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

描述（由申请人提供）：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，我们创建了阀门使能细胞共培养（VEC 3）平台，这是一类新的微流体设备，旨在分析肿瘤细胞与肿瘤微环境中其他细胞之间的相互作用。该技术能够通过可溶性因子或空间分离的细胞群之间的物理接触来单独培养不同的细胞类型和细胞-细胞相互作用，同时保持对其单独培养环境的流体控制。此外，通过选择性阻断不同细胞群之间的特异性配体交换，该平台可用于鉴定相关目标配体的功能。 传统的细胞共培养技术和报道的微流体细胞共培养平台具有局限性，并且不能满足所有重要的细胞共培养需求。所提出的VEC 3细胞共培养平台通过引入简单、稳健且用户友好的阀门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活细胞成像。迄今为止，作为原理的证明，VEC 3已被应用于动态观察海马神经元之间的突触形成，分析常氧和缺氧环境中的肿瘤-内皮相互作用，研究3D基质中的肿瘤-成纤维细胞相互作用，以及量化由各种分子介导的肿瘤-内皮交叉迁移。在拟议的研究中，我们将通过定量表征细胞微环境，包括细胞密度和均匀性，葡萄糖和氧浓度，以及细胞-细胞相互作用速率，进一步开发VEC 3。通过改进设计和性能工程，我们将开发优化的平台和操作方案，以提高基于VEC 3的检测的成功率。此外，我们还将实现新的功能，例如通过阻断两个细胞群之间特定配体的交换来控制细胞-细胞相互作用。更重要的是，我们将应用VEC 3来研究由各种配体和受体介导的肿瘤-内皮交叉迁移，并确定特定配体在细胞-细胞相互作用中的功能。因此，成功执行拟议的研究将导致一类新的通用，多功能的VEC 3微流体平台，广泛适用于癌症生物学。该装置将用于阐明肿瘤血管生成、血管内渗和转移的分子机制，最终可能导致更好的癌症治疗。总体定量里程碑是在使用VEC 3平台进行肿瘤血管生成、血管内注射和转移研究的试验中实现95%的成功率，并使用细胞通讯的定量参数。

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.