我们感谢加州大学洛杉矶分校的克伦普临床前成像技术中心, 帮助他们的宠物/CT 影像的小鼠, 翻译病理核心实验室和统计核心在加利福尼亚大学洛杉矶 '大卫. 格芬医学院为他们的协助与肿瘤标本的制备和分析。为供资, 大卫 b. 沙克尔福德得到了 CTSI 和 KL2 翻译科学奖赠款号码 KL2TR000122 和 UL1TR000124 在加州大学洛杉矶分校医学院和国防部肺癌研究计划的翻译研究伙伴关系 W81XWH-13-1-0459 和 ACS RSG-16-234-01-TBG。肖恩 t. 贝利得到了 NIH T32 训练补助金 HL072752 通过加州大学洛杉矶分校医学院。安东尼. 琼斯得到了加州大学洛杉矶分校肿瘤细胞生物学训练计划 (USHHS Kirschstein 机构国家研究服务奖 T32 CA009056) 的支持。Gihad Abdelhady 由 NIH/癌症多样性补充 R01CA208642 支持。

《议定书》中所述的所有程序都已由洛杉矶加利福尼亚大学的机构动物护理和使用委员会 (IACUC) 批准。
1. 小鼠18的 F 葡萄糖 PET 和 CT 成像
注意事项: 在处理放射性物质时使用防护设备。在处理放射性物质时遵循所有适用的监管程序。
<ol><li>在18F 葡萄糖注射液之前, 将笼子与老鼠放在温暖的床上, 37 °c 1 小时, 以减少18葡萄糖的褐色脂肪消耗。 注: 禁食 4-16 小时的小鼠可以帮助减少18葡萄糖的心肌消耗。</li>
	<li>称量鼠标并记录它的重量。</li>
	<li>麻醉老鼠使用 2-3% 异氟醚在氧气 0.5-2 升/分 2-3 分钟使用麻醉室保持在37°c。通过捏脚趾确保老鼠被麻醉;如果老鼠被麻醉, 就不会有反应。将眼膏涂抹于眼部, 以防麻醉时有任何干燥。</li>
	<li>稀释18F 葡萄糖 (109 分钟放射性半衰期) 在未育盐水在被调整的朽烂校正的注射集中 70-75 µCi/100 µL。 注: 按照 PET 扫描仪制造商推荐的18F 剂量进行最佳扫描仪成像。</li>
	<li>用一根28克针的胰岛素注射器绘制 70-75 µCi, 用剂量校验仪测量放射性剂量, 并记录测量和时间。将注射器放在注射器支架上。 注: 根据制造商的协议, 每种剂量的18F 葡萄糖的放射性量是用剂量校验仪测量的, 该校准器按照标准参考材料 (如 cesium-137) 进行校准。读取的时间也记录下来, 以确定衰变校正。</li>
	<li>刺到鼠标尾部的远端, 用嘉测量老鼠的血糖。</li>
	<li>用温水浸泡的纱布将尾巴加热 1-2 分钟。用70% 异丙醇擦拭尾部, 在注射前扩张尾静脉。管理100µL 18F 葡萄糖 (整个体积在注射器) 与丸注射通过侧尾静脉和记录注射时间。用剂量校验仪测量注射器中的剩余剂量, 记录测量和时间。 注: 注射器中会留有一定量的探针。使用胰岛素注射器是首选的注射器连接到针通过鲁尔锁, 由于减少的剂量被困在注射器/针后注射已被管理。</li>
	<li>将注射的老鼠放在 1.5-2% 异氟醚37摄氏度以下的麻醉室中, 使探针在 PET 扫描之前通过鼠标的系统循环分布在1小时内。 注: 在扫描前使膀胱无效可能是有益的, 以便能更容易地在小鼠的下侧翼植入肿瘤的18F 葡萄糖 pet 可视化。</li>
	<li>1小时后, 将鼠标放置在鼻锥异氟醚麻醉下的成像腔内, 并在37摄氏度处, 将其四肢固定在仰卧位的医用胶带上。</li>
	<li>将成像室放在 PET/CT 扫描仪中。</li>
	<li>获取 pet 和 ct 扫描, 如 pet/ct 扫描仪手册28所述。 注: PET 图像是为600s 获得 150-650 凯文的能量窗口, 重建使用最大似然期望最大化与修正的光子衰减, 探测器正常化和放射性同位素衰变 (散射校正是未应用)。五十年代使用 50 kVp、200µA X 射线源和平板探测器, 以连续模式获取 CT 图像, 并使用 Feldkamp 算法进行重建。</li>
	<li>在 PET/CT 完成后, 从成像室取出鼠标, 并允许它在笼子里恢复。监视鼠标直到它完全恢复知觉并能维持胸骨卧床。</li>
	<li>通过单击 "文件", 然后打开"", 然后选择相应的文件, 将重建的 PET/CT 图像导入到酰胺软件中。</li>
	<li>将 PET 数据转换为每克注射剂量的单位 (%ID/克), 方法是在对注射器留下的剩余剂量进行核算后, 或通过进入该主体的标准摄取值 (SUV) 的单位, 在注射时输入剂量。重量。为此, 请右键单击宠物数据集, 然后在 "基本信息" 选项卡上找到%ID/g 字段. 输入先前记录的%ID/g。</li>
	<li>绘制肿瘤和正常组织 (肝脏、肌肉、肺、心脏、脑和皮下脂肪) 的感兴趣区域 (ROIs)。为此, 请单击 "编辑", 选择 "添加 roi", 选择 " roi" 形状, 并为 roi 提供一个名称。画 ROIs 在肿瘤和组织, 并调整其尺寸, 以覆盖所有3轴感兴趣的组织。 注意: 为了解释在宠物探针体内动物之间的差异, 肿瘤 roi 值可以进一步归一化到肝脏的 roi 值, 一个很好的灌注器官和最小的酵活动, 代表18F 葡萄糖在循环。对同一小鼠进行肿瘤和正常组织的 ROI 分析。肺肿瘤病变通常由18f-葡萄糖 PET 识别, 因为18f 葡萄糖保留在正常肺是相对较低的。CT 也用于鉴别病灶, 特别是18的非狂热的病变。对孤立肺部的体外活体分析也有助于肿瘤病灶的定位。</li>
</ol>2. 18F 葡萄糖显影
<ol><li>通过以下步骤 1.1-1.12, 将鼠标准备成像与18F 葡萄糖, 除了现在, 稀释18氟葡萄糖在无菌盐水中, 在调整衰变校正注射浓度1000µCi/200 µL。 注: 更高剂量的18F 葡萄糖用于显影, 以考虑到额外的样品处理时间和最佳的磷板检测。</li>
