肝细胞癌原位小鼠模型中的无创PET/MR成像
Summary
在这里，我们提出了一种方案来创建有和没有肝动脉结扎的原位肝细胞癌异种移植物，并使用[18 F]氟咪唑（[18 F]FMISO）和[18 F]氟脱氧葡萄糖（[18F]FDG）对肿瘤缺氧进行非侵入性正电子发射断层扫描（PET）成像。
Abstract
肝细胞癌（HCC）的临床前实验模型概括了人类疾病，是研究肿瘤发生和评估新治疗方法的重要工具。使用正电子发射断层扫描 （PET） 的非侵入性全身成像可在分子水平上实时提供对组织体内特征的关键见解。我们在这里提出了一个原位HCC异种移植物创建的方案，有和没有肝动脉结扎（HAL）诱导肿瘤缺氧，并使用[18 F]氟咪唑（[18 F]FMISO）和[18 F]氟脱氧葡萄糖（[18F]FDG）PET/磁共振（MR）成像评估其体内肿瘤代谢。使用缺氧标记物[18 F]FMISO可以很容易地观察到肿瘤缺氧，并且发现接受HAL的HCC小鼠的[18 F]FMISO摄取高于非HAL组，而[18F]FDG无法区分两组之间的肿瘤缺氧。HAL肿瘤在缺氧反应中也表现出更高水平的缺氧诱导因子（HIF）-1α表达。HAL肿瘤的定量显示，基于标准化价值摄取（SUV）方法的[18F] FMISO摄取增加了2.3倍。
Introduction
肝细胞癌 （HCC） 是全球第六大确诊癌症和第三大常见癌症死因，2020 年新增病例超过 900，000 例，死亡800，000 例 1.主要危险因素是肝硬化，这是病毒感染（乙型和丙型肝炎病毒）、酗酒、糖尿病和非酒精性脂肪性肝炎2 的结果。肝细胞癌的管理相当复杂，有几种治疗选择，包括手术切除、热消融或化学消融、移植、经动脉化疗栓塞、放疗和化疗，具体取决于疾病分期2，3。肝细胞癌是一种化疗难治性肿瘤，在根治性治疗后，多达 70% 的患者会复发2。
尽管肿瘤异质性很高，但肝细胞癌与两种常见结局有关：（i）肝细胞癌非常缺氧，（ii）肿瘤缺氧与更大的肿瘤侵袭性和治疗失败有关。HCC细胞不受控制的增殖导致血管形成前的高耗氧率，从而产生缺氧微环境。低肿瘤内氧水平会触发一系列影响肿瘤侵袭性和治疗反应的生物反应。缺氧诱导因子（HIF）通常被认为是对缺氧反应的基本转录调节因子2，3。因此，检测缺氧的能力对于可视化肿瘤组织和识别需要侵入性手术的难以接近的部位至关重要。它还有助于更好地了解导致肿瘤侵袭性的分子变化并改善患者的治疗结果。
使用正电子发射断层扫描（PET）的分子成像通常用于许多癌症（包括HCC）的诊断和分期。特别是，联合使用涉及 [18F] 氟脱氧葡萄糖 （[18F]FDG） 和 [11C] 乙酸盐的双示踪 PET 成像可显著提高肝细胞癌诊断的总体敏感性4，5。另一方面，缺氧成像可以通过使用常用的缺氧标记物[18 F]氟米索硝唑（[18F]FMISO）来实现。在临床实践中，缺氧的无创评估对于区分各种类型的肿瘤和放射治疗计划的区域非常重要6。
临床前成像已成为对不同疾病的小鼠模型进行无创和纵向评估不可或缺的工具。稳健且高度可重复的HCC模型代表了人类HCC病理生理学的临床前和转化研究以及新疗法评估的重要平台。结合PET成像，可以阐明体内行为，以在任何给定时间点提供分子水平的重要见解。在这里，我们描述了用于生成肝动脉结扎（HAL）原位HCC异种移植物的方案，并使用[18 F]FMISO和[18F]FDG PET / MR分析其体内肿瘤代谢。HAL的掺入使得转基因或化学诱导的HCC小鼠异种移植物成为研究体内肿瘤缺氧的合适模型，因为HAL可以有效阻断动脉血液供应以诱导肿瘤内缺氧7，8。此外，与使用匹莫硝唑的离体免疫组织化学染色不同，缺氧引起的肿瘤代谢变化可以很容易地使用PET成像进行无创可视化和准确定量，从而能够纵向评估治疗反应或测量耐药性的出现3，7，8.我们这里显示的方法允许创建一个强大的缺氧HCC模型，以及使用PET / MR成像对肿瘤缺氧进行无创监测，以研究体内HCC生物学。
Protocol
Representative Results
Discussion
在这项研究中，我们描述了使用皮下肿瘤对肝原位HCC异种移植物进行HAL的程序，以及使用[18 F]FMISO和[18F]FDG PET / MR对原位异种移植物中肿瘤缺氧进行无创监测的方法。我们的兴趣在于各种癌症和疾病模型的代谢成像，用于早期诊断和治疗反应评估11，13，14，15。迄今为止，HAL HCC异种移?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢香港抗癌信托基金、香港研究资助局合作研究基金（CRF C7018-14E）对小动物影像实验的支持。我们亦感谢香港大学分子成像及医学回旋加速器中心（MIMCC）提供[18 F]FMISO及[18F]FDG的支持。
Materials
| 0.9% sterile saline | BBraun | N/A | 0.9% sodium chloride intravenous infusion, 500 mL |
| 10# Scalpel blade | RWD Life Science Co.,ltd | S31010-01 | Animal surgery tool |
| 10% povidone-iodine solution | Banitore | 6.425.678 | For disinfection |
| 25G needle with a 1 mL syringe | BD PrecisionGlide | N/A | 1 mL syringe with 25G needle for cell suspensions injections |
| 5 mL syringe | Terumo | SS05L | 5 mL syringe Luer Lock |
| 70% Ethanol | Merck | 1.07017 | For disinfection |
| Automated Cell Counter | Invitrogen | AMQAF2000 | For automated cell counting |
| Buprenorphine | HealthDirect | N/A | Subcutaneous injection (0.05-0.2 mg/kg/12 hours) for analgesic after surgery |
