作者想感谢 Stichting Hemelaere 和职业妇女福利互助会国际协会支持这项工作。

这项研究由动物实验伦理学委员会 (09/23 和幼儿发展 12/28) 批准。所有商业细节都可以在材料表中找到。
1. F98 GB 鼠细胞模型
<ol>
	<li>培养 F98 GB 细胞, 从 ATCC 获得, 在单分子膜中使用 Dulbecco 氏改良的鹰培养基, 10% 小牛血清, 1% 青霉素, 1% 链霉素, 1% l-谷氨酰胺, 和0.1% 两性霉素 b, 并在 co2孵化器 (5% co2和37° c) 中放置。</li>
	<li>在雌性 Fischer F344 鼠脑中接种胶质瘤细胞 (体重170克)。<ol>
		<li>使用无菌器械, 随时佩戴无菌手套。</li>
		<li>通过注射74毫克/千克氯胺酮和11毫克/千克嗪 intrapertioneally (IP) 与胰岛素注射器 (1 毫升, 29 克) 的混合物, 麻醉大鼠。通过对肢体的撤退反射没有反应来证实麻醉。把老鼠固定在一个立体定位装置里, 用鼻子和耳朵的点。放置一个卡波姆眼凝胶, 以防止在麻醉下的眼睛干燥。</li>
		<li>把老鼠从眼睛的水平刮到头骨的后面, 用聚维酮碘消毒皮肤。</li>
		<li>通过2厘米的中线头皮切口露出颅骨, 并使1毫米孔 (金刚石钻头) 2 mm 后部和2.5 毫米侧向右前额半球的 bregma。</li>
		<li>插入一个 stereotactically 引导胰岛素针 (29 克) 和注入5µL 细胞悬浮 (2万 F98 GB 细胞) 3 毫米深使用微量泵控制器 (设置: 注入 (I50), 率 1 nL/秒 (001 私人))。</li>
		<li>慢慢取出注射器, 用骨蜡缝合切口。缝合皮肤, 用聚维酮碘消毒。</li>
		<li>用红灯稳定动物后的体温。监测鼠的觉醒, 直到它已经恢复足够的意识, 以维持胸骨卧床。在完全恢复之前, 不要将动物归还给其他动物的公司。将所有动物置于环境控制条件下 (12 小时正常光照/暗循环、20-24 ° c 和40-70% 相对湿度) 与食物和水的ad 随意。确保密切跟踪动物的体重, 食物, 水的摄入量, 他们的活动和正常行为。使用致命剂量戊巴比妥钠安乐动物 (160 毫克/千克), 如果下降20% 体重是观察或当正常行为严重恶化 (如, 缺乏梳理)。</li>
	</ol>
	</li>
</ol>
2. 肿瘤生长的确认
注: 使用 T2-weighted mri、动态造影增强 mri (DCE mri) 和造影增强 T1-weighted mri 对肿瘤生长8天 post-inoculation 进行评估。当肿瘤达到 2.5 x 2.5 x 2.5 mm3的大小时, 选择用于治疗的大鼠。
<ol>
	<li>首先, 将30克针连接到一个60厘米长的管子上, 在侧尾静脉中放置静脉注射。麻醉的大鼠通过鼻锥与2% 异氟醚混合氧 (0.3 升/分)。当老鼠对肢体的撤退反射没有反应时, 确认麻醉。用加热的毯子盖住老鼠, 把它们放在 MRI 床上。使用卡波姆眼凝胶防止干燥。</li>
	<li>将床放置在支架上, 用固定的鼠脑表面线圈, 并将床置于72毫米大鼠全身发射机线圈中。</li>
	<li>执行定位扫描, 然后进行 T2-weighted 自旋回波扫描, 以评估肿瘤的生长。T2-MRI 序列细节: TR/TE 3661/37.1 毫秒, 109 µm 各向同性内平面分辨率, 切片厚度600µm, 4 平均数, TA 9 分钟四十五年代。</li>
	<li>如果肿瘤在 T2-weighted 获得证实, 注射钆造影剂进入静脉置管 (mri 造影剂; 0.4 毫升/千克) 后, 三十年代开始的 DCE mri 采集。使用单片 (1 mm 切片厚度) 中的快速低角度拍摄 (FLASH) 序列在12分钟内获取 DCE-MRI。使用 (312 µm2) 的平面空间分辨率和 1.34 s 的时间分辨率。</li>
	<li>使用图像序列分析工具, 选择一个感兴趣的区域 (ROI) 在可疑的肿瘤区域, 以绘制信号强度随着时间的推移。随后, 分析产生的 DCE 曲线的形状以确认胶质母细胞瘤的存在 (图 3)。</li>
	<li>最后, 获得一个对比度增强的 T1-weighted 自旋回波序列。T1-MRI 序列细节: TR/TE 1539/9.7 毫秒, 117 µm 各向同性内平面分辨率, 切片厚度600µm, 3 平均数, TA 4 分钟十五年代. 典型的对比度增强 T1-weighted MR 图像显示在图 2中。</li>
	<li>在 T1-weighted 序列完成后, 动物可以在连续的监督下醒来, 直到它恢复完全意识。</li>
</ol>
3. 目标体积选择的多模成像
注: 为了能够执行 MRI 引导的 F98 GB 大鼠模型的3D 保形照射 PET 引导子促进, 3 成像模式需要执行。首先, 注入示, 然后在示踪剂摄取过程中进行 MRI, 随后进行静态 PET 采集和治疗计划 CT。
<ol>
	<li>麻醉的动物使用鼻子锥与2% 异氟醚混合氧 (0.3 升/分)。当老鼠对肢体的撤退反射没有反应时, 确认麻醉。使用卡波姆眼凝胶, 以防止在麻醉下干燥。</li>
	<li>将导管 (26 克) 插入尾静脉, 使 37 MBq 的 PET 放射性示踪剂在200µL 盐水中溶解。分别在 PET 采集前注入18f-FET 或18f 扎、30分钟或2小时。</li>
	<li>在 PET 获得前15分钟, 在尾静脉静脉注射 MRI 造影剂 (0.4 毫升/千克)。</li>
	<li>将大鼠放在一个 in-house 的多模床上, 用钩环紧固件固定, 在成像和微辐照时保持一定的位置 (图 1)。</li>
	<li>修复三多模态标记 (充满水的毛细血管) 在头骨的右侧。将大鼠, 仍然固定在多模床上, 在 MRI 扫描仪的动物持有人, 修复大鼠脑表面线圈和位置这一设置在一个72毫米的大鼠全身发射机线圈。执行定位扫描, 然后进行对比增强的 T1-weighted 自旋回波序列。</li>
	<li>运送动物执行18f-FET 或18f-扎 PET 捕获。在列表模式下获取30分钟静态 PET 扫描。扫描应获得30分钟后, 18f-FET 注入或2小时后, 18f 扎注入。</li></ol>将所有 PET 扫描重建为 200 x 200 x 64 矩阵, 由2D 最大似然期望最大化 (MLEM) 算法使用60次迭代和一个体素大小 0.5 x 0.5 x 1.157 mm。
	<li>将该动物放在多模床上, 固定在四轴微型器机器人定位台上的塑料支架上。使用 1 mm 和 20 x 20 cm (1024 x 1024 像素) 非晶硅平板探测器的铝滤光片进行高分辨率的 CT 扫描。重建 CT 图像的各向同性的体素大小为0.2 毫米, 分别修复 70 kV 和0.4 毫安的管电压和管电流。共获得360预测超过360°。</li>

