近红外光免疫疗法为小鼠模型的胸膜传播
Summary
近红外光免疫疗法（NIR-PIT）是一种新兴的癌症治疗策略，利用抗体光吸收剂（IR700Dye）结合光和NIR光来破坏癌细胞。在这里，我们提出了一种方法，评估NIR-PIT的抗肿瘤作用，在小鼠模型的胸膜传播肺癌和恶性胸膜间皮瘤使用生物发光成像。
Abstract
光免疫疗法的疗效可以更准确地评估与矫名小鼠模型比皮下模型。可用于评估肺癌或恶性胸膜间皮瘤等胸内疾病的治疗方法。
近红外光免疫疗法（NIR-PIT）是最近开发的癌症治疗策略，它结合了肿瘤靶向抗体的特异性与光吸收物（IR700Dye）暴露在NIR光下后引起的毒性。NIR-PIT的功效已报告使用各种抗体：然而，只有少数报告在矫名模型中显示了这种策略的治疗效果。在本研究中，我们展示了一个使用NIR-PIT治疗的胸膜传播肺癌模型的疗效评估示例。
Introduction
尽管进行了数十年的研究，癌症仍然是导致死亡的主要原因之一。原因之一是放射治疗和化疗是高侵入性技术，这可能限制其治疗效果。细胞或分子靶向疗法，这是侵入性较小的技术，正受到越来越多的关注。光免疫疗法是一种通过结合免疫治疗和光疗协同提高治疗效果的治疗方法。免疫疗法通过增加肿瘤微环境的免疫原性，减少免疫调节抑制，提高肿瘤免疫力，导致肿瘤在体内的破坏。光疗通过光敏剂和光线的组合破坏原发性肿瘤，从肿瘤细胞中释放出的肿瘤特异性抗原增强肿瘤免疫力。肿瘤可以选择性地使用光敏剂治疗，因为它们是特定和选择性的目标细胞。光疗模式包括光动力疗法（PDT）、光热疗法（PTT）和光化学疗法1。
近红外光免疫疗法（NIR-PIT）是一种最近开发的抗肿瘤光疗方法，结合了光化学疗法和免疫疗法1，2。NIR-PIT 是一种分子靶向疗法，通过将近红外硅邻苯二甲酸酯染料 IRdye 700DX （IR700） 与单克隆抗体 （mAb） 结合，针对特定细胞表面分子。目标细胞的细胞膜在用NIR光（690纳米）3照射时被破坏。
使用靶向光疗法的概念，结合传统的光敏剂和抗体或靶向PDT是超过30年的历史4，5。先前的研究试图通过将常规PDT制剂与抗体结合来瞄准它们。然而，由于光敏剂6，7的疏水性，这些结合体被困在肝脏中，因此成功有限。此外，NIR-PIT的机制与传统的PDT机制完全不同。传统的光敏剂产生氧化应激，这种应激来自能量转换，这种能量转换吸收光能，脱位到兴奋状态，过渡到地面状态，并导致凋亡。然而，NIR-PIT通过光化学反应8将光敏剂聚合在膜上，直接破坏细胞膜，从而导致快速坏死。NIR-PIT 在很多方面优于传统的目标 PDT。传统光敏剂的消亡系数较低，需要将大量光敏剂附着在单个抗体分子上，从而可能降低结合亲和力。大多数传统光敏剂是疏水剂，因此很难将光敏剂与抗体结合，而不影响其免疫反应或体内目标积累。传统光敏剂通常吸收可见范围内的光线，减少组织渗透。
关于NIR-PIT针对内胸肿瘤，如肺癌和恶性胸膜间皮瘤（MPM）细胞的几项研究已经报告了9，10，11，12，13，14，15，16，17。然而，只有少数报告描述了NIR-PIT在胸膜传播MPM或肺癌模型9，10，11，12的疗效。皮下肿瘤异种模型被认为是标准肿瘤模型，目前广泛用于评估新疗法18的抗肿瘤效果。然而，皮下肿瘤微环境是不允许发展一个适当的组织结构或条件，适当地回顾一个真正的恶性表型19，20，21，22。理想情况下，应建立正位疾病模型，以便更精确地评估抗肿瘤效应。
在这里，我们展示了一种在小鼠模型的胸膜传播肺癌，这是使用NIR-PIT治疗的功效评估方法。通过将肿瘤细胞注射到胸腔中，并使用荧光酶发光进行确认，生成胸腔传播小鼠模型。小鼠接受静脉注射，注射与IR700和NIR照射到胸部的mAb。使用荧光酶发光评估治疗效果。
Protocol
所有活体实验均按照名古屋大学动物护理和使用委员会实验室动物资源的护理和使用指南（批准 #2017-29438，#2018-30096，#2019-31234，#2020-20104）进行。在名古屋大学动物中心购买并饲养了六周大的同源性裸体小鼠。在小鼠进行手术时，他们麻醉时会用异黄素（介绍：4-5%，维持2-3%）：爪子被用钳子压住， 以确认麻醉的深度。 1. IR700 与 mAb 的结合 孵化 mAb …
Representative Results
抗多普兰素抗体NZ-1与IR700结合产生NZ-1-IR700。我们确认了 NZ-1 和 IR700 在 SDS-PAGE（图 8）上的绑定。Luciferase表达H2373（H2373-luc）是由通过用荧光酶基因10转染恶性间皮瘤细胞（H2373）来制备的。 我们麻醉了8-12周大的雌性同源性同源性裸鼠，并将1×10个H2373-luc细胞注射到胸腔中。将肿瘤细胞注射到小鼠体内的当天被指示为第1天。 <…
Discussion
在这项研究中，我们展示了一种测量NIR-PIT对MPM胸膜传播模型的治疗效果的方法。用NIR-PIT进行了高度选择性的细胞杀戮;因此，正常组织几乎没有损坏23，24，25。通过这种选择性细胞杀伤，NIR-PIT在传播的9、26型中被证明是安全的。但是，某些步骤可能采用替代…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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Materials
| 0.25w/v% Trypsin-1mmol/l EDTA 4Na Solution with Phenol Red | Wako | 209-016941 | for cell culture |
