磁共振引导的高强度聚焦超声产生热疗：小鼠横纹肌肉瘤模型中的可行治疗方法
1Institute of Medical Science,University of Toronto, 2Lunenfeld-Tanenbaum Research Institute,Mount Sinai Hospital, 3The Wilfred and Joyce Posluns Centre for Image-Guided Innovation and Therapeutic Intervention,The Hospital for Sick Children, 4Institute of Biomedical Engineering,University of Toronto, 5Leslie Dan Faculty of Pharmacy,University of Toronto, 6Departments of Radiology and Clinical Neurosciences,University of Calgary, 7Department of Radiation Oncology,University of Toronto, 8Department of Medical Imaging,University of Toronto, 9Department of Pediatric Surgery,University of Toronto, 10Department of Surgery,University of Toronto
Summary
这里介绍的是一种协议，使用由磁共振引导的高强度聚焦超声产生的受控热疗来触发横纹肌肉瘤小鼠模型中温度敏感脂质体的药物释放。
Abstract
磁共振引导的高强度聚焦超声（MRgHIFU）是一种产生局部热疗的既定方法。考虑到实时成像和声能调制，这种模式可以在定义的区域内实现精确的温度控制。这种非侵入性、非电离技术正在探索许多热应用，例如热疗产生，以从热敏脂质体载体中释放药物。这些药物可以包括化疗，如阿霉素，由于剂量限制性全身副作用，即心脏毒性，需要靶向释放。多柔比星是治疗多种恶性肿瘤的主要药物，通常用于复发或复发性横纹肌肉瘤 （RMS）。RMS是儿童和年轻人中最常见的实体性软组织颅外肿瘤。尽管进行了积极的多模式治疗，但RMS存活率在过去30年中保持不变。为了探索解决这一未满足需求的解决方案，开发了一种实验方案，以评估使用MRgHIFU作为药物释放热疗源的免疫功能正常的同系RMS小鼠模型中热敏脂质体阿霉素（TLD）的释放。
Introduction
横纹肌肉瘤 （RMS） 是一种骨骼肌肿瘤，最常见于儿童和年轻人1.局部疾病通常采用多模式治疗，包括化疗、电离放疗和手术。多药化疗方案的使用在儿科患者中更为普遍，与成人患者相比，结局有所改善2;然而，尽管正在进行研究努力，在最具侵略性的疾病中，5 年生存率仍保持在 30% 左右3，4。化疗标准治疗是一种多药方案，包括长春新碱、环磷酰胺和放线菌素 D。在疾病复发或复发的情况下，使用替代化疗，包括标准（游离）多柔比星 （FD） 和异环磷酰胺1。虽然所有这些化疗都具有全身毒性，但阿霉素的心脏毒性施加了终生剂量限制5-7。为了增加递送到肿瘤的药物量并尽量减少全身毒性，已经开发了替代制剂，包括脂质体包封。这些可以是非热敏性阿霉素，已被批准用于治疗乳腺癌和肝细胞癌，也可以是热敏性阿霉素，其临床试验正在进行中8，9，10，11，12，13。递送脂质体包封药物的替代方法，例如多泡脂质体和配体靶向脂质体已经得到评估，并显示出治疗肿瘤的希望9。在这项研究中，热量的增加具有多因素影响，包括药物释放14。热疗（HT）与磁共振引导的高强度聚焦超声（MRgHIFU）和热敏脂质体阿霉素（TLD）的组合是一种新颖的多模式治疗方法，用于使用这种有毒但有效的药物治疗RMS，同时最大限度地减少剂量限制毒性并可能增加对肿瘤的免疫反应。
阿霉素在>39°C的温度下从TLD迅速释放，远高于人体平均体温37°C，但不足以引起组织损伤或消融;这在43°C时开始发生，但随着温度接近60°C15，发生得更快。已经使用了多种方法在体内产生HT，包括激光，微波，射频消融和聚焦超声，其中许多是侵入性加热方法16。MRgHIFU是一种无创、非电离的加热方法，有助于在原位目标组织内精确设置温度。磁共振（MR）成像至关重要地提供了实时成像，其中可以使用计算机软件来计算整个治疗过程中组织的测温测量值;随后，该数据可用于实时控制超声治疗以达到并保持所需的温度设定点17。MRgHIFU已在各种组织类型中进行了测试，可用于从轻度激素疗法到消融术的各种温度治疗，以及临床上成功治疗疼痛的骨转移18。此外，HT已被证明可引起肿瘤细胞毒性，调节蛋白表达并改变肿瘤微环境中的免疫反应19，20，21，22。一项研究将轻度激素疗法与TLD相结合，然后用MRgHIFU消融，在协同R1大鼠模型23中，导致肿瘤核心坏死和药物输送到外围。传统上，放疗被用作辅助疗法，以破坏肿瘤细胞并减少局部疾病复发。然而，它的使用受到终生剂量和脱靶损伤的限制1.因此，HT的独特之处在于它可以引起一些相同的效果而没有相同的毒性或限制。
