心内注射人前列腺癌细胞以创建骨转移异种移植小鼠模型
Summary
在这里，我们提出了一种用于心内注射人前列腺癌细胞的方案，以生成具有骨转移病变的小鼠模型。
Abstract
作为最常见的男性恶性肿瘤，前列腺癌（PC）的死亡率排名第二，主要是由于65%-75%的骨转移率。因此，了解前列腺癌骨转移的过程和相关机制对于开发新的治疗方法至关重要。为此，骨转移的动物模型是必不可少的工具。在这里，我们报告了通过心内注射前列腺癌细胞 来 生成骨转移小鼠模型的详细程序。生物发光成像系统可以确定前列腺癌细胞是否已准确注射到心脏中并监测癌细胞转移，因为它在监测转移性病变发展方面具有很大的优势。该模型复制播散癌细胞的自然发育，在骨骼中形成微转移，并模仿前列腺癌骨转移的病理过程。为进一步探索本病的分子机制和 体内 治疗效果提供了有效的工具。
Introduction
前列腺癌是112个国家中男性最常见的癌症，在人类发展指数较高的国家中排名第二1，2。前列腺癌患者的死亡大多是由转移引起的，约65%-75%的病例会发生骨转移3，4。因此，迫切需要预防和治疗前列腺癌骨转移，以改善前列腺癌患者的临床结果。骨转移动物模型是探索前列腺癌骨转移各阶段涉及的多阶段过程和分子机制，从而确定治疗靶点和开发新疗法不可或缺的工具5。
生成前列腺癌骨转移实验动物模型的最常见方法包括原位、骨干内（如胫骨内）和心内注射前列腺癌细胞。原位注射的骨转移模型是通过将前列腺癌细胞直接注射到小鼠的前列腺中而生成的6，7。该实验动物模型与前列腺癌骨转移具有非常相似的临床特征。然而，转移主要发生在腋窝淋巴结和肺，而不是骨8，9。前列腺癌的胫内注射模型直接将前列腺癌细胞注射到胫骨中，骨（胫骨）肿瘤形成率高10，11;但是，骨皮层和骨髓腔很容易受损。此外，胫骨注射方法不能刺激前列腺癌骨转移的病理过程，其中癌细胞通过循环定植骨骼。为了研究癌细胞骨转移率较高的循环、血管外渗和远处转移，已经开发出一种通过将前列腺癌细胞直接注射到小鼠左心室的心内注射技术8，12，13。这使其成为骨转移研究的有价值的动物模型8。心内注射法显示骨转移率约为75%9，14，远高于原位注射法。因此，心内注射是生成前列腺癌骨转移动物模型的理想方法。
这项工作旨在描述建立前列腺癌骨转移小鼠模型的过程，让读者可视化模型的建立。目前的工作提供了详细的过程，预防措施和说明性图片，以通过在无胸腺小鼠中心内注射人前列腺癌细胞 来 生成骨转移异种移植模型。该方法为进一步探索前列腺癌骨转移的分子机制和 体内 治疗效果提供了有效的工具。
Protocol
6至8周龄雄性BALB / c无胸腺小鼠（n = 10）在12小时光照/黑暗循环条件下，在无特异性（SPF）动物室中单独通风的小鼠笼子（5只小鼠/笼）中，自由获得SPF饲料和无菌水。小鼠在实验前适应性喂养一周。所有动物实验均经上海中医药大学动物福利委员会批准。 1. 细胞制备 在前列腺癌细胞注射当天，用预冷无菌PBS（pH 7.4）洗涤在10cm细胞培养皿中培养的80%-90%汇?…
Representative Results
生物发光成像在监测心内注射模型的转移性病变发展方面具有巨大的优势。癌细胞注射后不久（24小时内），使用生物发光成像来可视化进入一般循环的癌细胞（图3A）。当癌细胞被正确注射到动脉循环中时，全身都会看到明显的生物发光信号。来自仅在注射部位（心脏）显示生物发光信号的小鼠数据应从最终数据收集中排除。细胞注射后2周观察到后肢的转移性病变（<strong …
Discussion
心内注射人前列腺癌细胞产生骨转移是探索前列腺癌骨转移的功能和机制以及评价治疗效果的理想小鼠模型。研究表明，骨损伤最有可能发生在胫骨近端和股骨远端17，这可能是由于它们的高血管化和代谢活性。
由于骨转移是乳腺癌患者中经常观察到的转移性病变，因此通过心内注射乳腺癌细胞产生的骨转移模型也常用于乳腺癌研究18?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家重点研发计划（2018YFC1704300和2020YFE0201600）、国家自然科学基金（81973877和82174408）、上海中医药大学预算内的研究项目（2021LK047）和上海市医院中药制剂产业转化协同创新中心的资助。
Materials
| 1 mL syringes and needles | Shandong Weigao Group Medical Polymer Co., Ltd | 20200411 | The cells were injected into the ventricles of mice |
| Anesthesia machine | Shenzhen RWD Life Technology Co., Ltd | R500IP | Equipment for anesthetizing mice |
| Automatic cell counter | Shanghai Simo Biological Technology Co., Ltd | IC1000 | For counting cells |
| BALB/c athymic mice | Shanghai SLAC Laboratory Animal Co, Ltd. | Male | 6-8 week old, male mice |
| Bioluminescence imaging system | Shanghai Baitai Technology Co., Ltd | Vieworks | For tracking the tumor growth and pulmonary metastasis if the injected cells are labeled by luciferase |
| Centrifuge tube (15 mL, 50 mL) | Shanghai YueNian Biotechnology Co., Ltd | 430790, Corning | |
| EDTA solution | Wuhan Xavier Biotechnology Co., Ltd | G1105 | For decalcification of bone tissure |
| F-12 medium | Shanghai YueNian Biotechnology Co., Ltd | 21700075, GIBCO | Cell culture medium |
| Formalin solution | Shanghai YueNian Biotechnology Co., Ltd | BL539A | For fixing the specimen of each mouse |
| Isoflurane | Shenzhen RWD Life Technology Co., Ltd | VETEASY | For anesthesia |
| Lipofectamine 2000 | Shanghai YueNian Biotechnology Co., Ltd | 11668027, Thermo fisher | Plasmid transfection reagent |
| PC-3 cell line | Cell Bank of Chinese Academy of Sciences | TCHu 158 | Prostate cancer cell line |
| Phosphate-buffered saline | Beyotime Biotechnology | ST447 | Wash the human osteosarcoma cells |
| Trypsin (0.25%) | Shanghai YueNian Biotechnology Co., Ltd | 25200056, Gibco | For detaching the cells |
| Vector (pLV-luciferase) | Shanghai YueNian Biotechnology Co., Ltd | VL3613 | Plasmid for transfection |
| X-ray imaging system | Brook (Beijing) Technology Co., Ltd | FX PRO | For obtaining x-ray images to detect tumor growth |
| μCT80 | Shenzhen Fraun Technology Service Co., Ltd | Scanco Medical AG,Switzerland | For detection of bone destruction. The mico-CT is equipped with 3DCalc, cone reconstruction, and μCT Ray V3.4A model visualization software. |
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Cite This Article
Chang, J., Sun, X., Ma, X., Zhao, P., Shi, B., Wang, Y., Han, X., Yang, Y. Intra-Cardiac Injection of Human Prostate Cancer Cells to Create a Bone Metastasis Xenograft Mouse Model. J. Vis. Exp. (189), e64589, doi:10.3791/64589 (2022).