠 LUAD 细胞系, 被注入自体免疫 B6129SF1/J 鼠 (图 3D)。博莱霉素预处理自体小鼠早期病变主要为1和2级, 分化良好, 类似于KrasG12D/+的原位肿瘤;p53-/- GEMMs33。所有嫁接 LUADs 生长在近端和远端肺 (图 3B,图 3E,图 3G; 星号 (远端), 星 (近端))。从肺癌 biospecimens (从活检或外科切除) 中建立 PDXs 是相对无效的, 即使人类细胞被皮移植到严重 immunodeficient 的动物, 如点头免疫伽玛 (核供应核)。鼠标应变34。为了评估我们的方法在 PDXs 中的潜在应用, 我们还注射了 H2030 细胞系的高度转移细胞亚群, 称为 H2030-BrM3 细胞35, intratracheally 在核型核型小鼠, 已预先适应车辆或博莱 霉 素。我们注意到, 核供应的小鼠可能更容易受到博莱霉素的毒性效应 (未显示数据), 并且在使用这种菌株时, 每只老鼠的剂量低于0.02 单位是可取的。然而, 我们的议定书还大大增加了核供应国 (图 3F,图 3G) 肺中的植入和肿瘤负担。
接下来, 我们使用这个协议来测试裸小鼠的 H2030 细胞系限制细胞数量的植入率。用博莱霉素治疗的小鼠注射 5 x 105, 5 x 104, 或 5 x 103 H2030 细胞。85.7-100% 小鼠在7和10星期之间开发了肺肿瘤在所有稀释测试了 (桌 2)。我们的结论是, 如果小鼠与博莱霉素有预适应, 肺中肿瘤细胞数量相对较少的植入是可能的。总的来说, 我们的协议可以针对几株小鼠和各种 LUAD 细胞线模型, 以提高癌细胞的植入率从0-16.7% 到 71.4-100% (表 2)。
最后, 为了评价 LUAD 细胞的生长动力学和从博莱霉素预适应肺转移的传播, 我们用我们的协议描述了高度转移 H2030-BrM3 细胞的行为。在经车处理的小鼠原位植入后, 3 天注射后的生物发光观察肿瘤细胞的磨损, 此后或在终点 (35 天) 无法检测到肺部肿瘤生长 (图 4A,图 4B)。相反, 我们发现肺癌的小鼠在注射前7天的早期治疗后的肺肿瘤生长 (图 4A)。肿瘤扩张39-57 天后, 与高肺肿瘤负担相关的发病率和发光强度限制了传播疾病的分析 (未显示数据)。因此, 我们在尸检的远处器官的影像转移。在有肺肿瘤负担57天的动物中, 也可以在可变频率的脑、肝、肾、肾上腺和骨骼后肢等组织中检测到小的病变 (图 4C)。因此, 我们得出结论, 临床相关部位的自发传播疾病可以通过这种原位方法产生。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56650/56650fig1.jpg"> 图 1: 为肿瘤细胞植入的肺部预条件的方法.使用的协议示意图。肺损伤是通过注射博莱霉素 intratracheally 进行的。LUAD 细胞在14天后注入小鼠气管。小鼠随后被成像每周为发光监测肿瘤植入, 生长和远处转移。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56650/56650fig1large.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/56650/56650fig2.jpg"> 图 2: 博莱霉素导致瞬变肺纤维化和 ECM 重塑.三色和博莱霉素治疗小鼠14天后注射 (纤维化峰值) 和51天后注射 (纤维化分辨率) 在肿瘤细胞缺失的典型图像注射。刻度条 = 500 µm.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56650/56650fig2large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56650/56650fig3.jpg"> 图 3: 用博莱霉素对小鼠进行预处理后, 多小鼠模型 LUAD 细胞株的肺植入增加.A) 用车或博莱霉素治疗裸小鼠, 注射 5x105 H2030 细胞。根据生物发光的测定, 在终点 (35 天规范化为0天后) 的相对肺肿瘤负担。对于每个组, 显示一个肿瘤负荷中位数的小鼠。每组 n=6-7 小鼠。B) 从35天起, 用车辆或博莱霉素对肺部进行预治疗的代表性 H & E 图像。星号 = 远端;星 = 近端。C) 裸小鼠注射 1x105 PC9 细胞治疗。肿瘤负担和代表性图像的测量和显示为每组 n=7 小鼠。DE) 用车或博莱霉素治疗 B6129SF1/J 小鼠, 注射 1x105 368T1 KrasG12D/+ p53-/ (KP T 不符合) 细胞。肿瘤负担, 代表性图像和 H & Es 被测量和描述为 A 和 B. n = 每组7只老鼠。以 1x 105 H2030-BrM3 细胞为载体, 用车载或博莱霉素对其进行预处理。肿瘤负担, 代表性图像和 H & Es (9 天和10天后注射) 的测量和显示, 在 A 和 B. n=3-6 小鼠每组。缩放条 = 500 µm. 嵌入 = 肿瘤细胞嫁接区域的高放大倍数。缩放栏的插入 = 100 µm 值计算的曼-惠特尼测试。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56650/56650fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56650/56650fig4.jpg"> 图 4: LUAD 细胞嫁接在博莱霉素治疗小鼠转移到多个远端器官.A) 用车或博莱霉素治疗裸小鼠, 注射 5x105 H2030-BrM3 细胞。每周使用生物荧光测量肺肿瘤负担。显示0天的背总光子通量正常化。B) 肿瘤负担由肺的生物发光从一个终点 (35 天后注射)。对于每个组, 显示一个肿瘤负荷中位数的小鼠。每组 n=6-7 小鼠。C) 有代表性的图像的器官有可检测的转移从同一鼠标在尸检 (左)。表与指示的频率的小鼠转移在远端器官在终点 (天39到57后植入) (右)。在 1.5 x 104光子/(秒厘米2 steradian) 以上的视觉发光信号的前体器官被算作阳性转移.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56650/56650fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>鼠标应变</td>
			<td>单位/鼠标</td>
		</tr>
<tr>
<td>裸</td>
			<td>0.02</td>
		</tr>
<tr>
<td>Nsg</td>
			<td>0.02</td>
		</tr>
<tr>
<td>B6129SF1/J</td>
			<td>0.005</td>
		</tr>
</tbody></table>表 1:测试的小鼠菌株推荐剂量的博莱霉素的清单。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td rowspan="2">鼠标应变</td>
			<td rowspan="2">单元格线</td>
			<td rowspan="2">注入的细胞数</td>
			<td colspan="2">% 植入</td>
			<td rowspan="2">p值</td>
		</tr>
<tr>
<td>车辆</td>
			<td>博莱 霉 素</td>
		</tr>
<tr>
<td>裸</td>
			<td>H2030</td>
			<td>5x105
</td>
			<td>0% (0/7)</td>
			<td>100% (6/6)</td>
			<td>0.0006</td>
		</tr>
<tr>
<td>裸</td>
			<td>H2030</td>
			<td>5x104
</td>
			<td>北达科他州</td>
			<td>85.7% (6/7)</td>
			<td>戒毒</td>
		</tr>
<tr>
<td>裸</td>
			<td>H2030</td>
			<td>5x103
</td>
			<td>北达科他州</td>
			<td>85.7% (6/7)</td>
			<td>戒毒</td>
		</tr>
<tr>
<td>裸</td>
			<td>PC-9</td>
			<td>1x105
</td>
			<td>0% (0/7)</td>
			<td>85.7% (6/7)</td>
			<td>0.0023</td>
		</tr>
<tr>
<td>Nsg</td>
			<td>H2030-BrM3</td>
			<td>1x105
</td>
			<td>16.7% (1/6)</td>
			<td>100% (3/3) *</td>
			<td>0.0476</td>
		</tr>
<tr>
<td>B6129SF1/J</td>
			<td>368T1</td>
			<td>1x105
</td>
			<td>0% (0/7)</td>
			<td>71.4% (5/7) *</td>
			<td>0.0105</td>
		</tr>
</tbody></table>表 2:植入频率 (%) 的摘要表, 使用指定的鼠标株、细胞线和细胞数.植入是由体内发光成像确定的, 经体外影像证实, 注射后37至50天的组织组织学. * 阳性植入在10天注射后的体内发光证实. p值由单侧费舍尔的精确测试计算。不适用;北达科他州 = 未确定。