4. RT 治疗计划
<ol>
	<li>使用临床前治疗计划系统 (PCTPS) 进行治疗计划。将规划 ct 导入 PCTPS, 并将 ct 图像手动分割成三不同的组织类别: 骨骼、软组织和空气。这种手工分割的基础上定义三不同的灰度值门限的规划 CT。应选择这些人工选择的灰度阈值, 使大脑中的空气消失, 颅骨的骨厚度为非零。一旦定义了这些阈值, 材料密度由 PCTPS 分配给骨骼、软组织和空气 (图 4)。</li>
	<li>如果只需要 mri 引导, 用 PCTPS 进行 mri 扫描和 co-register。<ol>
		<li>使用刚体变换 (三平移和三旋转), 多模标记, 和头骨。通过在 MRI 上用黑色信号覆盖头颅 CT 增加的信号强度, 可以实现精确的融合 (图 5)。</li>
		<li>在 T1-weighted MRI 上选择造影剂肿瘤中心的照射靶, 请参阅图 6和图 7。</li>
	</ol>
	</li>
	<li>当额外的宠物信息必须包括在内, 包括一个 CT/MRI/pet co-registration 使用生物医学图像量化软件 (BIQS)。<ol>
		<li>使用 BIQS 中的轮廓工具实现 PET/MRI 图像融合 (图 8)。在 co-registration 之后, 在 BIQS (图 9) 中, 选择增加的 PET 示踪剂摄取中心的目标, 并使用下列变换将坐标手动输入 PCTPS: x → x、Y → z 和 z → y。</li>
		<li>选择指定的剂量、弧数、弧位、圆弧的旋转范围和准直器大小 (图 10)。</li>
		<li>对于 MRI 引导 RT, 请使用以下设置: 规定剂量 20, 3 弧定位在沙发角-45 °, 0 °, 45 °与弧旋转120°, 和准直器大小 5 x 5 毫米。</li>
		<li>对于 PET MRI 引导 RT, 请使用以下设置: 使用3弧和 5 x 5 mm 准直器的规定剂量为 20, 额外的5子升压使用 3 non-coplanar 弧和 1 x 1 mm 准直仪。选择一个120°旋转的所有弧形, 同时改变沙发的位置 (-45 °, 0 °, 和45°)。</li>
	</ol>
	</li>
	<li>计算在动物体内的剂量分布和光束传递参数, 使用 PCTPS 将规定的剂量传递给目标。在实际照射之前, 在不同的沙发位置测试圆弧旋转, 以防止辐照期间发生碰撞。</li>
	<li>对于实际辐照, 选择一个0.15 毫米的铜过滤器, 设置 x 射线电压到220伏, 设置 x 射线电流为13毫安, 并在龙门位置正确的准直仪。通过将适当的光束传递参数从 PCTPS 转移到微器来执行 RT。</li>
	<li>在这些过程中, 大鼠保持连续的异氟醚麻醉 (2% 异氟醚, 混合氧0.3 升/分)。在最后一个弧线的执行之后, 动物可以在持续的监督下醒来, 直到它恢复全意识。</li>
</ol>
5. 剂量容积直方图 (DVHs)
注意: 要比较实际剂量传递到肿瘤靶体积和周围正常的脑组织, 计算 DVHs。
<ol>
	<li>绘制一个 volume-of-interest (VOI) 周围的肿瘤和正常的大脑 T1-weighted 对比增强磁共振图像计算的平均, 最大和最低剂量 (图 11)。</li>
	<li>作为对肿瘤体积和正常脑组织体积的最大、平均和最小剂量的替代物, 计算 d2、d50和 d90。D 代表由 x% 所接收的量的剂量, 用下标表示, 并且可以从产生的 DVH。</li>
</ol>
6. 八卦和假化疗
<ol>
	<li>为了模拟治疗患者的胶质母细胞瘤, 管理伴随化疗使用 IP 注射29毫克/千克在盐水中溶解与25% 砜 (亚甲基亚砜) 每天一次5天从辐照日起24,25. 使用1毫升, 29 克胰岛素注射器来管理注射。</li>
	<li>对于控制组, 管理步骤6.1 中的注入而不进行任何的八卦。</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+RECIST+to+PERCIST:+Evolving+Considerations+for+PET+response+criteria+in+solid+tumors.">From RECIST to PERCIST: Evolving Considerations for PET response criteria in solid tumors.</a> J Nucl Med. 50 (5), 122S-150S (2009).</li><li>Daisne, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+volume+in+pharyngolaryngeal+squamous+cell+carcinoma:+comparison+at+CT,+MR+imaging,+and+FDG+PET+and+validation+with+surgical+specimen.">Tumor volume in pharyngolaryngeal squamous cell carcinoma: comparison at CT, MR imaging, and FDG PET and validation with surgical specimen.</a> Radiology. 233 (1), 93-100 (2004).</li><li>Grosu, A. -. L., Weber, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+for+radiation+treatment+planning+of+brain+tumours.">PET for radiation treatment planning of brain tumours.</a> Radiother Oncol. 96 (3), 325-327 (2010).</li><li>Banissi, C., Ghiringhelli, F., Chen, L., Carpentier, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treg+depletion+with+a+low-dose+metronomic+temozolomide+regimen+in+a+rat+glioma+model.">Treg depletion with a low-dose metronomic temozolomide regimen in a rat glioma model.</a> Cancer Immunol Immunother. 58, 1627-1634 (2009).</li><li>Robinson, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+alternative+temozolomide+schedules+on+glioblastoma+O(6)-methylguanine-DNA+methyltransferase+activity+and+survival.">Effect of alternative temozolomide schedules on glioblastoma O(6)-methylguanine-DNA methyltransferase activity and survival.</a> Br J Cancer. 103, 498-504 (2010).</li><li>Espa&#241;a, S., Marcinkowski, R., Keereman, V., Vandenberghe, S., Van Holen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DigiPET:+sub-millimeter+spatial+resolution+small-animal+PET+imaging+using+thin+monolithic+scintillators.">DigiPET: sub-millimeter spatial resolution small-animal PET imaging using thin monolithic scintillators.</a> Phys Med Biol. 59 (13), 3405 (2014).</li></ol>