	<li>弄死老鼠通过致命的吸入异氟醚在5% 或由 CO2 (IACUC 批准的程序)。 注: 不应使用颈椎脱位, 因为这会损害肺组织。</li>
	<li>将鼠标钉在腹面上, 然后用70% 乙醇喷出, 然后在切口前把它涂在头发上。</li>
	<li>应用中线切口切开胸腔, 切开隔膜, 取出胸腔壁。小心切除涎腺来暴露气管。将牛头犬钳放在气管上, 尽可能靠近下颚, 确保气管紧固。将23克针连接到3毫升注射器内的气管内的牛头犬钳和注射〜2毫升的 OCT: PBS (1:1) (最佳切割温度: 磷酸盐缓冲盐水) 解决方案。 注意: OCT 是非常粘性的, 并与 PBS 混合, 以允许更容易注射到肺部。</li>
	<li>从气管中取出针头, 用镊子钳住注射点, 防止 OCT 的任何泄漏: PBS 解决方案。</li>
	<li>小心地从胸腔取出肺部, 并将左叶与肺的其余部分分开。将左叶放置在标记的 cryomold 中, 填充几滴 OCT。一旦肺叶在模具内, 用 OCT 填充 cryomold 顶端。</li>
	<li>用右半边肺重复同样的步骤。 注: 如果心脏中的18f-葡萄糖信号是高的, 或者肺肿瘤位于心脏附近, 那么去除心脏可能是有益的, 以防止任何示踪物泄漏。或者, 整个肺部可以嵌入一个单一的 cryomold。在使用 OCT 时, 避免气泡是很重要的。</li>
	<li>使用长钳, 将准备好的 cryomold 放在含有干冰和异戊烷混合物的密闭细胞挤出聚苯乙烯泡沫容器中。 注: 此混合物应在大约-70 °c 之前放置 cryomold 在它冻结。硬化后, OCT 化合物将变为白色。如果同时处理多个样品, cryomolds OCT 中的冷冻样品可以暂时存放在干冰上。</li>
	<li>从 cryomold 中移除冻结块, 然后将其装入到恒温器进行切片。用切片刀片 (34°/80 mm, 高剖面) 将块以4µm 的厚度分段。将组织部分转移到已储存在室温下的玻璃滑梯上。</li>
	<li>将试样幻灯片放到荧光粉成像板上。把盘子放在卡带里, 轻轻地关上它, 防止幻灯片移动。将卡带存放在-20 摄氏度的冰箱中, 用于平板曝光, 一般一夜之间。 注: 在使用前, 盘子和卡带需要预冷至-20 摄氏度。将样品放置在摄氏-80 摄氏度也可以接受。</li>
	<li>曝光后, 从盘子中取出幻灯片, 阅读图像阅读器上的印版。</li>
	<li>这些幻灯片可以包装在塑料包装和存储在-80 °c, 或者他们可以准备为苏木精 & 伊红染色或免疫组化。</li>
</ol>3. 为组织学采集肺组织
<ol><li>按照步骤 2.2-2.4, 除了现在, 而不是使用 OCT: PBS 解决方案, 注入 2-3 毫升10% 正常缓冲福尔马林修复肺部。</li>
	<li>从气管中取出针头, 用镊子钳住注射点, 以防甲醛泄漏。小心地将肺部从胸腔取出, 放入50毫升圆锥管, 其中20毫升10% 正常缓冲福尔马林, 用于 16-24 h, 以确保完整的固定。 注: 修复肺部可保留最佳解剖特征。</li>
	<li>第二天, 将固定肺部从福尔马林转移到70% 乙醇, 并准备将肺部放置在纸巾盒中。</li>
	<li>用剪刀仔细解剖裂片, 在5裂片之间的分枝处切开, 1-5, 如图 1D所示, 并将其放置在组织盒中的非重叠方向上, 准备肺进行组织学。将泡沫垫轻轻放在肺组织上, 保持方向完好。</li>
	<li>将经解剖的肺裂片贮存在70% 乙醇中, 直至石蜡嵌入。</li>
	<li>石蜡-嵌入盒内的组织和削减4µm 厚切片染色, 使用标准程序。</li>
</ol>4. 使用商用软件进行组织分割和量化
<ol><li>图像苏木精和伊红 (H & E) 使用商业多光谱成像系统在1.25X 倍的放大染色肺部部分。</li>
	<li>将图像转换为数字图像立方体并加载 (单击文件, 然后在负载光谱库中) 预制作的用于 H & E 的光谱库。 注: 光谱库是通过从单个染色的肺切片获取光谱图像, 在一个开放源码的专有光谱库中保存的, 仅有一条染有苏木素, 另一部分仅染色的。文件 (. csl)。成像系统有操作软件在每个波长获取一个图像 (由承购协议定义)。这些图像 ("图像立方体") 存储在开源的专有多光谱文件格式 (. im3) 中。利用形态学软件从图像立方体中提取光谱, 并将其存储在不同的光谱库文件中。</li>
	<li>通过点击unmix按钮, 光谱 unmix 整个肺部的伪彩色 H & E 图像。 注: 分解不到1秒。利用形态学图像分析软件对各组织类型的像素数量进行量化。</li>
	<li>通过将多光谱数据转换为等效的3色 (红色、绿色、蓝色) 图像, 通过使用眼睛的波长依赖颜色响应卷积混合与每个图像的强度来可视化每个图像立方体。 注意: 结果3图像显示为标准的24位彩色图像。</li>
	<li>通过在用户选择的颜色 (如红色、绿色、紫色等) 中缩放图像, 并将其他伪彩色纯图像添加到一个标准的24位彩色图像中, 伪彩色图像。</li>
	<li>使用所有分析的默认设置。 注意: 一般而言, 由于这种分割依赖于对结果的视觉评估 (例如, 评估在训练集之外的图像的分割效果如何), 因此将图像正确划分为训练是很重要的,测试和验证集。</li>
	<li>开始与 2-3 图像作为训练集 (10-15 的人体活检样本), 培训, 直到结果看起来不错, 然后将该算法应用到另一个 2-3 图像。然后, 将生成的算法应用于完整的验证集。 注: 很可能需要进行一些再培训。</li>
	<li>通过计算每只小鼠裂片 1-5 的红色伪彩色肿瘤的总像素计数, 分析全肺切片中的肿瘤面积。 注: 正常组织为伪色绿色, 血/血管呈伪色粉红色, 如图 3所示。每个治疗组的平均肿瘤负担是通过测量治疗组中每只老鼠的总像素计数来计算的。</li>
</ol>

