人前列腺癌的临床前原位小鼠模型
Summary
Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.
Abstract
To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.
Introduction
前列腺癌是美国男性中癌症死亡（9％）的第二最常见的原因，旁边的肺和支气管（28％）1的癌症。根据最近的数据，估计是220，800初诊前列腺癌病例和27，540死亡将发生在2015年1。早期前列腺癌的五个年相对存活率为> 99％，而晚期转移性疾病是只有28％1。治疗晚期转移性疾病的一个主要挑战是缺乏对本病的基本倾向的转移到其它器官，尤其是对骨骼，这是前列腺癌的一个频繁的部位分子机制的认识。因此，有一个明确的需要研究这些前列腺肿瘤的分子组成，以发展对发展有效的治疗方案，以先进的转移性疾病2,3。
前列腺肿瘤展览HIGħ生物异质没有明确定义的途径发展。转移往往与肿瘤的侵袭性4事先没有迹象发生。此临床异质性是由于前列腺癌的分子多样性。了解这些致命肿瘤的分子组成是设计出更好的诊断和治疗策略对本病的关键。因此，前列腺癌的研究目前主要集中在了解和预防转移。
临床前活体小鼠模型提供了多种选择，以了解前列腺癌的进展，以先进的转移性疾病的分子机制。此外，这些模型是对这种疾病的新的治疗策略的临床前评估重要。最常用的动物模型包括转基因小鼠模型，尾静脉注射，心内注入和人类原位小鼠模型。转基因研究是时间consumi纳克以及与人类的小鼠的前列腺癌的发展的相关性表明变性11。在自发转移小鼠模型，将细胞直接注射到循环和虽然，他们具有快速的周转时间，它们不能被用来研究在原发肿瘤或转移级联5的初始步骤。原位异种移植模型有显影骨转移灶，前列腺癌转移的常见部位的限制。然而，人类的原位前列腺癌异种移植的小鼠模型是充分表征的，并广泛用于研究原发性肿瘤发展，肿瘤和器官的微环境中，转移性疾病和使用的实验性药物用于治疗性干预6的初始相位之间的串扰的分子事件，7,8-11。
Protocol
Representative Results
Discussion
这份手稿描述了建立人体原位前列腺癌异种移植小鼠模型的详细过程。该模型是由人前列腺癌细胞系PC3M吕克-C 6的直接植入建立到免疫缺陷小鼠的背侧前列腺叶。肿瘤被允许开发在实验的过程中。在实验过程中的肿瘤生长每周监测由非侵入性生物发光成像系统。
在建立异种移植肿瘤模型中最重要的因素是整个肿瘤细胞的植入实现的一致性。以获得统计学显著的结果，各试验组…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We thank Dr. Roger Erickson for his support and assistance with the preparation of the manuscript. This work was supported by the National Cancer Institute at the National Institutes of Health through grant numbers RO1CA160079, RO1CA138642, UO1CA184966 and VA funded program project number 1P1 BX001604.
Materials
| PC3 prostate cancer cell line | ATCC | CRL-1435 | |
| Minimum Essential Medium (MEM) | GIBCO,Life Technology | 11095-080 | |
| PBS | GIBCO,Life Technology | 10010-023 | |
| FBS | GIBCO,Life Technology | 10437-028 | |
| Zeocin | Invitrogen,Life Technology | R250-01 | |
| Trypsin | GIBCO,Life Technology | 25300-54 | |
| IVIS | Xenogen-Caliper | ||
| Insulin Syringes (300ul, 28.5g) | Becton Dickinson | 309300 | |
| Mice | Charles River Laboratories, Inc | ||
| Alcohol Swabs | MEDEquip Depot | 326895 BD | |
| PVP Iodine Prep Pad | MEDEquip Depot | C12400PDI | |
| Surgical CatGut Chromic Suture | Demetech | CC224017F0P | |
| Matrigel | Corning | 354248 |
Riferimenti
- Siegel, R. L., Miller, K. D., Jemal, A. Cancer statistics, 2015. CA Cancer J Clin. 65 (1), 5-29 (2015).