| 1mL syringe | TERUMO | SS-01T | for mice experiment |
| 30G needle | Nipro | 1907613 | for mice experiment |
| BALB/cSlc-nu/nu | Japan SLC | ||
| Collidal Blue Staining Kit | Invitrogen | LC6025 | use for gel protein staining |
| Coomassie (bradford) Plus protein assay | Thermo Fisher Scientific Inc (Waltham, MA, USA) | PI-23200 | for measuring the APC concentration |
| Dimethyl sulfoxide (DMSO) | Wako | 043-07216 | use for conjugation of IR700 |
| D-Luciferin (potassium salt) | Cayman Chemical | 14681 | for bioluminescence imaging and DLIT |
| GraphPad Prism7 | GraphPad software | for statistical analysis | |
| Image Studio | Li-Cor Biosciences | for analyzing 700 nm fluorescent image | |
| IRDye 700DX Ester Infrared Dye | LI-COR Bioscience (Lincoln, NE, USA) | 929-70011 | |
| isoflurane | Wako | 095-06573 | for mice anesthesia |
| IVIS Spectrum CT | PerkinElmer | for capturing bioluminescent image and DLIT | |
| Living Image | PerkinElmer | for analyzing bioluminescent image and DLIT | |
| Na2HPO4 | SIGMA-ALDRICH (St. Louis, MO, USA) | S9763 | use for conjugation of IR700 |
| NIR Laser | Changchun New Industries Optoelectronics Technology | MRL-III-690R | for NIR irradiation |
| Novex WedgeWell 4 to 20%, Tris-Glycine, 1.0 mm, Mini Protein Gel, 12 well | Invitrogen | XP04202BOX | use for SDS-PAGE |
| NuPAGE LDS Sample Buffer (x4) | Invitrogen | NP0007 | use for SDS-PAGE |
| Optical power meter | Thorlabs (Newton, NJ, USA) | PM100 | for measuring the output of the NIR laser |
| PBS(-) | Wako | 166-23555 | |
| Pearl Trilogy imaging system | Li-Cor Biosciences | for capturing 700 nm fluorecent image | |
| Penicilin-Streptomycin Solution (x100) | Wako | 168-23191 | for cell culture |
| Puromycin Dihydrochloride | ThermoFisher | A1113803 | for luciferase transfection |
| RediFect Red-Fluc-Puromycin Lentiviral Prticles | PerkinElmer | CLS960002 | for luciferase transfection |
| RPMI-1640 with L-glutamine and Phenol Red | Wako | 189-02025 | for cell culture |
| Sephadex G25 column (PD-10) | GE Healthcare (Piscataway, NJ, USA) | 17-0851-01 | use for conjugation of IR700 |
| UV-1900i | Shimadzu | for measuring the APC concentration |
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Cite This Article
Yasui, H., Nishinaga, Y., Taki, S., Takahashi, K., Isobe, Y., Sato, K. Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination. J. Vis. Exp. (168), e61593, doi:10.3791/61593 (2021).