Turk Amy N, Byer Stephanie J, Zinn Kurt R, Carroll Steven L
Department of Pathology, University of Alabama at Birmingham, USA.
J Vis Exp. 2011 Mar 7(49):2558. doi: 10.3791/2558.
Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome(1)and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop. Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period(2). However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)(2). Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types. Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 1(3) and most commonly arise in the sciatic nerve(4). Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.
虽然体外筛选对于初步鉴定候选治疗药物至关重要，但必须在合适的动物模型中对药物抑制肿瘤生长的能力进行严格评估。最适合此目的的动物模型类型是一个激烈讨论的话题。一些证据表明，在转基因小鼠中研究药物对肿瘤影响的临床前试验对临床结果的预测性更强(1)，因此候选治疗药物通常在这些模型中进行测试。不幸的是，许多肿瘤类型没有转基因模型。此外，转基因模型往往还有其他局限性，如小鼠肿瘤对人类肿瘤的模拟程度、肿瘤表型的不完全外显率以及无法预测肿瘤何时会发生等问题。因此，如果没有合适的转基因肿瘤模型，许多研究人员会在临床前试验中使用异种移植模型（将人类肿瘤细胞移植到免疫缺陷小鼠中）。即使有转基因模型，它们也常常与异种移植模型结合使用，因为后者有助于快速确定治疗范围。此外，这种结合可以比较药物在转基因肿瘤和真正的人类肿瘤细胞中的有效性。从历史上看，异种移植通常是通过皮下注射肿瘤细胞（异位异种移植）来进行的。这种技术快速且可重复，相对便宜，并且在治疗期间可以持续定量肿瘤生长(2)。然而，皮下空间并非大多数肿瘤的正常微环境，因此由于宿主组织和肿瘤基因缺乏器官特异性表达等因素，异位异种移植获得的结果可能会产生误导。因此，强烈建议用将人类肿瘤细胞移植到其起源组织（原位异种移植）的研究来取代或补充异位移植研究(2)。不幸的是，由于许多肿瘤类型尚未开发出原位异种移植方法，这一建议的实施常常受到阻碍。恶性外周神经鞘瘤（MPNSTs）是一种高度侵袭性的肉瘤，可散发或与1型神经纤维瘤病相关(3)，最常见于坐骨神经(4)。在这里，我们描述了一种技术上简单的方法，即将萤火虫荧光素酶标记的人类MPNST细胞原位异种移植到免疫缺陷小鼠的坐骨神经中。我们还讨论了评估个体动物移植程序成功与否以及随后无偏随机分组到研究组的方法。