作者没有利益冲突披露

为实现对大鼠脑胶质瘤靶向的精确照射, 微器的 on-board CT 引导是不够的。脑肿瘤是很难看到的, 由于缺乏足够的软组织对比, 即使使用对比增强。因此, 需要包括 MRI 来允许更精确的照射。利用 7 T 系统的序贯 MR 采集和微器的 ct 采集, 我们能够将剂量靶向脑内增强的肿瘤组织, 并利用规划 ct 计算剂量计划。这是可行的后, 图像融合和剂量计算使用 PCTPS17。然而, 应该牢记的是, MRI 容易出现几何失真, 而这在本协议中没有得到纠正。此外, 在把这一辐射协议翻译到身体的其他部分之前, 还需要进一步的研究。应考虑使用低 kV 能量光子的精确组织分割的重要性。虽然在大鼠大脑中分割成三组织类可能是足够的, 但需要在大鼠的胸或腹区域中显示更多的组织类, 以提供精确的剂量计算。为了避免在不同的成像系统之间的运输过程中, 我们使用了一个多模态床, 最大限度地减少头部的移动 (图 1)。然而, 在将本议定书应用于其他身体部位时, 无论是胸腹还是腹部区域, 都需要额外的努力。特别是小动物对受呼吸运动或肠道转运影响的器官的照射仍然具有挑战性。
尽管有 labor-intensive 的协议, 但也证明了将 PET 引导的子促进的结合是可行的。核成像技术的优势, 如 PET, 是能够图像的异质性肿瘤, 这使得目标新陈代谢高活性或抗辐射的部分肿瘤。我们能够增加剂量, 特别是针对最生物活性或最缺氧区的肿瘤使用18f-FET pet 或18f-扎 pet, 分别。协议中的关键步骤是图像 co-registration。目前, 没有任何软件能够自动 co-register 临床前 MRI 或 CT 与 PET 图像的高准确度和重现性。通常, 神经肿瘤中的 PET 示踪剂在正常大脑中的摄取率较低, 这使得注册过程复杂化。为了融合三成像模式 (CT/MRI/PET), 我们喜欢的 BIQS, 而不是 PCTPS, 这是目前尚未开发, 以方便地结合多种成像模式。此外, BIQS 有更多的刚性匹配的智能工具。一个主要的帮助也是使用多床, 防止动物在不同的影像采集之间移动。然而, 手动 co-registration 是费时和增加的麻醉时间的动物。一旦实现了图像注册, 通过在目标坐标上应用简单的转换, 将 BIQS 的坐标导出到 PCTPS 是可行的。
这不仅是重要的精确目标的 (生物) 肿瘤体积: 保留周围正常的脑组织也必须考虑到。后者在目前的动物放疗实验中往往被忽视, 但对模型的临床意义也很重要。这是通过应用多个 non-coplanar 弧来实现的。据我们所知, 在小动物的多弧颅照射从来没有应用过。关于光束的使用, 这种方法与临床图像引导的共形 RT 是非常相似的, 由于电弧治疗的使用, 目标最终得到规定的剂量, 而正常组织只接受一小部分。因此, 第一步是要尽量减少前临床和临床医学 RT 技术17之间的差距。这种微器的限制是, 龙门旋转限制在120°。将弧形旋转与长沙发位置的改变结合起来, 进一步增加了围绕肿瘤靶的正常脑组织的保留。
这一方法是将生物成像方法纳入放射治疗指南的一个重要步骤。然而, 需要新的发展, 以简化前临床图像融合, 并纳入剂量绘画 (DPBN) 的临床应用。利用目前的微器, 我们现在可以应用子增压;然而, 由于剂量计算、龙门旋转和准直器设计的限制, DPBN 还不能实现。最后, 发展紧凑的前临床 PET 扫描仪提供毫米的空间分辨率是希望的26 , 这些设备可能提供一个非常优雅的解决方案, 将宠物融入小动物辐射平台。
我们证明了该模型的适用性, MRI 和 PET 引导下的照射和化疗的大鼠胶质母细胞瘤和未来的研究新的治疗方法的胶质母细胞瘤。此外, PET 引导子促进的应用是第一步, 将电视台纳入小动物肿瘤模型的放射治疗规划。

