Mercatali Laura, La Manna Federico, Miserocchi Giacomo, Liverani Chiara, De Vita Alessandro, Spadazzi Chiara, Bongiovanni Alberto, Recine Federica, Amadori Dino, Ghetti Martina, Ibrahim Toni
Osteoncology and Rare Tumors Center, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, 47014 Meldola, Italy.
Biomedical and Neuromotor Sciences Department, University of Bologna, 40123 Bologna, Italy.
Int J Mol Sci. 2017 Jul 29;18(8):1655. doi: 10.3390/ijms18081655.
Although bone metastases represent a major challenge in the natural history of breast cancer (BC), the complex interactions involved have hindered the development of robust in vitro models. The aim of this work is the development of a preclinical model of cancer and bone stromal cells to mimic the bone microenvironment. We studied the effects on osteoclastogenesis of BC cells and Mesenchymal stem cells (MSC) cultured alone or in combination. We also analyzed: (a) whether the blockade of the Epithelial Growth Factor Receptor (EGFR) pathway modified their influence on monocytes towards differentiation, and (b) the efficacy of bone-targeted therapy on osteoclasts. We evaluated the osteoclastogenesis modulation of human peripheral blood monocytes (PBMC) indirectly induced by the conditioned medium (CM) of the human BC cell line SCP2, cultured singly or with MSC. Osteoclastogenesis was evaluated by TRAP analysis. The effect of the EGFR blockade was assessed by treating the cells with gefitinib, and analyzed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Western Blot (WB). We observed that SCP2 co-cultured with MSC increased the differentiation of PBMC. This effect was underpinned upon pre-treatment of the co-culture with gefitinib. Co-culture of SCP2 with MSC increased the expression of both the bone-related marker Receptor Activator of Nuclear Factor κB (RANK) and EGFR in BC cells. These upregulations were not affected by the EGFR blockade. The effects of the CM obtained by the cells treated with gefitinib in combination with the treatment of the preosteoclasts with the bone-targeted agents and everolimus enhanced the inhibition of the osteoclastogenesis. Finally, we developed a fully human co-culture system of BC cells and bone progenitor cells. We observed that the interaction of MSC with cancer cells induced in the latter molecular changes and a higher power of inducing osteoclastogenesis. We found that blocking EGFR signaling could be an efficacious strategy for breaking the interactions between cancer and bone cells in order to inhibit bone metastasis.
尽管骨转移是乳腺癌（BC）自然病程中的一个重大挑战，但其中涉及的复杂相互作用阻碍了强大的体外模型的开发。这项工作的目的是开发一种癌症和骨基质细胞的临床前模型，以模拟骨微环境。我们研究了单独培养或联合培养的BC细胞和间充质干细胞（MSC）对破骨细胞生成的影响。我们还分析了：（a）上皮生长因子受体（EGFR）通路的阻断是否改变了它们对单核细胞分化的影响，以及（b）骨靶向治疗对破骨细胞的疗效。我们评估了由人BC细胞系SCP2单独培养或与MSC共同培养的条件培养基（CM）间接诱导的人外周血单核细胞（PBMC）的破骨细胞生成调节。通过抗酒石酸酸性磷酸酶（TRAP）分析评估破骨细胞生成。通过用吉非替尼处理细胞来评估EGFR阻断的效果，并用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐（MTT）法和蛋白质免疫印迹（WB）进行分析。我们观察到，与MSC共培养的SCP2增加了PBMC的分化。用吉非替尼对共培养物进行预处理后，这种效应得到了加强。SCP2与MSC的共培养增加了BC细胞中骨相关标志物核因子κB受体激活剂（RANK）和EGFR的表达。这些上调不受EGFR阻断的影响。用吉非替尼处理细胞获得的CM与用骨靶向药物和依维莫司处理前破骨细胞相结合的效果增强了对破骨细胞生成的抑制作用。最后，我们开发了一种BC细胞和骨祖细胞的完全人源共培养系统。我们观察到，MSC与癌细胞的相互作用在后者中诱导了分子变化以及更高的诱导破骨细胞生成的能力。我们发现，阻断EGFR信号传导可能是打破癌症与骨细胞之间的相互作用以抑制骨转移的有效策略。