RMS的临床前动物模型包括免疫功能低下宿主中的同源免疫功能模型和患者来源的异种移植物（PDX）。虽然免疫功能低下的模型允许人类肿瘤的生长，但它们缺乏适当的肿瘤微环境，并且研究免疫反应的能力有限24。FGFR4 激活突变是预后不良的有希望的标志物，也是成人和儿童 RMS1，25 的潜在治疗靶点。在Gladdy实验室开发的同源RMS模型中，肿瘤能够在免疫功能正常的宿主中生长，该宿主对肿瘤产生先天性和适应性免疫反应26。由于HT影响免疫应答，观察小鼠免疫应答的变化是该肿瘤模型的宝贵优势。为了测试肿瘤对TLD的反应与FD相比，以及肿瘤对化疗和激素疗法的免疫反应的变化，开发并采用一种方案使用MRgHIFU和TLD治疗 体内 同系鼠RMS肿瘤，这是本研究的重点。
Protocol
研究是在表型基因组学中心（TCP）和大学健康网络（UHN）动物资源中心（ARC）动物研究设施的监督兽医的监督兽医的监督下按照动物护理委员会批准的动物使用方案进行的。所有涉及动物的程序，不包括MRgHIFU，都是在生物安全柜（BSC）中进行的，以尽量减少动物暴露于外部空气或易感染。 1.小鼠育种 注意：共有65只小鼠（菌株B6.129S2-Trp53tm1Tyj /…
Representative Results
使用MRgHIFU生成的热疗方案，后肢肿瘤能够在治疗期间持续加热到所需的设定温度（图4 显示了代表性治疗，10或20分钟，n = 65）。为了认为治疗成功，整个治疗过程中的ROI必须保持在39°C以上，在整个治疗过程中变化<6°C，并且不加热脱靶组织。此外，根据直肠探头或起始直肠温度加上食管探头温度的变化，核心温度必须保持在39°C以下（补充图2）。一旦MRgHIFU的?…
Discussion
本文开发的方案用于使用MRgHIFU靶向后肢肿瘤进行轻度激素疗法治疗，并从体内脂质体中释放包封的药物 。 在试点研究期间，该协议中遇到了几个关键步骤，优化这些关键步骤可以提高试点研究的治疗成功率。首先是完全去除要超声处理的区域上的毛发。皮毛内的任何气体捕获都会阻止超声波束通过并阻止超声波进入目标组织1.其次，小鼠定位对于成功治疗至关重要;肿瘤?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢该项目的资金来源和参与人员，包括：C17研究补助金，加拿大研究生奖学金，安大略省学生机会信托基金和詹姆斯J.哈蒙德基金。
Materials
| 1.5mL Eppendorf tubes | Eppendorf | 22363204 | |
| 1kb plus DNA Ladder | Froggabio | DM015-R500 | |
| 2x HS-Red Taq (PCR mix) | Wisent | 801-200-MM | |
| 7 Tesla MRI BioSpec | Bruker | T184931 | 70/30 BioSpec, Bruker, Ettlingen, Germany |
| C1000 Thermal cycler | Biorad | 1851148 | |
| Clippers | Whal Peanut | 8655 | |
| Compressed ultrasound gel | Aquaflex | HF54-004 | |
| Convection heating device | 3M Bair Hugger | 70200791401 | |
| Depiliatory cream | Nair | 61700222611 | Shopper's Drug Mart |
| DMEM | Wisent | 219-065-LK | |
| DNeasy extraction kit | Qiagen | 69504 | |
| DPBS | Wisent | 311-420-CL | |
| Drug injection system | Harvard Apparatus | PY2 70-2131 | PHD 22/2200 MRI compatible Syringe Pump |
| Eye lubricant | Optixcare | 50-218-8442 | |
| F10 Media | Wisent | 318-050-CL | |
| FBS | Wisent | 081-105 | |
| Froggarose | FroggaBio | A87 | |
| Gel Molecular Imager | BioRad | GelDocXR | |
| Glutamax | Wisent | 609-065-EL | |
| Heat Lamp | Morganville Scientific | HL0100 | Similar to this product |
| Intravascular Polyethylene tubing (0.015" ID x 0.043" OD, 20G) | SAI infusion | PE-20-100 | |