高级别胶质瘤是最常见和最积极的恶性脑肿瘤在成人中中位数生存1年, 尽管目前的治疗方式。护理标准包括最大的手术切除, 其次是联合外照射治疗 (RT) 和莫 (八卦), 其次是维护八卦1,2,3。自从15年前引入了 "八卦" 以来, 在治疗这些肿瘤方面没有取得明显的进展。因此, 实施新的治疗策略是迫切的, 但应该首先在小动物肿瘤治疗模型 (主要是老鼠和老鼠) 中进行研究。含肿瘤的啮齿动物模型可以用来调查新的和复杂的辐射协议的功效, 可能与其他 (新的) 治疗剂结合, 以评估辐射反应或调查无线电保护剂。临床前辐射研究的一个主要优势是能够在受控实验条件下使用大的群体, 由于啮齿类动物的寿命较短而导致数据产量加快。临床前的发现应该被翻译成一个比当前实践中更快、更有效的方法4。
在过去的几十年中, 小动物辐射实验通常是通过固定辐射源实现的5,6,7,如, 137Cs 和60Co, 同位素, 或线性用于人类临床用途的加速器, 适用于单辐射场与 MV X 射线6,8,9,10,11。但是, 这些设备无法达到毫米精度, 这是小动物所需的12。此外, MV X 射线具有不适合辐照小目标的特性, 例如在光束入口区域的 air-tissue 界面上的剂量积聚, 以动物大小本身的顺序4,6 ,8,9,10,11。后者使它相当具有挑战性, 提供一个统一的剂量, 以肿瘤, 而保留周围的正常脑组织4,8,9,10,11。因此, 目前尚不清楚当前的动物研究在何种程度上仍然适用于现代 RT 实践12。在这方面, 最近开发的三维 (3D) 保形小动物微辐照有望弥合先进的3D 图像制导 RT 技术之间的技术差距, 如强度调制辐射疗法 (放射) 或用于人和当前小动物辐照的共形弧形4,13。这些平台利用千伏 (kV) X 射线源获得尖锐的 penumbras, 并避免剂量积聚。这些平台包括一个计算机控制的动物定位阶段, 一个用于成像和辐射处理的 kV X 射线源, 一个旋转龙门组件, 允许从不同角度进行辐射传递, 以及一个准直系统来形成辐射光束4. 2011年, 在根特大学的临床前成像实验室安装了微器 (图 1)。该系统类似于现代的人类放疗实践, 并使广泛的临床前实验, 如辐射与其他疗法的协同作用, 复杂的辐射方案, 和图像引导 sub-target 促进研究。
这些微辐照的治疗计划是基于 CT, 它相当于人类规划系统14,15。对于 CT 成像, on-board x 射线检测器与治疗期间使用的同一个 kV x 射线管相结合。CT 成像是使用, 因为它允许准确的动物定位, 并提供必要的信息, 个人辐射剂量计算通过分割。然而, 由于 CT 成像的软组织对比度较低, 小动物的脑部肿瘤, 如高等级胶质瘤, 不能轻易被描述。因此, 为了精确的目标体积划分, 多成像是必要的。与 CT 相比, MRI 提供了极大的软组织对比。这使得更容易可视化病变边界, 这将导致更好地划定目标体积, 有助于更好地照射病灶和避免周围组织, 如 图 24所示, 16。另外一个优点是 MRI 使用电离辐射, 与使用电离辐射的 CT 不同。MRI 的主要缺点是相对较长的采集时间和较高的操作成本。重要的是要注意, MRI 扫描不能用于剂量计算, 因为他们没有提供所需的电子密度信息, 虽然在这方面正在取得进展, 也随着最近的发展加速器先生。因此, 联合 CT/MRI 数据集是规划恶性胶质瘤照射的选择方法, 同时包含靶向 (MRI-based 体积) 和剂量计算 (ct 电子密度) 所需的信息。
为了减少小动物照射与临床常规的差距, mri 显然需要与微器的工作流程相结合, 需要在 mri 与 CT 之间进行正确的定位, 而这远非微不足道。本文讨论了 F98 脑胶质瘤的 MRI 引导3D 适形照射的方案, 该方法已在最近的17中发表。
虽然在微器的工作流程中纳入 CT 和 MRI 是小动物照射研究的一个明显的进步, 但这些解剖成像技术并不总是允许对目标体积进行完整的定义。CT 和 MRI 对脑组织的病理改变表现为增加含水量 (水肿) 和血脑屏障渗漏或造影增强。然而, 对比增强和超强区域的 T2-weighted MRI 并不总是准确的测量肿瘤程度。肿瘤细胞的检测远远超出了对比增强12的边缘。此外, 这些技术都不能识别肿瘤中最具侵袭性的部分, 这可能是治疗性抵抗和肿瘤复发的原因。因此, 从像 PET 这样的分子成像技术中获得的额外信息可能会增加 RT 目标体积定义的值, 因为这些技术能够使生物通路在体内12、18中可视化, 19。
在 2000年, 凌et al.引入了生物靶体积 (电视台) 的概念, 将解剖和功能成像集成到放疗工作流中, 导致他们所说的多维适形放疗20。这就创造了一种可能性, 通过给目标区域提供一个非均匀剂量的 PET 图像来改善剂量靶向。最广泛使用的肿瘤分期 PET 示踪和监测治疗反应是 fluor-18 (18F) 标记葡萄糖 (葡萄糖), 这可视化为葡萄糖代谢21。在头颈部癌, 以前的研究表明, 使用18f-葡萄糖 PET 导致更好的估计实际肿瘤体积, 由病理标本, 与 CT 和 MRI 相比,22。在原发性脑肿瘤, 葡萄糖是不有用的, 由于非常强烈的背景信号, 从正常的大脑, 氨基酸, 如11c-蛋氨酸和最近的18F-fluoroetthyltyrosine (FET), 已被调查的 GTV在氨基酸 PET 和 MRI-based GTVs 之间经常标记的区别的分界23。然而, 目前还没有对这一发现的意义进行调查的前瞻性试验。在本研究中, 我们选择了氨基酸示踪剂18f-FET 和缺氧示踪剂18f-fluoroazomycin-苷 (18f-扎)。18选择 f-FET 和18f-扎, 因为增加的氨基酸摄取量与 GB 肿瘤的增殖率密切相关, 而缺氧 PET 示踪剂的摄取与抗 (化疗) 放疗相关,18,23. 通过对小鼠 F98 GB 肿瘤的 PET 定义部分给予额外的辐射剂量, 优化了微器的亚容积升压。