<ol><li>Shackelford, D. B., Shaw, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+LKB1-AMPK+pathway%3A+metabolism+and+growth+control+in+tumour+suppression%5BTitle%5D)+AND+%22Nature+Reviews+Cancer%22%5BJournal%5D)">The LKB1-AMPK pathway: metabolism and growth control in tumour suppression.</a> Nature Reviews Cancer. 9 (8), 563-575 (2009).</li>
<li>Shaw, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+LKB1+tumor+suppressor+negatively+regulates+mTOR+signaling%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">The LKB1 tumor suppressor negatively regulates mTOR signaling.</a> Cancer Cell. 6 (1), 91-99 (2004).</li>
<li>Shackelford, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((mTOR+and+HIF-1alpha-mediated+tumor+metabolism+in+an+LKB1+mouse+model+of+Peutz-Jeghers+syndrome%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">mTOR and HIF-1alpha-mediated tumor metabolism in an LKB1 mouse model of Peutz-Jeghers syndrome.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (27), 11137-11142 (2009).</li>
<li>Faubert, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Loss+of+the+tumor+suppressor+LKB1+promotes+metabolic+reprogramming+of+cancer+cells+via+HIF-1alpha%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Loss of the tumor suppressor LKB1 promotes metabolic reprogramming of cancer cells via HIF-1alpha.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (7), 2554-2559 (2014).</li>
<li>Hemminki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+molecular+basis+and+clinical+aspects+of+Peutz-Jeghers+syndrome%5BTitle%5D)+AND+%22Cellular+and+Molecular+Life+Sciences%22%5BJournal%5D)">The molecular basis and clinical aspects of Peutz-Jeghers syndrome.</a> Cellular and Molecular Life Sciences. 55, 735-750 (1999).</li>
<li>Hemminki, A., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/9428765">A serine/threonine kinase gene defective in Peutz-Jeghers syndrome.</a> Nature. 391 (6663), 184-187 (1998).</li>
<li>Sanchez-Cespedes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+role+for+LKB1+gene+in+human+cancer+beyond+the+Peutz-Jeghers+syndrome%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">A role for LKB1 gene in human cancer beyond the Peutz-Jeghers syndrome.</a> Oncogene. 26 (57), 7825-7832 (2007).</li>
<li>Sanchez-Cespedes, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inactivation+of+LKB1%2FSTK11+is+a+common+event+in+adenocarcinomas+of+the+lung%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Inactivation of LKB1/STK11 is a common event in adenocarcinomas of the lung.</a> Cancer Research. 62 (13), 3659-3662 (2002).</li>
<li>Ding, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Somatic+mutations+affect+key+pathways+in+lung+adenocarcinoma%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Somatic mutations affect key pathways in lung adenocarcinoma.</a> Nature. 455 (7216), 1069-1075 (2008).</li>
<li>Ylikorkala, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vascular+abnormalities+and+deregulation+of+VEGF+in+Lkb1-deficient+mice%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Vascular abnormalities and deregulation of VEGF in Lkb1-deficient mice.</a> Science. 293 (5533), 1323-1326 (2001).</li>
<li>Bardeesy, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Loss+of+the+Lkb1+tumour+suppressor+provokes+intestinal+polyposis+but+resistance+to+transformation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Loss of the Lkb1 tumour suppressor provokes intestinal polyposis but resistance to transformation.</a> Nature. 419 (6903), 162-167 (2002).</li>
<li>Miyoshi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gastrointestinal+hamartomatous+polyposis+in+Lkb1+heterozygous+knockout+mice%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Gastrointestinal hamartomatous polyposis in Lkb1 heterozygous knockout mice.</a> Cancer Research. 62 (8), 2261-2266 (2002).</li>
<li>Jishage, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+Lkb1%2C+the+causative+gene+of+Peutz-Jegher%27s+syndrome%2C+in+embryogenesis+and+polyposis%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Role of Lkb1, the causative gene of Peutz-Jegher's syndrome, in embryogenesis and polyposis.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (13), 8903-8908 (2002).</li>
<li>Shackelford, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unravelling+the+connection+between+metabolism+and+tumorigenesis+through+studies+of+the+liver+kinase+B1+tumour+suppressor%5BTitle%5D)+AND+%22Journal+of+Carcinogenesis%22%5BJournal%5D)">Unravelling the connection between metabolism and tumorigenesis through studies of the liver kinase B1 tumour suppressor.</a> Journal of Carcinogenesis. 12, 16(2013).</li>
<li>Jackson, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+lung+tumor+initiation+and+progression+using+conditional+expression+of+oncogenic+K-ras%5BTitle%5D)+AND+%22Genes+%26+Development%22%5BJournal%5D)">Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras.</a> Genes & Development. 15 (24), 3243-3248 (2001).</li>
<li>Ji, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LKB1+modulates+lung+cancer+differentiation+and+metastasis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">LKB1 modulates lung cancer differentiation and metastasis.</a> Nature. 448 (7155), 807-810 (2007).</li>
<li>Momcilovic, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heightening+energetic+stress+selectively+targets+LKB1-deficient+non-small+cell+lung+cancers%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Heightening energetic stress selectively targets LKB1-deficient non-small cell lung cancers.</a> Cancer Research. 75 (22), 4910-4922 (2015).</li>
<li>Wislez, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+mammalian+target+of+rapamycin+reverses+alveolar+epithelial+neoplasia+induced+by+oncogenic+K-ras%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Inhibition of mammalian target of rapamycin reverses alveolar epithelial neoplasia induced by oncogenic K-ras.</a> Cancer Research. 65 (8), 3226-3235 (2005).</li>
<li>Liang, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TSC1+loss+synergizes+with+KRAS+activation+in+lung+cancer+development+in+the+mouse+and+confers+rapamycin+sensitivity%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">TSC1 loss synergizes with KRAS activation in lung cancer development in the mouse and confers rapamycin sensitivity.</a> Oncogene. 29 (11), 1588-1597 (2010).</li>
<li>Hudes, G., et al. <a target="_blank" href="http://www.nejm.org/doi/full/10.1056/NEJMoa066838">Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma.</a> The New England Journal of Medicine. 356 (22), 2271-2281 (2007).</li>
<li>Wander, S. A., Hennessy, B. T., Slingerland, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Next-generation+mTOR+inhibitors+in+clinical+oncology%3A+how+pathway+complexity+informs+therapeutic+strategy%5BTitle%5D)+AND+%22The+Journal+of+Clinical+Investigation%22%5BJournal%5D)">Next-generation mTOR inhibitors in clinical oncology: how pathway complexity informs therapeutic strategy.</a> The Journal of Clinical Investigation. 121 (4), 1231-1241 (2011).</li>
