Campbell J Preston, Merkel Alyssa R, Masood-Campbell S Kathryn, Elefteriou Florent, Sterling Julie A
Department of Pharmacology, Vanderbilt University, USA.
J Vis Exp. 2012 Sep 4(67):e4260. doi: 10.3791/4260.
Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio, that has since been transfected for GFP expression and re-cloned by our group. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis, in certain instances intratibial or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception. This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.
骨转移在包括乳腺癌、前列腺癌和肺癌在内的多种恶性肿瘤中很常见。肿瘤一旦在骨中形成，就会导致显著的发病率和死亡率。因此，非常有必要了解控制骨中肿瘤形成、生长和活性的分子机制。已经建立了几种体内模型来研究这些事件，每种模型都有其特定的优点和局限性。最常用的模型是将肿瘤细胞经心内注射直接注入无胸腺（裸）BalbC小鼠的动脉血供中。该方法可应用于许多不同的肿瘤类型（包括PC-3前列腺癌、肺癌和小鼠乳腺脂肪垫肿瘤）；然而，在本手稿中，我们将重点关注乳腺癌模型MDA-MB-231。在这个模型中，我们使用了一个高度骨选择性的克隆株，最初来自圣安东尼奥市蒙迪博士的研究小组，此后我们的小组对其进行了绿色荧光蛋白（GFP）表达转染并重新克隆。该克隆株是一种骨转移变异株，具有高骨嗜性，很少转移到肺、肝或肾上腺。虽然经心内注射最常用于骨转移研究，但在某些情况下，经胫骨或乳腺脂肪垫注射更合适。经心内注射通常在使用人类肿瘤细胞时进行，目的是监测转移的后期阶段，特别是癌细胞在骨中停滞、存活、增殖并形成发展为癌症诱导性骨病的肿瘤的能力。如果关注肿瘤转移到骨后癌细胞与骨的关系，即大致与已确立的转移性骨病相关，则进行经胫骨注射。这些模型都不能重现肿瘤细胞在栓塞和进入循环之前转移过程的早期步骤。如果监测原发肿瘤的生长或从原发部位到骨的转移，那么通常首选乳腺脂肪垫接种；然而，很少有肿瘤细胞系会持续从原发部位转移到骨，4T1骨优先克隆株（一种小鼠乳腺癌）是个例外。本手稿详细介绍了接种程序，并突出了接种后分析的关键步骤。具体来说，它包括细胞培养、经心内和经胫骨接种的肿瘤细胞接种程序，以及关于通过X射线、荧光和组织形态计量分析进行每周监测的简要信息。