<table>
	<tbody>
		<tr>
			<td>GB RAT model</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>F98 Glioblastoma cell line</td>
			<td>ATCC</td>
			<td>CRL-2397</td>
			<td></td>
		</tr>
		<tr>
			<td>Fischer F344/Ico crl Rats</td>
			<td>Charles River</td>
			<td>N/A</td>
			<td>http://www.criver.com/products-services/basic-research/find-a-model/fischer-344-rat</td>
		</tr>
		<tr>
			<td>Micropump system</td>
			<td>World Precision Instruments</td>
			<td>UMP3</td>
			<td>Micro 4: https://www.wpiinc.com/products/top-products/make-selection-ump3-ultramicropump/#tabs-1</td>
		</tr>
		<tr>
			<td>Stereotactic frame</td>
			<td>Kopf</td>
			<td>902</td>
			<td>Model 902 Dual Small Animal Stereotaxic frame</td>
		</tr>
		<tr>
			<td>diamant drill</td>
			<td>Velleman</td>
			<td>VTHD02</td>
			<td>https://www.velleman.eu/products/view/?id=370450</td>
		</tr>
		<tr>
			<td>Bone wax</td>
			<td>Aesculap</td>
			<td>1029754</td>
			<td>https://www.aesculapusa.com/products/wound-closure/hemostatic-bone-wax</td>
		</tr>
		<tr>
			<td>Insulin syringe Microfine</td>
			<td>Beckton-Dickinson</td>
			<td>320924</td>
			<td>1 mL, 29G</td>
		</tr>
		<tr>
			<td>InfraPhil IR lamp</td>
			<td>Philips</td>
			<td>HP3616/01</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethilon</td>
			<td>Ethicon</td>
			<td>662G/662H</td>
			<td>FS-2, 4-0, 3/8, 19 mm</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>14040-091</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilline-streptomycine</td>
			<td>Invitrogen</td>
			<td>15140-148</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030-032</td>
			<td></td>
		</tr>
		<tr>
			<td>Fungizone</td>
			<td>Invitrogen</td>
			<td>15290-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25300-062</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>14040-224</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcons</td>
			<td>Thermo Scientific</td>
			<td>178883</td>
			<td>175 cm2 nunclon surface, disposables for cell culture with filter caps</td>
		</tr>
		<tr>
			<td>Cell freezing medium</td>
			<td>Sigma-aldrich</td>
			<td>C6164</td>
			<td>Cell Freezing Medium-DMSO, sterile-filtered, suitable for cell culture, endotoxin tested</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Animal irradiation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-irradiator</td>
			<td>X-strahl</td>
			<td>SARRP</td>
			<td></td>
		</tr>
		<tr>
			<td>software for irradiation</td>
			<td>X-strahl</td>
			<td>MuriPlan</td>
			<td>pre-clinical treatment planning system (PCTPS), version 2.0.5.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Small animal PET</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microPET system possibility 1</td>
			<td>Molecubes</td>
			<td>B-Cube</td>
			<td>http://www.molecubes.com/b-cube/</td>
		</tr>
		<tr>
			<td>microPET system possibility 2</td>
			<td>TriFoil Imaging, Northridge CA</td>
			<td>FLEX Triumph II</td>
			<td>http://www.trifoilimaging.com</td>
		</tr>
		<tr>
			<td>PET tracers</td>
			<td>In-house made</td>
			<td></td>
			<td>18F-FDG, 18F-FET, 18F-FAZA, 18F-Choline</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Small animal MRI</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microMRI system</td>
			<td>Bruker Biospin</td>
			<td>Pharmascan 70/16</td>
			<td>https://www.bruker.com/products/mr/preclinical-mri/pharmascan/overview.html</td>
		</tr>
		<tr>
			<td>Dotarem contrast agent</td>
			<td>Guerbet</td>
			<td></td>
			<td>MRI contrast agent, Dotarem 0,5 mmol/ml</td>
		</tr>
		<tr>
			<td>rat whole body transmitter coil</td>
			<td>Rapid Biomedical</td>
			<td>V-HLS-070</td>
			<td></td>
		</tr>
		<tr>
			<td>rat brain surface coil</td>
			<td>Rapid Biomedical</td>
			<td>P-H02LE-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Water-based heating unit</td>
			<td>Bruker Biospin</td>
			<td>MT0125</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G Needle for IV injection</td>
			<td>Beckton-Dickinson</td>
			<td>305128</td>
			<td>30 G</td>
		</tr>
		<tr>
			<td>PE 10 tubing (60 cm/injection)</td>
			<td>Instech laboratories, Inc</td>
			<td>BTPE-10</td>
			<td>BTPE-10, polyethylene tubing 0.011 x .024 in (0.28 x 60 mm), non sterile, 30 m (98 ft) spool, Instech laboratories, Inc Plymouth meeting PA USA- (800) 443-4227- http://www.instechlabs.com</td>
		</tr>
		<tr>
			<td>non-heparinised micro haematocrit capillaries</td>
			<td>GMBH</td>
			<td>7493 21</td>
			<td>these capillaries are filled with water to create markers visible on MRI and CT</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>isoflurane: Isoflo</td>
			<td>Zoetis</td>
			<td>B506</td>
			<td>Anaesthesia</td>
		</tr>
		<tr>
			<td>ketamine: Ketamidor</td>
			<td>Ecuphar</td>
			<td></td>
			<td>Anaesthesia</td>
		</tr>
		<tr>
			<td>xylazine: Sedaxyl</td>
			<td>Codifar NV</td>
			<td></td>
			<td>Anaesthesia</td>
		</tr>
		<tr>
			<td>catheter</td>
			<td>Terumo</td>
			<td>Versatus-W</td>
			<td>26G</td>
		</tr>
		<tr>
			<td>Temozolomide</td>
			<td>Sigma-aldrich</td>
			<td>T2577-100MG</td>
			<td>chemotherapy</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-aldrich</td>
			<td>276855-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe Microfine</td>
			<td>Beckton-Dickinson</td>
			<td>320924</td>
			<td>1 mL, 29G</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Image analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PMOD software</td>
			<td>PMOD technologies LLC</td>
			<td>PFUS (fusion tool)</td>
			<td>biomedical image quantification software (BIQS), version 3.405, https://www.pmod.com/web/?portfolio=22-image-processing-pfus</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Anesthesia-equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthetic movabe unit</td>
			<td>ASA LTD</td>
			<td>ASA 0039</td>
			<td>ASA LTD, 5 valley road, Keighley, BD21 4LZ</td>
		</tr>
		<tr>
			<td>Oxygen generator</td>
			<td>Veterinary technics Int.</td>
			<td>7F-3</td>
			<td>BDO-Medipass, Ijmuiden</td>
		</tr>
	</tbody>
</table>