| Isoflurane | Sigma | 792632 | |
| M25FV24C Cell line | Gladdy Lab | N/A | |
| Microliter Syringe | Hamilton | 01-01-7648 | |
| Molecular Imager Gel Doc XR | Biorad | 170-8170 | |
| Mouse holder | The 3D printing material used was ABS-M30i, and it was printed on FDM Fortus 380mc machine | N/A | Dimensions: length = 43 mm, outer radius = 15 mm, inner width (where the mouse would sit) = 20.7 mm. |
| MyRun Machine | Cosmo Bio Co Ltd | CBJ-IMR-001-EX | |
| Nanodrop 8000 Spectrophotometer | Thermo Scientific | ND-8000-GL | |
| p53 primers | Eurofins | N/A | Custom Primers |
| PCR tubes | Diamed | SSI3131-06 | |
| Penicillin/Streptomycin | Wisent | 450-200-EL | |
| Proteus software | Pichardo lab | N/A | |
| Respiratory monitoring system | SAII | Model 1030 | MR-compatible monitoring and gating system for small animals |
| Small Bore HIFU device, LabFUS | Image Guided Therapy | N/A | LabFUS, Image Guided Therapy, Pessac, France Number of elements 8 frequency 2.5 MHz diameter 25 mm radius of curvature 20 mm Focal spot size 0.6 mm x 0.6 mm x 2.0 mm Motor: axes 2 Generator: Number of channels 8 Maximum electrical power/channel Wel 4 Maximum electrical power Wel 32 Bandwidth 0.5 – 5 MHz Control per channel: Freq., Phase and. amplitude Measurements per channel: Vrms, Irms, cos(theta) Duty Cycle at 100% power % 100% for 1 min. Transducer: Number of elements 8 frequency 2.5 MHz diameter 25 mm radius of curvature 20 mm Focal spot size 0.6 mm x 0.6 mm x 2.0 mm |
| SYBR Safe | ThermoFisher Scientific | S33102 | |
| TAE | Wisent | 811-540-FL | |
| Tail vein catheter (27G 0.5" ) | Terumo Medical Corp | 15253 | |
| Thermal probes | Rugged Monitoring | L201-08 | |
| Trypan blue | ThermoFisher Scientific | 15250061 | |
| Trypsin | Wisent | 325-052-EL | |
| Ultrasound Gel | Aquasonic | PLI 01-08 |
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Cite This Article
Wunker, C., Piorkowska, K., Keunen, B., Babichev, Y., Wong, S. M., Regenold, M., Dunne, M., Nomikos, J., Siddiqui, M., Pichardo, S., Foltz, W., Waspe, A. C., Gerstle, J. T., Drake, J. M., Gladdy, R. A. Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model. J. Vis. Exp. (191), e64544, doi:10.3791/64544 (2023).