Watch this Scientific Journal Video about Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment at JoVE.com

Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

本文介绍一种通过对气道损伤进行预适应来显著提高肺癌细胞原位植入的方法。这种方法也可以应用于研究肺微环境中的间质相互作用, 转移传播, 肺癌的共同发病, 并更有效地产生病人的移植。

博莱霉素对小鼠气管的预处理提高原位肺癌细胞植入的效率

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of ...

pre-conditioning-airways-mice-with-bleomycin-increases-efficiency

Nanyang Technological University

Research

JoVE Journal

Medicine

13.5K 次观看.  Yale University School of Medicine. 本文介绍一种通过对气道损伤进行预适应来显著提高肺癌细胞原位植入的方法。这种方法也可以应用于研究肺微环境中的间质相互作用, 转移传播, 肺癌的共同发病, 并更有效地产生病人的移植。

视频: Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

闭塞性细支气管炎在原位肺移植小鼠模型的发展

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

科研

医学

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

无创性气管内插管，以研究小鼠肺的病理生理

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure.
The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.

We describe a non-invasive multimodal imaging approach based on Micro-CT and fluorescence molecular tomography for longitudinal assessment of the mouse lung fibrosis model induced by double intratracheal instillation of bleomycin.

基于显微 CT 和荧光分子层析成像的多模态成像方法对博莱霉素诱导的小鼠肺纤维化的纵向评价

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which ...