pet and mri guided irradiation of a glioblastoma rat model using a micro-irradiator

几十年来, 小动物的辐射研究大多是使用相当粗糙的实验设置, 应用简单的单技术, 而没有能力针对特定或界定肿瘤体积。辐射的交付使用固定的辐射源或线性加速器生产 megavoltage (MV) x-射线。这些设备无法达到小动物所需的毫米精度。此外, 高剂量传递到健康的周围组织阻碍反应评估。为了增加小动物研究和人类之间的翻译, 我们的目标是模仿在老鼠模型中对人脑胶质瘤的治疗。为了在临床前设置更精确的照射, 最近开发了精确的图像引导小动物辐射研究平台。类似于人类规划系统, 这些微辐照的治疗计划是基于计算机断层扫描 (CT)。然而, 低软组织对比 CT, 使它非常具有挑战性的定位目标在某些组织, 如大脑。因此, 与 CT 相比, 磁共振成像 (MRI) 具有优异的软组织对比度, 能够更精确地描述靶点的照射。在过去的十年中, 生物成像技术, 如正电子发射断层扫描 (PET) 获得了辐射治疗治疗指导的兴趣。PET 使如, 葡萄糖消耗, 氨基酸运输, 或缺氧, 目前在肿瘤的可视化。以更高的剂量靶向肿瘤的高度增殖或耐药部位, 可以获得生存的好处。这个假说导致了生物肿瘤容量 (电视台) 的介绍, 除常规总目标容量 (GTV)、临床目标容量 (中视) 和计划的目标容量 (PTV) 之外。
在根特大学的前临床影像实验室, 有一个微型器, 一个小动物宠物, 和一个 7 T 小动物的 MRI 是可用的。其目的是将 MRI 引导的照射和 PET 引导子在胶质瘤大鼠模型中的增强。