<li>Pourdehnad, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myc+and+mTOR+converge+on+a+common+node+in+protein+synthesis+control+that+confers+synthetic+lethality+in+Myc-driven+cancers%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Myc and mTOR converge on a common node in protein synthesis control that confers synthetic lethality in Myc-driven cancers.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (29), 11988-11993 (2013).</li>
<li>Hsieh, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+translational+landscape+of+mTOR+signalling+steers+cancer+initiation+and+metastasis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The translational landscape of mTOR signalling steers cancer initiation and metastasis.</a> Nature. 485 (7396), 55-61 (2012).</li>
<li>Momcilovic, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heightening+energetic+stress+selectively+targets+LKB1-deficient+non-small+cell+lung+cancers%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Heightening energetic stress selectively targets LKB1-deficient non-small cell lung cancers.</a> Cancer Research. 75 (22), 4910-4922 (2015).</li>
<li>Frese, K. K., Tuveson, D. A. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/17687385">Maximizing mouse cancer models.</a> Nature Reviews Cancer. 7 (9), 645-658 (2007).</li>
<li>Shackelford, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LKB1+inactivation+dictates+therapeutic+response+of+non-small+cell+lung+cancer+to+the+metabolism+drug+phenformin%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">LKB1 inactivation dictates therapeutic response of non-small cell lung cancer to the metabolism drug phenformin.</a> Cancer Cell. 23 (2), 143-158 (2013).</li>
<li>Momcilovic, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+Inhibition+of+EGFR+and+Glutaminase+Induces+Metabolic+Crisis+in+EGFR+Mutant+Lung+Cancer%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Targeted Inhibition of EGFR and Glutaminase Induces Metabolic Crisis in EGFR Mutant Lung Cancer.</a> Cell Reports. 18 (3), 601-610 (2017).</li>
<li>Preclinical PET Imaging Systems | GA PET/X-ray & G8 PET/CT. Perkin Elmer. , Available from: 
 <a target="_blank" href="http://www.perkinelmer.com/preclinical-pet-imaging/index.html">http://www.perkinelmer.com/preclinical-pet-imaging/index.html</a> (2018).</li>
<li>Goodwin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+distinct+metabolic+phenotype+of+lung+squamous+cell+carcinoma+defines+selective+vulnerability+to+glycolytic+inhibition%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">The distinct metabolic phenotype of lung squamous cell carcinoma defines selective vulnerability to glycolytic inhibition.</a> Nature Communications. 8, 15503(2017).</li>
<li>Fueger, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+animal+handling+on+the+results+of+18F-FDG+PET+studies+in+mice%5BTitle%5D)+AND+%22Journal+of+Nuclear+Medicine%22%5BJournal%5D)">Impact of animal handling on the results of 18F-FDG PET studies in mice.</a> Journal of Nuclear Medicine. 47 (6), 999-1006 (2006).</li>
<li>Dandekar, M., Tseng, J. R., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reproducibility+of+18F-FDG+microPET+studies+in+mouse+tumor+xenografts%5BTitle%5D)+AND+%22Journal+of+Nuclear+Medicine%22%5BJournal%5D)">Reproducibility of 18F-FDG microPET studies in mouse tumor xenografts.</a> Journal of Nuclear Medicine. 48 (4), 602-607 (2007).</li>
<li>Luker, G. D., Luker, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+imaging%3A+current+applications+and+future+directions%5BTitle%5D)+AND+%22Journal+of+Nuclear+Medicine%22%5BJournal%5D)">Optical imaging: current applications and future directions.</a> Journal of Nuclear Medicine. 49 (1), 1-4 (2008).</li>
<li>Vander Heiden, M. G., Cantley, L. C., Thompson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Understanding+the+Warburg+effect%3A+the+metabolic+requirements+of+cell+proliferation%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Understanding the Warburg effect: the metabolic requirements of cell proliferation.</a> Science. 324 (5930), 1029-1033 (2009).</li>
<li>Vander Heiden, M. G., DeBerardinis, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Understanding+the+intersections+between+metabolism+and+cancer+biology%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Understanding the intersections between metabolism and cancer biology.</a> Cell. 168 (4), 657-669 (2017).</li>
<li>Zhang, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glycine+decarboxylase+activity+drives+non-small+cell+lung+cancer+tumor-initiating+cells+and+tumorigenesis%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Glycine decarboxylase activity drives non-small cell lung cancer tumor-initiating cells and tumorigenesis.</a> Cell. 148 (1-2), 259-272 (2012).</li>
<li>Possemato, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+genomics+reveal+that+the+serine+synthesis+pathway+is+essential+in+breast+cancer%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Functional genomics reveal that the serine synthesis pathway is essential in breast cancer.</a> Nature. 476, 346-350 (2011).</li>
<li>Sullivan, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Supporting+aspartate+biosynthesis+is+an+essential+function+of+respiration+in+proliferating+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Supporting aspartate biosynthesis is an essential function of respiration in proliferating cells.</a> Cell. 162 (7360), 552-563 (2015).</li>
<li>Hassanein, M., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/25971659">Preclinical evaluation of 4-[(18)F]fluoroglutamine PET to assess ASCT2 expression in lung cancer.</a> Molecular Imaging and Biology. 18 (1), 18-23 (2016).</li>
<li>Qu, W., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/22173839">Preparation and characterization of L-[5-11C]-glutamine for metabolic imaging of tumors.</a> Journal of Nuclear Medicine. 53 (1), 98-105 (2012).</li>
<li>Venneti, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glutamine-based+PET+imaging+facilitates+enhanced+metabolic+evaluation+of+gliomas+in+vivo%5BTitle%5D)+AND+%22Science+Translational+Medicine%22%5BJournal%5D)">Glutamine-based PET imaging facilitates enhanced metabolic evaluation of gliomas in vivo.</a> Science Translational Medicine. 7 (274), 217(2015).</li>
<li>Gambhir, S. S. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/12209157">Molecular imaging of cancer with positron emission tomography.</a> Nature Reviews Cancer. 2 (9), 683-693 (2002).</li>
<li>Keng, P. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micro-chemical+synthesis+of+molecular+probes+on+an+electronic+microfluidic+device%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Micro-chemical synthesis of molecular probes on an electronic microfluidic device.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (3), 690-695 (2012).</li>
<li>Lazari, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ELIXYS+-+a+fully+automated%2C+three-reactor+high-pressure+radiosynthesizer+for+development+and+routine+production+of+diverse+PET+tracers%5BTitle%5D)+AND+%22EJNMMI+Research%22%5BJournal%5D)">ELIXYS - a fully automated, three-reactor high-pressure radiosynthesizer for development and routine production of diverse PET tracers.</a> EJNMMI Research. 3 (1), 52(2013).</li>
</ol>

