Decoding novel therapeutic targets for the bone metastatic neuroblastoma using a tailored 3D in vitro engineered model recapitulating bone-like microenvironment

转移性癌症仍然是一个治疗挑战，大量患者未能对多模式治疗作出充分反应。神经母细胞瘤是发生在学龄前儿童的最常见的颅外实体肿瘤，单一靶向治疗仅具有有限的肿瘤特异性，且副作用严重，且由于靶分子的异质表达和肿瘤细胞的可塑性，常常失败。这种疾病的特异性反映了胚胎起源和儿童在一岁以内发病的情况，这往往阻碍了收集足够的肿瘤材料进行更全面的分子和细胞生物学分析的可能性。为了寻找新的药物靶点和提出更有效的治疗策略，需要更好地了解转移性疾病的分子。为了解决这个问题，需要3D体外模型来模拟肿瘤细胞浸润骨髓的天然结构。我们最近介绍了这样一个基于简化支架系统的模型，该系统具有定制的相互连接的微通道，并在原理验证研究中证实了其与神经母细胞瘤细胞生长的生物相容性。在这里提出的研究项目中，将利用生物医学工程技术、生物材料和3D打印技术的进步，建立一个更复杂的骨和转移性骨髓环境组成。由基质细胞、内皮细胞、造血干细胞和神经母细胞瘤细胞组成的多细胞3D共培养模型将使用共聚焦、双光子和电子显微镜进行表征，以评估其与天然骨髓的功能相似性。接下来，分子和细胞生物学方法将应用于更详细地研究转移性微环境如何维持神经母细胞瘤细胞的存活并形成它们对药物的反应。特别是，转录组学和表观遗传学分析将被执行，以揭示受3D生长条件影响的主要基因。随后将使用免疫细胞化学方法对其蛋白产物进行验证，以评估其在支架内的表达和分布。此外，免疫图谱将被破译，以研究哪些分子在人工骨髓生态位中更受青睐，并在体外确定它们的生物学作用。根据现有文献，将评估靶向致瘤效应物的潜力，并通过测量细胞活力和毒性来测试选定的阻滞剂。这个项目的结果将提高对转移性神经母细胞瘤如何与非肿瘤细胞相互作用的认识，并指出假定的新的药物靶点。这可以为更有效和量身定制的治疗弥散到骨髓中的肿瘤细胞开辟新的策略，并确定所提出的体外模型在研究更复杂的肿瘤相关过程中的适用性。

Metastatic cancers remain a therapeutic challenge with a significant number of patients failing to respond sufficiently to multimodal therapies. In Neuroblastoma, most frequent extracranial solid tumors developing in preschool children, single targeted therapies show only limited tumor specificity causing severe side effects and frequently fail due to heterogeneous expression of target molecules and tumor cell plasticity. Specificity of the disease that reflects embryonal origin and onset in children within their first years of life often impedes a possibility for collecting enough tumor material for more comprehensive molecular and cellular biology analyses. Better molecular understanding of the metastatic disease is required for finding new druggable targets and propose more effective therapeutic strategies. In order to address this issue, 3D in vitro models that mimic native structure of bone marrow infiltrated by tumor cells are required. We recently introduced such a model based on a simplified scaffold system with tailored interconnected micro-channels and confirmed in a proof-of-principle study its biocompatibility with neuroblastoma cell growth. In here proposed research project, a more complex composition of the bone and metastatic bone marrow environment will be established by leveraging advances of biomedical engineering technologies, biomaterials, and 3D printing technique. A multi-cellular 3D co-culture model made of stromal, endothelial, hematopoietic stem and neuroblastoma cells will be then characterized using confocal, two-photon and electron microscopy to evaluate its functional resemblance with native bone marrow. Next, molecular and cellular biology approaches will be applied to scrutinize more in details how metastatic microenvironment sustain neuroblastoma cells survival and shape their response to drugs. In particular, transcriptomics and epigenetic analyses will be performed to reveal main genes that are influenced by 3D growth conditions. Validations of their protein products will be done in following using immunocytochemistry approach to assess expression and distribution inside the scaffolds. As well, immunologic profiling will be deciphered to investigate which molecules are favored in the artificial bone marrow niche and their biological role will be determined in vitro. Based on the available literature, the potential for targeting pro-tumorigenic effectors will be evaluated, and selected blockers will be tested in vitro by measuring cell viability and toxicity. The results of this project will improve knowledge of how metastatic neuroblastoma interact with non-tumor cells and indicate putative new druggable targets. This could open new strategies for more effective and tailored treatments of tumor cells disseminated into the bone marrow and define applicability of proposed in vitro model for studying more sophisticated tumor-related processes.