VEC3-Valve Enabled Cell Co-Culture Platforms for Cancer Biology Study

描述(申请人提供)：越来越清楚的是，肿瘤微环境在肿瘤进展中起着关键作用，这表明需要开发技术来研究肿瘤细胞在不同微环境中的行为。为了实现这一目标，我们创建了瓣膜使能细胞共培养(VIEVE ENABLED CEL-COMPLATION，VIE3)平台，这是一种新型的微流控设备，旨在分析肿瘤微环境中肿瘤细胞与其他细胞之间的相互作用。该技术通过可溶性因子或空间上分离的细胞群体之间的物理接触，实现了不同细胞类型的单独培养和细胞间的相互作用，同时保持了对各自培养环境的流体控制。此外，通过选择性地阻断不同细胞群体之间的特定配体的交换，该平台可以用于识别相关感兴趣的配体的功能。传统的细胞共培养技术和已报道的微流控细胞共培养平台具有局限性，不能满足所有重要的细胞共培养需求。建议的ve3细胞共培养平台，通过引入一个简单、坚固和用户友好的气动或液压控制阀来可逆地分离或连接相邻的细胞培养室，不仅允许单独培养和处理单个细胞类型，而且还允许对细胞相互作用进行实时的活细胞成像。到目前为止，作为原理的证明，Vec3已经被应用于动态观察海马神经元之间的突触形成，分析常氧和低氧环境下肿瘤-内皮细胞的相互作用，在3D基质中研究肿瘤-成纤维细胞的相互作用，以及定量各种分子介导的肿瘤-内皮细胞的交叉迁移。在拟议的研究中，我们将通过定量描述细胞微环境，包括细胞密度和均匀性、葡萄糖和氧气浓度以及细胞与细胞的相互作用速率，来进一步开发ve3。通过改进的设计和性能工程，我们将开发优化的平台和操作方案，以提高基于ve3的检测的成功率。此外，我们将实施新的功能，如通过阻断两个细胞群体之间特定配体的交换来控制细胞与细胞之间的相互作用。更重要的是，我们将应用ve3来研究各种配体和受体介导的肿瘤-内皮细胞的交叉迁移，并确定特定配体在细胞-细胞相互作用中的功能。因此，这项研究的成功实施将导致一类新的多功能、多功能的ve3微流控平台广泛应用于癌症生物学。该设备将被用来阐明肿瘤血管生成、血管内皮细胞生长和转移的分子机制，最终可能导致更好的癌症治疗。总体的量化里程碑是在使用ve3平台进行具有细胞通讯定量参数的肿瘤血管生成、血管内和转移研究的分析中达到95%的成功率。 公共卫生相关性：细胞迁移是许多生物学过程的关键，而肿瘤-内皮细胞的交叉迁移对肿瘤血管生成至关重要。该研究项目的目标是开发新型微流控细胞共培养平台，以允许对调控细胞迁移和肿瘤-内皮相互作用的分子机制进行定量评估和鉴定。这将导致治疗各种疾病的新治疗方法，如癌症，这些疾病是由异常细胞迁移引起的。

DESCRIPTION (provided by applicant): It is becoming increasingly clear that the tumor microenvironment plays a key role in tumor progression, pointing to a need to develop technologies to study tumor cell behavior in different microenvironments. Toward this goal, we have created Valve Enabled Cell Co-Culture (VEC3) platforms, which are a new class of microfluidic devices designed for analyzing interactions between tumor cells and others in the tumor microenvironment. The technology enables separate culture of distinct cell types and cell-cell interactions through either soluble factors or physical contacts between spatially separated cell populations while maintaining fluidic control over their individual culture environment. In addition, through selective blockage of the exchange of specific ligands between distinct cell populations, the platform can be used to identify the functions of relevant ligands of interest. Traditional cell co-culture techniques and reported microfluidic cell co-culture platforms have limitations and cannot address all important cell co-culture needs. The proposed VEC3 cell co-culture platform, through the introduction of a simple, robust, and user-friendly pneumatically or hydraulically controlled valve to reversibly separate or connect adjacent cell culture chambers, not only allows for separate culture and treatment of individual cell types, but also permits real-time, live-cell imaging of cellular interactions. To date, as a proof of principle, VEC3 has been applied to observe dynamically synapse formation between hippocampal neurons, analyze tumor-endothelial interactions in normoxic and hypoxic environments, study tumor-fibroblasts interactions in 3D matrices, and quantify tumor-endothelial cross migration mediated by various molecules. In the proposed research we will further develop VEC3 through quantitative characterizations of cellular microenvironments, including cell density and uniformity, glucose and oxygen concentration, and cell-cell interaction rates. Through improved design and performance engineering, we will develop optimized platforms and operation protocols to increase the success rate of VEC3-based assays. In addition, we will implement new functions such as controlled cell-cell interactions via blockage of the exchange of specific ligands between two cell populations. More importantly, we will apply VEC3 to study tumor- endothelial cross migration mediated by various ligands and receptors and identify the functions of specific ligands in cell-cell interactions. Therefore, successful execution of the proposed research will lead to a new class of versatile, multifunctional VEC3 microfluidic platforms that are widely applicable to cancer biology. This device will be used to elucidate the molecular mechanisms underlying tumor angiogenesis, intravasation and metastasis, which could eventually lead to better cancer treatments. The overall quantitative milestone is to achieve 95% success rate in assays using the VEC3 platforms for tumor angiogenesis, intravsation and metastasis studies with quantitative parameters of cellular communication. PUBLIC HEALTH RELEVANCE: Cell migration is critical for many biological processes, and tumor-endothelial cross-migration is of fundamental importance to tumor angiogenesis. The goal of this research project is to develop novel microfluidic cell co- culture platforms that allow for the quantitative assessment and identification of the molecular mechanisms that regulate cell migration and tumor-endothelial interactions. This will lead to new therapeutic approaches for treating various diseases, such as cancer, that arise from aberrant cell migration.