- Andrieu, C., et al. Heat shock protein 27 confers resistance to androgen ablation and chemotherapy in prostate cancer cells through eIF4E. Oncogene. 29 (13), 1883-1896 (2010).
- Fusi, A., et al. Treatment options in hormone-refractory metastatic prostate carcinoma. Tumori. 90 (6), 535-546 (2004).
- Hughes, C., Murphy, A., Martin, C., Sheils, O., O’Leary, J. Molecular pathology of prostate cancer. J Clin Pathol. 58 (7), 673-684 (2005).
- Pavese, J., Ogden, I. M., Bergan, R. C. An orthotopic murine model of human prostate cancer metastasis. J Vis Exp. (79), e50873 (2013).
- Pettaway, C. A., et al. Selection of highly metastatic variants of different human prostatic carcinomas using orthotopic implantation in nude mice. Clin Cancer Res. 2 (9), 1627-1636 (1996).
- Rembrink, K., Romijn, J. C., van der Kwast, T. H., Rubben, H., Schroder, F. H. Orthotopic implantation of human prostate cancer cell lines: a clinically relevant animal model for metastatic prostate cancer. Prostate. 31 (3), 168-174 (1997).
- Kim, S. J., et al. Blockade of epidermal growth factor receptor signaling in tumor cells and tumor-associated endothelial cells for therapy of androgen-independent human prostate cancer growing in the bone of nude mice. Clin Cancer Res. 9 (3), 1200-1210 (2003).
- Kim, S. J., et al. Targeting platelet-derived growth factor receptor on endothelial cells of multidrug-resistant prostate cancer. J Natl Cancer Inst. 98 (11), 783-793 (2006).
- Park, S. I., et al. Targeting SRC family kinases inhibits growth and lymph node metastases of prostate cancer in an orthotopic nude mouse model. Cancer Res. 68 (9), 3323-3333 (2008).
- Zhang, J., et al. AFAP-110 is overexpressed in prostate cancer and contributes to tumorigenic growth by regulating focal contacts. J Clin Invest. 117 (10), 2962-2973 (2007).
- Park, S. I., Kim, S. J., McCauley, L. K., Gallick, G. E. Pre-clinical mouse models of human prostate cancer and their utility in drug discovery. Curr Protoc Pharmacol. Chapter 14, Unit 14.15 (2010).
- Johnson, L. C., et al. Longitudinal live animal micro-CT allows for quantitative analysis of tumor-induced bone destruction. Bone. 48 (1), 141-151 (2011).
- Steinbauer, M., et al. GFP-transfected tumor cells are useful in examining early metastasis in vivo, but immune reaction precludes long-term tumor development studies in immunocompetent mice. Clin Exp Metastasis. 20 (2), 135-141 (2003).
- Yang, M., et al. A fluorescent orthotopic bone metastasis model of human prostate cancer. Cancer Res. 59 (4), 781-786 (1999).
- Stephenson, R. A., et al. Metastatic model for human prostate cancer using orthotopic implantation in nude mice. J Natl Cancer Inst. 84 (12), 951-957 (1992).
- Hoffman, R. M. Orthotopic metastatic mouse models for anticancer drug discovery and evaluation: a bridge to the clinic. Invest New Drugs. 17 (4), 343-359 (1999).
- Wang, X., An, Z., Geller, J., Hoffman, R. M. High-malignancy orthotopic nude mouse model of human prostate cancer LNCaP. Prostate. 39 (3), 182-186 (1999).
- An, Z., Wang, X., Geller, J., Moossa, A. R., Hoffman, R. M. Surgical orthotopic implantation allows high lung and lymph node metastatic expression of human prostate carcinoma cell line PC-3 in nude mice. Prostate. 34 (3), 169-174 (1998).
- Kim, S. J., et al. Reduced c-Met expression by an adenovirus expressing a c-Met ribozyme inhibits tumorigenic growth and lymph node metastases of PC3-LN4 prostate tumor cells in an orthotopic nude mouse model. Clin Cancer Res. 9 (14), 5161-5170 (2003).
