Mercatali Laura, Spadazzi Chiara, Miserocchi Giacomo, Liverani Chiara, De Vita Alessandro, Bongiovanni Alberto, Recine Federica, Amadori Dino, Ibrahim Toni
Osteoncology and Rare Tumors Center, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST), IRCCS;
Osteoncology and Rare Tumors Center, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST), IRCCS.
J Vis Exp. 2017 Sep 13(127):56311. doi: 10.3791/56311.
The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.
骨微环境中肿瘤细胞与骨细胞之间的相互作用对于理解骨转移形成机制至关重要。我们建立了一种体外全人源临床前模型，用于乳腺癌细胞与向破骨细胞分化的单核细胞的共培养。我们从健康供体采集的外周血样本开始优化破骨细胞生成模型。外周血单个核细胞（PBMCs）首先通过密度梯度离心分离，高密度接种，并通过添加两种生长因子（GFs）诱导分化：核因子κB受体活化因子配体（RANKL）和巨噬细胞集落刺激因子（MCSF）。细胞在培养中放置14天，然后固定并通过下游分析进行检测。在溶骨性骨转移中，癌细胞到达骨的作用之一是诱导破骨细胞生成。因此，我们用乳腺癌细胞共培养物挑战我们的模型，以研究癌细胞对生长因子的分化能力。研究癌细胞与破骨细胞相互作用的一种直接方法是基于使用从乳腺癌细胞培养物中收集并与新鲜培养基混合的条件培养基进行间接共培养。然后将这种混合物用于诱导破骨细胞分化。我们还优化了一种直接共培养方法，其中癌细胞和正在分化的单核细胞共享培养基并交换分泌因子。这是对原始间接共培养方法的重大改进，因为研究人员可以观察两种细胞类型的相互作用，并对癌细胞和破骨细胞进行下游分析。这种方法使我们能够研究药物对转移性骨微环境的影响，并接种除乳腺癌来源以外的细胞系。该模型还可用于研究其他疾病，如骨质疏松症或其他骨病。