| Cell Culture Dish (60 mm diameter) | Thermo Scientific | 150462 | For tumor tissue processing |
| Centrifuge | Sigma | 3-16KL, fixed-angle rotor 12311 | For cell suspensions collection |
| Centrifuge Conical Tube | Eppendorf | EP0030122151 | For cell suspensions collection |
| Culture media (Dulbecco’s modified Eagle’s medium) | Gibco | 10566024 | high glucose, GlutaMAX™ Supplement |
| Digital Caliper | RS PRO | 841-2518 | For subcutaneous tumor size measurement |
| Direct heat CO2 incubator | Techcomp Limited | NU5841 | For cell culture |
| Dose calibrator | Biodex | N/A | Atomlab 500 |
| DPBS (Dulbecco’s phosphate-buffered saline) | Gibco | 14287072 | For cell wash and injection |
| Eye lubricant | Alcon Duratears | N/A | Sterile ocular lubricant ointment, 3.5 g |
| Fetal bovine serum (FBS) | Gibco | A4766801 | Used for a broad range of cell types, especially sensitive cell lines |
| Forceps (curved fine and straight blunt) | RWD Life Science Co.,ltd | F12012-10 & F12011-13 | Animal surgery tool |
| Heating pad | ALA Scientific Instruments | N/A | Heat pad for mice during surgery |
| Insulin syringe | Terumo | 10ME2913 | 1 mL insulin syringe with needle for radiotracer injections |
| InterView fusion software | Mediso | Version 3.03 | Post-processing and image analysis software |
| Inverted microscope | Yu Lung Scientific Co., Ltd | BM-209G | For cells morphology visualization |
| Isoflurane | Chanelle Pharma | N/A | Iso-Vet, inhalation anesthetic, 250 mL |
| Ketamine | Alfasan International B.V. | HK-37715 | Ketamine 10% injection solution, 10 mL |
| Medical oxygen | Linde HKO | 101-HR | compressed gas, 99.5% purity |
| nanoScan PET/MR Scanner | Mediso | N/A | 3 Tesla MR |
| Needle holder | RWD Life Science Co.,ltd | F31026-12 | Animal surgery tool |
| Nucline nanoScan software | Mediso | Version 3.0 | Scanner operating software |
| Nylon Suture (6/0 and 5/0) | Healthy Medical Company Ltd | 000524 & 000526 | Animal surgery tool |
| Penicillin- Streptomycin | Gibco | 15140122 | Culture media for a final concentration of 50 to 100 I.U./mL penicillin and 50 to 100 µg/mL streptomycin. |
| Pentabarbital | AlfaMedic | 13003 | Intraperitoneal injection (330 mg/kg) to induce cessation of breathing of mice |
| Sharp scissors | RWD Life Science Co.,ltd | S14014-10 | Animal surgery tool |
| Spring Scissors | RWD Life Science Co.,ltd | S11005-09 | Animal surgery tool |
| Trypan Blue Solution, 0,4% | Gibco | 15250061 | For cell counting |
| Trypsin-ethylenediaminetetraacetic acid (EDTA, 0.25%), phenol red. | Gibco | 25200072 | For cell digestion |
| Xylazine | Alfasan International B.V. | HK-56179 | Xylazine 2% injection solution, 30 mL |
References
- Sung, H., et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians. 71 (3), 209-249 (2021).