凯文. 弗朗西斯是珀的雇员。詹姆斯. 曼斯菲尔德是纳斯达克 PerkinElmer (PKI) 股份的公开股东。作者没有其他可以透露的东西。

本文描述了一种基于成像的实验方法, 利用18的 qIHC PET/CT 成像与 mTOR, 以测量在肺肿瘤的代谢和分子反应后, 交付的 MLN0128。MLN0128 有效地降低了18的葡萄糖摄入量, 表明肿瘤有很大的代谢反应。通过将 pet/ct 成像与免疫组化相结合, 我们能够在3D 的 pet/ct 图像上对切片肿瘤进行空间记录, 并对细胞和分子水平的整个肿瘤进行详细检查。这使得有可能证实 MLN0128 抑制 mTOR 信号, 从而确认对肿瘤药物的靶向分子反应。最后, 利用定量组织学, 我们能够映射和分离不同的肿瘤病理, 如肿瘤的整体肿瘤肿块, 从鳞状细胞癌定义腺癌, 并补充 microPET 成像。
MicroPET 目前受空间分辨率的限制, 约为1毫米。此外, 在某些组织中的18F 葡萄糖保留可能受各种因素的影响, 包括血糖水平, 麻醉暴露的类型和持续时间, 环境温度和动物的一般健康, 这可能影响18葡萄糖药代动力学30。这些参数已针对该协议进行了优化, 但应针对每个动物模型进行优化。小鼠皮下肿瘤18f-葡萄糖成像的重现性研究表明, 平均%ID/g 的变异系数约为 15%, 表明单个小鼠的肿瘤治疗反应为18F-葡萄糖 PET 应该大于这个阈值被认为是可靠的和显著的31。
PET 示踪剂的细胞和甚至亚型分布可以通过组织显影来评估, 随后的切片染色并与 qIHC 共同注册。用 CT 联合注册 pet 可以将 pet 图像置于解剖环境中;这是非常宝贵的, 即使低软组织对比度。磁共振成像 (MRI) 可以克服 CT 对软组织造影的缺乏。此外, 荧光成像的生物标志物可以用来评估体内的糖酵解, 但在肺腔内的光子吸收和散射可能影响准确的定量或检测灵敏度32。总之, 利用全动物 PET/CT 图像定量组织学提供了一个准确的实时地图的肿瘤生物学治疗干预。
多光谱成像 (MSI) 适用于可能使用彩色图像的任何情况。至少, msi 提供了与彩色图像相同的信息, 对于某些应用程序, msi 可以提供比简单的宽频带三色 (RGB) 图像更详细的样本光谱属性信息。通常, msi 的局限性是彩色图像的限制, 只是 msi 速度较慢, 需要更多的时间来获取图像。利用形态学软件对图像进行重现、准确的分割结果, 并在材料表中进行了描述。还有额外的商用产品, 可用于组织分割和定量的组织学。
癌症代谢的复杂性超出了华宝效应和葡萄糖代谢33,34。肿瘤很可能会轻易地适应单一的药物治疗, 抑制糖酵解。对氨基酸代谢的依赖已经在癌症中得到了很好的记录, 预计肿瘤依赖于大量的氨基酸, 如谷氨酰胺, 甘氨酸, 丝氨酸, 以及其他代谢物, 如游离脂肪酸35,36, 37。除了18f-葡萄糖, 探针, 如18f 和11C 标记谷氨酰胺, 胆碱, 醋酸盐, 1-(2 '-脱氧-2 '-fluoroarabinofuranosyl) 胞嘧啶 (外交事务) 和氟胸苷 (外语教学) 已成功地用于图像氨基酸,核苷酸和脂质代谢的动物模型的癌症38,39,40,41。自动化和微型示踪放射化学技术加上高分辨率, 高灵敏度 pet 扫描仪将提高 pet 的可达性, 用于测量各种生物程序42,43。随着对新陈代谢的理解的增加, 宠物 radiotracers 的汇辑也将增加, 使研究人员和医生能够无创性肿瘤代谢的分布。
利用 PET/CT 成像和定量组织学解决了临床需要, 这是快速翻译板凳发现到临床使用。为了做到这一点, 研究人员必须能够准确地测量治疗反应以及获得的药物的抗性, 这是 PET/CT 成像所能实现的。此外, 对肺肿瘤的 PET/CT 和免疫组化分析作为患者的护理标准, 可直接翻译为临床实践。重要的是, PET/CT 成像容易识别治疗抗肿瘤, 研究人员可以在分子水平上隔离和审问, 以便更好地了解疾病的机制。这是一个迭代的过程, 使它能够更好地了解抗药性的机制和设计更有效的治疗策略, 为临床翻译。

我们的研究重点是调查和针对癌症的突变在肝激酶 B1 (LKB1, 也称为 STK11) 突变体癌1。LKB1 是一个主要的肿瘤抑制物, 压抑 mTOR 复杂 1 (mTORC1) 通过活化的 AMP 激酶 (AMPK), 导致调节生长和新陈代谢。因此, LKB1 的丧失导致了无节制的 mTORC1 活化, HIF1-alpha 的活化产生于酵代谢表型, 通常称为华宝效应2,3,4。LKB1 失活突变直接导致了一种罕见的家族性癌症前置综合征, 称为黑斑-黑斑息肉病综合征 (睡衣), 其特点是良性胃肠息肉的发展称为错构瘤5,6,7. 此外, LKB1 经常与致癌 KRAS 共同变异, 导致高代谢和侵袭性人肺肿瘤8,9。
Lkb1-related 疾病在小鼠中很容易被模仿。Lkb1 在小鼠体内的异型灭活导致错构瘤精确建模睡衣10、11、12、13的发展。此外, Lkb1 突变, 易于建模的小鼠准确重述癌症表型的肺, 皮肤, 胰腺和乳房14。Kras/Lkb1 在转基因小鼠肺组织中的联合突变, 采用 recombinase 介导的致癌 KrasG12D等位基因活化和 biallelic 删除 Lkb1, 结果形成侵袭性和转移型肺肿瘤15 ,16。KrasG12D的表征;Lkb1 (KL) 小鼠肺肿瘤显示, 这些肿瘤具有较高的 mTORC1 活化和高度酵, 使用直接代谢物的葡萄糖和乳酸的测量或测量的消耗量 [18F]-2-fluoro-2-deoxy-D-glucose (18F 葡萄糖) 通过正电子发射断层扫描 (PET) 与计算机断层扫描 (CT) 17。LKB1 突变体肿瘤的 mTORC1 超活化为检测构和催化激酶抑制剂 mTOR 治疗这些癌症提供了明确的依据。
在前一项研究中, 我们证明构 mTORC1 抑制剂雷帕霉素成功地抑制了胃肠道肿瘤的生长和糖酵解, 使用Lkb1+/-转基因小鼠模型睡衣3。目前, 作为一种治疗肾细胞癌的单剂治疗方法, 现已被批准, 但在 NSCLC18、19、20中表现出有限的疗效。mTORC1 是一种构的抑制剂, 可以通过开发下一代 mTOR 催化激酶抑制剂来改善, 从而对 mTOR 配合物1和 2 (分别 mTORC1 和 mTORC2)21进行更接近完全的抑制。MLN0128 等药物目前正在临床前研究和22、23的早期试验阶段进行评估。我们实验室最近的一项研究表明, MLN0128 是 mTOR 在肺癌 GEMMs15、16的人肺肿瘤细胞系和体内的一种有效抑制剂。MLN0128 抑制了这些小鼠的肺肿瘤生长和葡萄糖代谢24。
在本研究中, 我们利用有条件活化的液氧-停止-液-KRASG12D癌基因15,25引发肺癌的典型的腺病毒诱导小鼠模型。这些 KrasG12D小鼠与有 floxed 等位基因的小鼠交叉, Lkb1 (Lkb1l) 产生 KrasG12D;Lkb1l/升(吉隆坡) 小鼠16。在 recombinase 的腺或慢病毒北疆的鼻腔传递后, KL 小鼠在肿瘤诱导后4周内形成早期病变。6周后, KL 小鼠的肿瘤从性腺瘤肿瘤转变为典型的肺癌的恶性、侵袭性肿瘤表型, 8-10 周, 小鼠以100% 显性16,26, 发展为弗兰克癌。
PET/CT 成像和定量免疫组化可用于确定的分子和代谢反应, 以及治疗反应, 在肿瘤后交付的靶向治疗, 如 MLN012817, 26,27。这里描述的是一个实验性的协议, 利用18f-葡萄糖 PET 成像测量代谢反应的 MLN0128-targeted 治疗。通过定量组织学耦合 PET 成像, 可以测量 mTOR 抑制的分子反应以及肿瘤负担和肿瘤组织学的定量。