为了模拟在临床前模型中对胶质母细胞瘤照射的人类治疗方法, 必须纳入 MRI 引导放射治疗。使用 PCTPS 和微器界面, 我们能够在 T1-weighted MRI17上用多形 non-coplanar 弧照射大鼠 F98 胶质母细胞瘤。将刚体变换与多床结合, 用于 MRI 与规划 CT 的图像配准。在 T1-weighted MRI (图 7) 中, 在造影增强肿瘤区的中心选择了等照射。
对五种不同的动物 (图 12) 计算了靶体积和正常脑组织容积的平均、最小和最大剂量的剂量分布和累积 DVHs。根据临床照射协议和最佳剂量分布的相似性, 选择了三 non-coplanar 弧的剂量计划。应用后者, 90% 的目标体积得到了期望的剂量, 同时最小化剂量的正常脑组织17。
在证实了 MRI 引导下 F98 大鼠胶质瘤模型的可行性后, 我们试图将 PET-based 子促进纳入 RT 规划的临床前工作流程。我们能够结合3成像模式, 执行第一次 MRI, 然后 PET, 最后 CT, 而老鼠是固定在一个 in-house 制成多模态床 (图 1)。对于这些模式的 co-registration, 我们使用了 BIQS, 从而为刚性匹配提供了更多的工具 (图 8)。应用简单的转换, 基于 MR 的和基于 PET 的等 (图 9) 都可以转移到 PCTPS。在图 13中, 显示了 PCTPS 中剂量计算后辐照照射的 MRI 和 PET-based 等。为了照射整个对比度增强音量, 我们选择了 5 x 5 准直器和三弧旋转120°。为了提高在18f-FET pet 上确定的最新陈代谢活性肿瘤部分, 或在18f 扎 pet 上发现的最缺氧肿瘤部分, 选择并使用1毫米直径的准直器进行了5的剂量。再次, 3 弧旋转120°适用。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56601/56601fig1.jpg"> 
图 1: 微器集成了 kV X 射线管、旋转龙门、计算机控制的机器人工作台、用于形状光束的准直系统以及平板 CT 探测器.该动物被放置在一个4毫米厚的 PVC 多模态床, 以防止在多个影像采集之间的运动, 如 MRI 扫描后, 规划 CT, 这有助于图像融合.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56601/56601fig1large.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/56601/56601fig2.jpg"> 
图 2: 确认胶质母细胞瘤.T1-weighted mri, T2-weighted mri, 和一个 F98 GB 大鼠的 DCE mri。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56601/56601fig2large.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/56601/56601fig3.jpg"> 
图 3: DCE 曲线.使用图像序列分析工具, 可以在 DCE MRI 扫描中选择 ROI, 以绘制一段时间的信号强度。随后, 分析产生的 DCE 曲线的形状能够证实胶质母细胞瘤的存在。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56601/56601fig3large.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/56601/56601fig4.jpg"> 
图 4: CT 分割.基于 CT 的分割是通过手工定义一些阈值来准确区分空气与肺部组织, 脂肪组织, 骨骼和其他组织在图像中。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56601/56601fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56601/56601fig5.jpg"> 
图 5: MRI-CT 融合.通过在 MRI 上用黑色信号覆盖头颅 CT 增加的信号强度, 可以实现精确的融合。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56601/56601fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56601/56601fig6.jpg"> 
图 6: 锥束 CT.CT 上没有可见肿瘤, 因此无法选择肿瘤中心的等。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56601/56601fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56601/56601fig7.jpg"> 
图 7: 对比度增强的 T1-weighted MRI.对比增强的 T1-weighted MRI 清楚地直观地说明了鼠 F98 脑肿瘤。对比度增强的中心被选作 RT 规划的等。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56601/56601fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56601/56601fig8.jpg"> 
图 8: MRI-PET 融合.利用 BIQS 中的轮廓工具, 实现了 PET/MRI 图像融合。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56601/56601fig8large.jpg" target="_blank">请点击这里查看更大版本的这个数字。</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56601/56601fig9.jpg"> 
图 9: MRI-PET 靶选择.在 T1-weighted MRI (左) 对比增强的中心选择照射靶。在18F-FET PET (右) 上增加的信号的中心选择了子升压的目标。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56601/56601fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/56601/56601fig10.jpg"> 
图 10: 放疗计划.为计算放疗计划, 选择等、规定剂量、弧数、电弧位置、圆弧旋转范围和准直器尺寸。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56601/56601fig10large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/56601/56601fig11.jpg"> 
图 11: DVH 计算.画一个 volume-of-interest (VOI) 周围的肿瘤 T1-weighted 对比增强磁共振图像计算 DVH 在这个体积。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56601/56601fig11large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/56601/56601fig12.jpg"> 
图 12: 使用对比度增强的 T1-weighted MRI 和三 non-coplanar arcsto 的剂量计划提供20的目标容量.在右侧, 给出了肿瘤体积的累积剂量体积直方图 (DVH) 和经对比增强的 T1-weighted MRI 描述的正常脑组织。此图已从 Bolcaen et al.中修改20 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56601/56601fig12large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/56601/56601fig13.jpg"> 
图 13: 为辐照而选择的 MRI 和 PET 引导等.在轴向、冠状和矢状位视图的 CT 图像中, 用剂量计划向靶区 (黄区) 提供20。在造影增强 MRI 上发现的等是可见的 (绿色), 在18F-FET PET 上确定的新陈代谢活性肿瘤部分的等也可见 (红色)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56601/56601fig13large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator at JoVE.com

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

PET 与 MRI 引导下微器对大鼠胶质瘤模型的照射

在过去, 小动物照射通常是没有能力靶界定肿瘤体积。其目的是模仿大鼠的人脑胶质瘤的治疗。使用小动物辐照平台, 我们进行了 MRI 引导3D 共形照射与 PET-based 子促进在临床前设置。

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

pet-mri-guided-irradiation-glioblastoma-rat-model-using-micro

Nanyang Technological University

Research

JoVE Journal

Cancer Research

9.4K 次观看.  Ghent University Hospital. 在过去, 小动物照射通常是没有能力靶界定肿瘤体积。其目的是模仿大鼠的人脑胶质瘤的治疗。使用小动物辐照平台, 我们进行了 MRI 引导3D 共形照射与 PET-based 子促进在临床前设置。

