4D Microfluidic Platforms for Targeting Breast Cancer:Lymphatic Interactions

摘要：淋巴转移是乳腺癌细胞最初扩散的主要途径，其在局部淋巴结的存在是临床上最重要的单一负面预后因素。一旦进入淋巴结，乳腺癌细胞就会进入血液循环并扩散到远处。在这种情况下，对我们来说，开发模型至关重要，这将使我们能够定义肿瘤细胞进入淋巴管的初始入侵机制，并开发针对这些机制的筛选药物的平台。我们的工作假设是，3D模型概括了乳腺癌细胞和淋巴微血管内皮细胞之间的相互作用，可用于识别与侵入淋巴管和淋巴管生成相关的药物通路。我们的基本原理是，体外鉴定的途径将概括患者样本中存在的途径的至少一个子集，从而使我们能够为新的治疗策略定义可用药的节点。对我们的研究翻译至关重要的是筛选平台，该平台可用于在3D中可重复地培养乳腺和淋巴细胞，并实时测试和量化活细胞对治疗干预的动态反应。我们的具体目标是：1)建立最佳条件来概括乳腺癌细胞与淋巴微血管内皮细胞之间的体内相互作用；2)设计、制造和测试可用于成像、治疗靶点识别和药物筛选的新型微流控平台；3)对于原理验证研究，确定候选治疗靶点并在患者样本中进行验证；4)对靶向候选治疗靶点的药物进行初步筛选。我们将使用创新的4D （3D +时间）培养来模拟人乳腺癌细胞与人淋巴微血管内皮细胞之间的相互作用。我们将构建新的微流控平台，用于新型3D培养的长期生长，通过共聚焦显微镜对培养物进行成像，分析这些培养物中动态相互作用改变的生化和免疫化学途径，确定潜在的治疗靶点并筛选针对这些靶点和相关途径的药物。有效的筛选要求我们的模型具有高度的可重复性，并且我们使用的平台允许我们在多种浓度下测试许多不同的药物。我们在构建可用于细胞培养的小型平台方面拥有丰富的经验，包括作为患者的假体装置。我们的研究应该加速识别有效的药物，可能是针对个别患者的药物。

DESCRIPTION (provided by applicant): Abstract Lymphatic metastasis is the predominant route of the initial spread of breast cancer cells and their presence in regional lymph nodes is the single most clinically important negative prognostic factor. Once in lymph nodes, breast cancer cells can find their way into the circulation and spread to distant sites. This being the case it is critically important for us to develop models that will allow us to define mechanisms fo the initial invasion of tumor cells into lymph vessels and to develop platforms for screening drugs that target those mechanisms. Our working hypothesis is that 3D models that recapitulate the interactions between breast carcinoma cells and lymphatic microvascular endothelial cells can be used to identify druggable pathways associated with invasion into lymphatic vessels and lymphangiogenesis. Our rationale is that the pathways identified in vitro will recapitulate at leas a subset of those present in patient samples and thereby allow us to define druggable nodes for novel therapeutic strategies. Critical to translation of our studies will be screening platforms tht can be used to reproducibly grow breast and lymphatic cells in 3D and test and quantify dynamic responses of live cells to therapeutic interventions in real-time. Our Specific Aims are to: 1) Establish optimal conditions for recapitulating in vivo interactions between breast carcinoma cells and lymphatic microvascular endothelial cells; 2) Design, fabricate and test novel microfluidic platforms that can be used for imaging, identification of therapeutic targets and drug screening; 3) For proof-of-principle studies, Identify a candidate therapeutic target and validate in patient samples; and 4) Perform initial screening of agents that target the candidate therapeutic target. We will use innovative 4D (3D + time) cultures to model the interactions between human breast carcinoma cells and human lymphatic microvascular endothelial cells. We will construct novel microfluidic platforms for long-term growth of the novel 3D cultures, image the cultures by confocal microscopy, analyze biochemical and immunochemical pathways that are altered by the dynamic interactions in these cultures, identify potential therapeutic targets and screen drugs directed against those targets and associated pathways. Effective screening requires that our models be highly reproducible and that we use platforms that allow us to test many different drugs at multiple concentrations. We have extensive experience in constructing miniaturized platforms that can be used for cell culture, including as prosthetic devices in patients. Our studies should accelerate the identification of effective drugs potentially ones targeted to individual patients.