<table><tbody><tr><td>G8 PET/CT</td><td>Perkin Elmer</td><td>CLS139564</td><td>Used for 18F-FDG PET and CT imaging of mice</td></tr><tr><td>Axio Imager.M2</td><td>Zeiss</td><td>490020-0003-000</td><td>Acquiring images of FFPE lung tumor sections</td></tr><tr><td>Inform software</td><td>Perkin Elmer</td><td>CLS135781</td><td>Morphometric used for image analysis of tumor pathologies</td></tr><tr><td>Glut1 antibody</td><td>Alpha Diagnostics</td><td>GT12-A</td><td>IHC staining of FFPE lung tumor sections</td></tr><tr><td>Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP&amp;trade; Rabbit mAb</td><td>Cell Signaling Technologies</td><td>4858</td><td>IHC staining of FFPE lung tumor sections</td></tr><tr><td>MX35 Premier microtome blades&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>3051835</td><td>Microtome blades for sectioning tissue for autoradiography</td></tr></tbody></table>

utilizing 18f-fdg pet/ct imaging and quantitative histology to measure dynamic changes in the glucose metabolism in mouse models of lung cancer

晚期肿瘤的一个标志是转换到有氧糖酵解, 这是容易测量的 [18F]-2-氟-2-脱氧 d-葡萄糖正电子发射断层扫描 (18f-葡萄糖 PET) 成像。KRAS原癌基因和LKB1肿瘤抑制因子的共同突变是肺癌的常见事件, 驱动高代谢, 酵肿瘤的生长。调节这些肿瘤生长和代谢的关键途径是雷帕霉素 (mTOR) 通路的机械靶, 可以有效地利用选择性催化 mTOR 激酶抑制剂进行靶向。mTOR 抑制剂 MLN0128 抑制 Kras 和 Lkb1 共同突变的小鼠的糖酵解, 称为 KL 小鼠。对 KL 小鼠的治疗反应首先用18MLN0128 PET 和计算机断层扫描 (CT) 成像在分娩前后进行测量。通过利用18的 F 葡萄糖 PET/CT, 研究人员能够测量的动态变化的基因工程鼠模型 (GEMMs) 肺癌后的治疗干预与靶向治疗。其次是体外显影和定量免疫组化 (qIHC) 分析应用形态学软件。使用 qIHC 可以检测和量化的生物标志物的变化, 在治疗后, 以及独特的肿瘤病理特征的特点。PET 成像与定量组织学的耦合是在小鼠疾病模型中识别体内代谢和治疗反应的有效策略。