免疫与感染

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

在小鼠气管插管快速的改进方法

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

小鼠肺损伤的诱导,通过博霉素内切注射

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

小鼠骨髓移植程序研究克隆造血症

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced pulmonary fibrosis.&#160; However, distribution of intratracheally-administered agents to the distal mouse lung is often asymmetric, with lung parenchymal concentrations increased in the smaller (but equally accessible) left lung of the mouse.&#160; Described in this report is a novel intrabronchial (IB) approach to cannulate the left and/or right lungs of living mice non-operatively.&#160; It is also demonstrated how this approach can be used to selectively administer agents to one lung or adapted (via dose-adjusted IB delivery) to improve the left-right symmetry of lung delivery of experimental agents, thereby improving models of diffuse lung disease such as bleomycin-induced pulmonary fibrosis.

Intratracheal (IT) administration of experimental agents in mice often results in asymmetric delivery to the distal lungs.&#160; In this report, we describe a direct intrabronchial (IB) approach to cannulate each lung in living mice non-operatively.&#160; This approach can be used to selectively administer agents to one lung or may be adapted to improve the symmetric agent delivery to both lungs.

直接注射内管理,改善小鼠肺内的选择性代理沉积

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced ...

Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo

The inflammatory response caused by early lung injury is one of the important causes of the development of idiopathic pulmonary fibrosis (IPF), which is accompanied by the activation of inflammatory cells such as macrophages and neutrophils, as well as the release of inflammatory factors including TNF-&#945;, IL-1&#946;, and IL-6. Early inflammation caused by activated pulmonary interstitial macrophages (IMs) in response to IL-33 stimulation is known to play a vital role in the pathological process of IPF. This protocol describes the adoptive transfer of IMs stimulated by IL-33 into the lungs of mice to study IPF development. It involves the isolation and culture of primary IMs from host mouse lungs, followed by the adoptive transfer of stimulated IMs into the alveoli of bleomycin (BLM)-induced IPF recipient mice (which have been previously depleted of alveolar macrophages by treatment with clodronate liposomes), and the pathological evaluation of those mice. The representative results show that the adoptive transfer of IL-33-stimulated macrophages aggravates pulmonary fibrosis in mice, suggesting that the establishment of the macrophage adoptive transfer experiment is a good technical means to study IPF pathology.

Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo

This protocol describes the isolation of pulmonary interstitial macrophages (IMs) and their adoptive transfer after IL-33 stimulation of the lung alveoli in a mouse model, which can facilitate the in vivo study of idiopathic pulmonary fibrosis (IPF).

将IL-33刺激的巨噬细胞过继转移到博来霉素诱导的小鼠模型中，以研究其对体内特发性肺纤维化的影响

The inflammatory response caused by early lung injury is one of the important causes of the development of idiopathic pulmonary fibrosis (IPF), which is ...

A Mouse Model of Pulmonary Fibrosis Induced by Nasal Bleomycin Nebulization

Pulmonary fibrosis is characteristic of several human lung diseases that arise from various causes. Given that treatment options are fairly limited, mouse models continue to be an important tool for developing new anti-fibrotic strategies. In this study, intrapulmonary administration of bleomycin (BLM) is carried out by nasal nebulization to create a mouse model of pulmonary fibrosis that closely mimics clinical disease characteristics. C57BL/6 mice received BLM (7 U/mL, 30 min/day) by nasal nebulization for 3 consecutive days and were sacrificed on day 9, 16, or 23 to observe inflammatory and fibrotic changes in lung tissue. Nasal aerosolized BLM directly targeted the lungs, resulting in widespread and uniform lung inflammation and fibrosis. Thus, we successfully generated an experimental mouse model of typical human pulmonary fibrosis. This method could easily be used to study the effects of the administration of various nasal aerosols on lung pathophysiology and validate new anti-inflammatory and anti-fibrotic treatments.

<meta charset="utf8"></head><body>鼻腔博来霉素雾化诱导的肺纤维化小鼠模型

Various animal models of pulmonary fibrosis have been established using bleomycin to clarify the pathogenesis of pulmonary fibrosis and find new drug targets. However, most pulmonary fibrosis models targeting lung tissue have uneven drug administration. Here, we propose a model of uniform pulmonary fibrosis induced by nasal bleomycin nebulization.

鼻腔博来霉素雾化诱导的肺纤维化小鼠模型

Pulmonary fibrosis is characteristic of several human lung diseases that arise from various causes. Given that treatment options are fairly limited, mouse ...

博莱霉素对小鼠气管的预处理提高原位肺癌细胞植入的效率

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闭塞性细支气管炎在原位肺移植小鼠模型的发展

无创性气管内插管，以研究小鼠肺的病理生理

基于显微 CT 和荧光分子层析成像的多模态成像方法对博莱霉素诱导的小鼠肺纤维化的纵向评价

在小鼠气管插管快速的改进方法

小鼠肺损伤的诱导,通过博霉素内切注射

小鼠骨髓移植程序研究克隆造血症

直接注射内管理,改善小鼠肺内的选择性代理沉积

将IL-33刺激的巨噬细胞过继转移到博来霉素诱导的小鼠模型中，以研究其对体内特发性肺纤维化的影响

鼻腔博来霉素雾化诱导的肺纤维化小鼠模型

鼻腔博来霉素雾化诱导的肺纤维化小鼠模型