- Chen, C., Lou, T. Hypoxia inducible factors in hepatocellular carcinoma. Oncotarget. 8 (28), 46691-46703 (2017).
- Lu, R. -. C., et al. Positron-emission tomography for hepatocellular carcinoma: Current status and future prospects. World Journal of Gastroenterology. 25 (32), 4682-4695 (2019).
- Larsson, P., et al. Adding 11C-acetate to 18F-FDG at PET examination has an incremental value in the diagnosis of hepatocellular carcinoma. Molecular Imaging and Radionuclide Therapy. 21 (1), 6-12 (2012).
- Huo, L., et al. Kinetic analysis of dynamic 11C-acetate PET/CT imaging as a potential method for differentiation of hepatocellular carcinoma and benign liver lesions. Theranostics. 5 (4), 371-377 (2015).
- Lopci, E., et al. PET radiopharmaceuticals for imaging of tumor hypoxia: A review of the evidence. American Journal of Nuclear Medicine and Molecular Imaging. 4 (4), 365-384 (2014).
- Mao, X., et al. Mechanisms through which hypoxia-induced caveolin-1 drives tumorigenesis and metastasis in hepatocellular carcinoma. 암 연구학. 76 (24), 7242-7253 (2016).
- Kung-Chun Chiu, D., et al. Hypoxia regulates the mitochondrial activity of hepatocellular carcinoma cells through HIF/HEY1/PINK1 pathway. Cell Death & Disease. 10 (12), 934 (2019).
- Li, Y., et al. Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97. World Journal of Gastroenterology. 7 (5), 630-636 (2001).
- Faustino-Rocha, A., et al. Estimation of rat mammary tumor volume using caliper and ultrasonography measurements. Lab Animal. 42 (6), 217-224 (2013).
- Liu, Q., Tan, K. V., Chang, H. C., Khong, P. L., Hui, X. Visualization and quantification of brown and beige adipose tissues in mice using [18F] FDG micro-PET/MR imaging. Journal of Visualized Experiments: JoVE. (173), e62460 (2021).
- Lin, W. -. H., et al. Hypoxia-activated cytotoxic agent tirapazamine enhances hepatic artery ligation-induced killing of liver tumor in HBx transgenic mice. Proceedings of the National Academy of Sciences. 113 (42), 11937-11942 (2016).
- Wong, T. L., et al. CRAF methylation by PRMT6 regulates aerobic glycolysis-driven hepatocarcinogenesis via ERK-dependent PKM2 nuclear relocalization and activation. Hepatology. 71 (4), 1279-1296 (2020).
- Yang, X., et al. Development of cisplatin-loaded hydrogels for trans-portal vein chemoembolization in an orthotopic liver cancer mouse model. Drug Delivery. 28 (1), 520-529 (2021).
- Shi, J., et al. Longitudinal evaluation of five nasopharyngeal carcinoma animal models on the microPET/MR platform. European Journal of Nuclear Medicine and Molecular Imaging. 49 (5), 1497-1507 (2021).
- Kilian, K., et al. Imaging of hypoxia in small animals with F fluoromisonidasole. Nukleonika. 61 (2), 219-223 (2016).
- Kawamura, M., et al. Evaluation of optimal post-injection timing of hypoxic imaging with 18F-Fluoromisonidazole-PET/CT. Molecular Imaging and Biology. 23 (4), 597-603 (2021).