18对 KL 小鼠进行了 f-葡萄糖 PET 成像, 证明这些小鼠的肿瘤高度酵, 如18的葡萄糖摄入量 (图 1A) 所示, 同意以前发表的研究26, 29。全肺切除术显示有若干肿瘤 (图 1B)。小鼠肺可以被分成5个分开的裂片代表在图 1C和1D。裂片 1-5 被标记在切片肺部染色的 H & E 或葡萄糖转运蛋白 1 (Glut1) (图 1D)。Glut1 是葡萄糖和18氟葡萄糖的主要转运体, 其表达和定位对肿瘤细胞的血浆膜直接相关的18f-葡萄糖 SUV29。Glut1 染色 (40X) 的高分辨率分析在18F-葡萄糖-热切肺肿瘤显示了高表达和本地化的转运器在血浆膜 (图 1D)。
由于 pet 成像的分辨率有限, 对 pet/CT 和组织显影进行了研究。显影更高的分辨率可以识别肿瘤的小肿瘤和/或非均质性18F 葡萄糖分布。在肿瘤诱导后, 对 KL 小鼠 (图 2A) 进行18显影 PET/CT 成像, 然后对这些小鼠的肺进行分离 (2B和2C)。如图 2B和2C所示, 显影发现两个额外的较小的肿瘤, 是阳性的18F 葡萄糖尚未容易看到的宠物。显影后, 具有组织的幻灯片也可用于生物标志物的免疫组化 (IHC) 染色。肿瘤的 H & E 染色证实了左叶肿瘤的存在 (图 2D)。
其次, 对 MLN0128-treated Kras G12D 进行了18f-葡萄糖 PET成像;Lkb1 老鼠为了利用18F 葡萄糖作为一个功能生物标志物的葡萄糖代谢在肺部肿瘤 (图 3)。我们发现, MLN0128 的治疗有力地抑制了 mTORC1 信号和糖酵解的表现, 减少了18的 F 葡萄糖消耗 (图 3A和3B)。这些结果同意评估 MLN0128 在吉隆坡小鼠的临床前研究, 以前我们的实验室17,27发表。最后, 对肿瘤进行 IHC 染色 (图 3C)。肿瘤被染色为 H & E 或抗体抗 phospho-S6, 这是一个保守的 mTORC1 基底, 并用于表明 mTORC1 活化 (P-S6)与失活 (S6)。图 3C显示 MLN0128 在 KL 肿瘤中对 P-S6 的抑制, 与使用车辆治疗的患者相比, 与以前发表的工作17一致。除了 KRAS, 致癌的驱动因素, 如表皮生长因子受体 (EGFR) 也支持酵代谢的肺肿瘤。因此, 我们测试了是否抑制组成性活性突变体 EGFR 与尼布抑制18F 葡萄糖代谢的小鼠移植。图 3D和3E显示, 人肺肿瘤线 HCC827, 这是一个 EGFR del19 突变, 显示了显著降低18的葡萄糖消耗后五天的尼布治疗。
最后, 对切片肺和肺肿瘤进行形态学组织分析, 以量化肿瘤总负担, 并区分包括组织亚型、坏死、正常肺组织血管和空气空间在内的肿瘤病理。吉隆坡 GEMMs 发展了一种复杂的病理异质疾病, 呈现出不同 histopathologies 的肺肿瘤。这些包括腺癌 (ADC) 和鳞状细胞癌 (SCC)-这种异质性使治疗这一癌症的一个艰巨的挑战。图 4a显示一个单一的 H & E 染色的肺叶与两个大肿瘤存在。图 4B中显示的放大图像越高, 就会发现正常的肺、血管和空域, 以及肿瘤坏死以及腺癌, 其特点是有明确的甲状腺结构和鳞状细胞癌。图 4用通知形态学软件表示肺叶和肿瘤的伪着色。图 4D显示正常肺、血管和个别病理的百分比, 如肿瘤坏死和肿瘤亚型, 分割分化良好的腺癌与鳞状细胞癌。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57167/57167fig1.jpg"> 图 1: 代谢活性KrasG12D;Lkb1 (KL) 突变型肺肿瘤为18F 葡萄糖阳性, 表达高水平葡萄糖转运蛋白 1 (Glut1).小组a和B显示了最大强度投射 [也称为3维 (3D) 图像] 的18F 葡萄糖-PET 和 CT 分析的一些葡萄糖狂热的吉隆坡小鼠窝藏鳞状肺肿瘤。显示 (a) 3D 重建和 (B) 肺肿瘤的横向, 矢状和冠状的看法作为 (T)。(C) 这一小组显示了在 a 和B的a组中, Glut1 的全肺组织学, 无论是为 H & E (顶层) 染色, 还是与特定的抗体 (下板)。肺裂片编号。刻度条 = 2 毫米 (D) 此图代表小鼠 (顶板) 中的裂片的方向和数量, 以及从 h & e (中间板) 面板C中显示的幻灯片中的 h & e 或 Glut1-stained 肿瘤的高分辨率40X 图像。染色的抗体特定的 Glut1 (底部面板)。刻度条 = 25 µm.<a href="https://www.jove.com/files/ftp_upload/57167/57167fig1large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57167/57167fig2.jpg"> 图 2: 18葡萄糖显影能识别新陈代谢活跃的小肿瘤.(A) 这18的 f-葡萄糖 PET/CT 图像显示了18的 f-葡萄糖-狂热的肿瘤在吉隆坡鼠标显示为最大强度投影图像。T1 和 T2 = 肿瘤, H = 心脏, B = 膀胱, K = 肾脏。(B) 本小组在鼠标右侧和左肺裂片的序列部分显示前体显影。左、右面板的肺部是相同的。左边板上的肺部是伪彩色橙色。右侧面板中的肺部颜色为黑白相间。肿瘤 (T1、T2 和 T3) 用箭头表示。(C) 这是一个放大视图的显影 pseudocolored 橙色 (顶部面板) 和黑白 (底部面板)。(D) 本小组显示B组所示左叶顶端切片的 H & E 染色。刻度条 = 200 µm<a href="https://www.jove.com/files/ftp_upload/57167/57167fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57167/57167fig3.jpg"> 图 3: mTOR 抑制剂 MLN0128 抑制18氟葡萄糖 PET 检测 KL 小鼠肺肿瘤的葡萄糖消耗.(A) 本小组展示了18个具有代表性的吉隆坡小鼠的 PET/CT 图像 (18氟葡萄糖狂热, 左) 或 MLN0128 (18f 葡萄糖非狂热, 右)。显示横向 (顶部面板)、日冕 (中间板) 和矢状 (下板) 视图。肿瘤用红线勾勒出来;H = 心脏, L = 肝脏。(B) 这个小组显示了车辆和 MLN0128-treated 肿瘤之间的 SUVmax (%ID/g) 的量化。(C) 本小组显示由车辆或 MLN0128 处理的吉隆坡小鼠全肺切片的 H & E 和 P-S6 染色。刻度条 = 25 µm. (D) 本小组显示有代表性的18F-葡萄糖-PET 和 CT 图像的 HCC827 EGFR (del19) 移植前和尼布后治疗。肿瘤 (T) 用箭头表示, K = 肾脏, B = 大脑。(E) 本小组显示了对尼布治疗前后 HCC827 移植的 SUVmax (%ID/g) 的量化。n = 10 肿瘤或小组。<a href="https://www.jove.com/files/ftp_upload/57167/57167fig3large.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/57167/57167fig4.jpg"> 图 4: 应用形态学软件对肿瘤的负担和肿瘤组织学进行量化. (A) 本小组显示一只小鼠肺叶的 H & E 染色, 并从 KL 老鼠身上收集到肿瘤。(B) 这些高分辨率图像显示鳞状细胞癌 (左上), 正常肺, 血管和空气空间 (右上角), 以及分化良好的乳癌 (左下) 和坏死 (右下)。(C) 本小组使用形态学软件显示了 pseudocoloring & 电子染色肺叶的情况。(D) 本小组显示个别肺叶及肿瘤病理的百分率, 由通知量度。<a href="https://www.jove.com/files/ftp_upload/57167/57167fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer at JoVE.com

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

在本协议中, 我们描述如何利用 [18F]-2-氟 2-脱氧 d-葡萄糖正电子发射断层扫描和计算机断层扫描 (18MLN0128 PET/CT) 成像, 以测量肿瘤代谢反应的靶向治疗在Kras/Lkb1突变小鼠肺癌模型及高分辨率体外显影成像与定量组织学的结合。

利用18f-葡萄糖 PET/CT 成像和定量组织学测量肺癌小鼠模型葡萄糖代谢的动态变化

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

utilizing-18f-fdg-petct-imaging-quantitative-histology-to-measure

Research

JoVE Journal

Cancer Research

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

17.9K 次观看.  University of California Los Angeles David Geffen School of Medicine. 在本协议中, 我们描述如何利用 [18F]-2-氟 2-脱氧 d-葡萄糖正电子发射断层扫描和计算机断层扫描 (18MLN0128 PET/CT) 成像, 以测量肿瘤代谢反应的靶向治疗在Kras/Lkb1突变小鼠肺癌模型及高分辨率体外显影成像与定量组织学的结合。