视频: PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

Endobronchial Ultrasound-guided Intratumoral Injection of Cisplatin for the Treatment of Isolated Mediastinal Recurrence of Lung Cancer

Isolated hilar and mediastinal recurrence (IMHR) following external beam radiation therapy (EBRT) in patients with lung cancer is common. These patients do not have many treatment options and are usually offered palliative chemotherapy or best supportive care. Endobronchial ultrasound (EBUS)-guided intratumoral injection of cisplatin (ITC) is a novel approach for these patients. The procedure is performed under conscious sedation. The lesion is located with a bronchoscopy using EBUS, and a 22-gauge EBUS needle is advanced through the working channel of the scope and locked in position. Under ultrasound guidance, the wall of the tracheobronchial tree is punctured and the needle is moved into the target lesion. The needle stylet is then removed and cisplatin (40 mg/40 mL) is injected into the lesion. One to two sites are treated per session. Details of the procedure are described in the protocol section of paper. At our center, 50 sites were treated in 36 patients (19 males, 17 females). The mean age of our cohort was 61.9 &#177;8.5 years. We performed final analyses on 35 patients and 41 sites. 24/35 (69%) had complete or partial response (responders), whereas 11/35 (31%) had stable or progressive disease (non-responders). Overall, survival in our group was 8 months (95% CI of 6-11 months), with patients who responded having significantly better survival than the ones who did not.

The management of isolated recurrent lung cancer in a previously-irradiated field is challenging. Here, we describe an endobronchial ultrasound (EBUS)-guided cisplatin injection for the management of patients with localized lung cancer recurrence in a previously-radiated field.

支气管内超声引导下顺铂瘤内注射对肺癌纵隔孤立复发的治疗

Isolated hilar and mediastinal recurrence (IMHR) following external beam radiation therapy (EBRT) in patients with lung cancer is common. These patients ...

科研

癌症研究

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

疲劳的小鼠模型的外围照射引起

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Whole-body PET/MRI of Pediatric Patients: The Details That Matter

Integrated PET/MRI is a hybrid imaging technique enabling clinicians to acquire diagnostic images for tumor assessment and treatment monitoring with both high soft tissue contrast and added metabolic information. Integrated PET/MRI has shown to be valuable in the clinical setting and has many promising future applications. The protocol presented here will provide step-by-step instructions for the acquisition of whole-body 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) PET/MRI data in children with cancer. It also provides instructions on how to combine a whole-body staging scan with a local tumor scan for evaluation of the primary tumor. The focus of this protocol is to be both comprehensive and time-efficient, which are two ubiquitous needs for clinical applications. This protocol was originally developed for children above 6 years, or old enough to comply with breath-hold instructions, but can also be applied to patients under general anesthesia. Similarly, this protocol can be modified to fit institutional preferences in terms of choice of MRI pulse sequences for both the whole-body scan and local tumor assessment.

This article provides comprehensive step-by-step instructions for the acquisition of whole-body 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) PET/MRI scans for cancer staging of pediatric patients. The protocol was developed for children above 6 years, or old enough to comply with breath-hold instructions, but can be used for general anesthesia patients as well.

儿科患者全身 PET/MRI: 物质的细节

Integrated PET/MRI is a hybrid imaging technique enabling clinicians to acquire diagnostic images for tumor assessment and treatment monitoring with both ...

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

胶质瘤移植中体内成像的荧光分子层析成像研究

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

超声引导组织定向细胞植入术在生物学相关转移性肿瘤移植中的应用

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. Although heterogeneous in presentation, a common denominator among these tumors is the overexpression of somatostatin receptors. 68Ga-DOTATATE is a somatostatin analog labeled with the positron emitter gallium-68 (68Ga). For well-differentiated neuroendocrine tumors, 68Ga-DOTATATE positron emission tomography (PET)/computed tomography (CT) imaging is used for diagnosis, determination of disease burden, and therapy selection.
This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiolabeling of 68Ga-DOTATATE is performed with a fully automated labeling module coupled to a germanium-68 (68Ge)/68Ga generator. Quality control of the final product evaluates radiochemical purity with instant thin-layer chromatography and solid-phase chromatography, and pH prior to patient injection. Periodic quality control is performed to determine 68Ge breakthrough, sterility, and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) content. Patient preparation includes patient instructions, a protocol for 68Ga-DOTATATE during treatment with somatostatin analogs, and intravenous administration of the radiopharmaceutical. For PET/CT imaging, the acquisition and reconstruction settings are described. For each step, radiation safety will be highlighted, as well as time constrictions due to the short half-life of 68Ga.
Fully automated in-house production and quality control of 68Ga-DOTATATE leads to very high success rates (95%) and produces two to four patient dosages per batch, depending on the yield of the generator. In conclusion, 68Ga-DOTATATE PET/CT imaging is a noninvasive and fast method of providing information on the tumor burden of neuroendocrine tumors (NETs) while also assisting in diagnosis and therapy selection.

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Well-differentiated neuroendocrine tumors overexpress somatostatin receptors which can be utilized for diagnostic imaging with the radiolabeled somatostatin analog 68Ga-DOTATATE. This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiation safety and time constrictions due to the short half-life of 68Ga are taken into account.

68型 Ga-DOTATATE 神经内分泌肿瘤的日常临床应用指南

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

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

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

研究使用正交乳腺癌模型的三阴性乳腺癌

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

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

毛尿胰腺腺腺腺癌超声引导正射植入

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

胶质母细胞瘤的3D球形模型

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

利用计算机图像分析提高4T1乳腺癌模型中肺转移的定量

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...