视频: Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

nog 小鼠多发性骨髓瘤异种移植的多模态生物发光和正电子发射成形断层扫描成像

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

科研

癌症研究

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

肝细胞癌原位小鼠模型中的无创PET/MR成像

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

多细胞球体的肺内接种以模拟原位 NSCLC

Construction of An Orthotopic Xenograft Model of Non-Small Cell Lung Cancer Mimicking Disease Progression and Predicting Drug Activities

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models based on subcutaneous inoculation of cancer cell suspensions do not recapitulate the tumor microenvironment in NSCLC. Herein we describe a murine orthotopic lung cancer xenograft model that employs the intrapulmonary inoculation of three-dimensional multicellular spheroids (MCS). Specifically, fluorescent human NSCLC cells (A549-iRFP) were cultured in low-attachment 96-well microplates with collagen for 3 weeks to form MCS, which were then inoculated intercostally into the left lung of athymic nude mice to establish the orthotopic lung cancer model.
Compared with the original A549 cell line, MCS of the A549-iRFP cell line responded similarly to anticancer drugs. The long-wavelength fluorescent signal of the A549-iRFP cells correlated strongly with common markers of cancer cell growth, including spheroid volume, cell viability, and cellular protein level, thus allowing dynamic monitoring of the cancer growth in vivo by fluorescent imaging. After inoculation into mice, the A549-iRFP MCS xenograft reliably progressed through phases closely resembling the clinical stages of NSCLC, including the expansion of the primary tumor, the emergence of neighboring secondary tumors, and the metastases of cancer cells to the contralateral right lung and remote organs. Moreover, the model responded to the benchmark antilung cancer drug, cisplatin with the anticipated toxicity and slower cancer progression. Therefore, this murine orthotopic xenograft model of NSCLC would serve as a platform to recapitulate the disease&#39;s progression and facilitate the development of potential anticancer drugs.

作者聚焦：建立小鼠非小细胞肺癌模型以开发抗癌药物纳米制剂

This study presents an orthotopic non-small cell lung cancer (NSCLC) model based on intrapulmonary inoculation of multicellular spheroids of fluorescent A549-iRFP cells. The model recapitulates clinical NSCLC stages and responds to cisplatin, according to dynamic in vivo monitoring of long-wavelength fluorescence.

模拟疾病进展和预测药物活性的非小细胞肺癌原位异种移植模型的构建

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

分析18葡萄糖 PET/CT 成像作为研究结核分枝杆菌感染和治疗非人类灵长类动物的工具

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

免疫与感染

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

采用微计算机断层扫描对肿瘤发展的评估和后续反应来治疗肺癌的小鼠模型

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

医学

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). Impaired uptake and metabolism of glucose in multiple organ systems due to insulin resistance is a hallmark of type 2 diabetes. The ability of this technique to extract an image-derived input function from the vena cava using an iterative deconvolution method eliminates the requirement of the collection of arterial blood samples. Fitting of tissue and vena cava time activity curves to a two-tissue, three compartment model permits the estimation of kinetic micro-parameters related to the 18F-FDG uptake from the plasma to the intracellular space, the rate of transport from intracellular space to plasma and the rate of 18F-FDG phosphorylation. This methodology allows for multiple measures of glucose uptake and metabolism kinetics in the context of longitudinal studies and also provides insights into the efficacy of therapeutic interventions.

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This study describes a protocol that uses 18F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of 18F-FDG into tissues.

确定葡萄糖代谢动力学研究<sup&gt; 18</sup&gt; F-FDG微PET / CT

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

功能成像在小鼠体内的棕色脂肪与FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

生物学

Gene Regulation and Targeted Therapy in Gastric Cancer Peritoneal Metastasis: Radiological Findings from Dual Energy CT and PET/CT

Gastric cancer remains fourth in cancer incidence worldwide with a five-year survival of only 20%-30%. Peritoneal metastasis is the most frequent type of metastasis that accompanies unresectable gastric cancer and is a definitive determinant of prognosis. Preventing and controlling the development of peritoneal metastasis could play a role in helping to prolong the survival of gastric cancer patients. A non-invasive and efficient imaging technique will help us to identify the invasion and metastasis process of peritoneal metastasis and to monitor the changes in tumor nodules in response to treatments. This will enable us to obtain an accurate description of the development process and molecular mechanisms of gastric cancer. We have recently described experiment using dual energy CT (DECT) and positron emission tomography/computed tomography (PET/CT) platforms for the detection and monitoring of gastric tumor metastasis in nude mice models. We have shown that weekly continuous monitoring with DECT and PET/CT can identify dynamic changes in peritoneal metastasis. The sFRP1-overexpression in gastric cancer mice models showed positive radiological performance, a higher FDG uptake and increasing enhancement, and the SUVmax (standardized uptake value) of nodules demonstrated an obvious alteration trend in response to targeted therapy of TGF-&#946;1 inhibitor. In this article, we described the detailed non-invasive imaging procedures to conduct more complex research on gastric cancer peritoneal metastasis using animal models and provided representative imaging results. The use of non-invasive imaging techniques should enable us to better understand the mechanisms of tumorigenesis, monitor tumor growth, and evaluate the effect of therapeutic interventions for gastric cancer.

This protocol describes the value of dual energy CT and PET/CT imaging methods in tumor imaging and efficacy evaluation. This article demonstrates the research methods and results acquired by dual energy CT and PET/CT to evaluate the gene regulation and targeted treatment of gastric cancer peritoneal metastasis.

胃癌腹膜转移的基因调控及靶向治疗: 双能量 ct 与 PET/ct 的影像学研究

Gastric cancer remains fourth in cancer incidence worldwide with a five-year survival of only 20%-30%. Peritoneal metastasis is the most frequent type of ...

利用¹⁸f-葡萄糖 PET/CT 成像和定量组织学测量肺癌小鼠模型葡萄糖代谢的动态变化

摘要

探索更多视频

nog 小鼠多发性骨髓瘤异种移植的多模态生物发光和正电子发射成形断层扫描成像

肝细胞癌原位小鼠模型中的无创PET/MR成像

模拟疾病进展和预测药物活性的非小细胞肺癌原位异种移植模型的构建

模拟疾病进展和预测药物活性的非小细胞肺癌原位异种移植模型的构建

模拟疾病进展和预测药物活性的非小细胞肺癌原位异种移植模型的构建

分析¹⁸葡萄糖 PET/CT 成像作为研究结核分枝杆菌感染和治疗非人类灵长类动物的工具

采用微计算机断层扫描对肿瘤发展的评估和后续反应来治疗肺癌的小鼠模型

确定葡萄糖代谢动力学研究

功能成像在小鼠体内的棕色脂肪与FDG micro-PET/CT

胃癌腹膜转移的基因调控及靶向治疗: 双能量 ct 与 PET/ct 的影像学研究