A Label-Free, Many-Parameter Benchtop Platform for Functionally-Preserved, Viable Cancer Stem Cell Isolation and Biomarker Discovery to Probe Urothelial Carcinoma of the Bladder

Nodexus之前开发的NX One系统利用节点-孔洞传感电子检测(非 基于标记的)和基于荧光的标记与低剪切微流控相结合的细胞检测 用于活细胞分离和单细胞分离的阀门。在这项提案中，我们强调了一种新的工具，即 将我们演示的节点-孔道检测(“NPS”)单细胞分离与无标记多标记相结合 快速崛起的癌症干细胞(CSC)空间的筛选，高效生物标记物的发现和 靶向治疗开发的功能研究将是变革性的，但很容易获得可行的、 使用任何单一的商业仪器都不可能在功能上保存活的单细胞。而当 该平台最终将适用于更多的癌症亚型，这是我们进入该领域的最初切入点 重点关注膀胱癌(UCB)。膀胱尿路上皮癌是最常见的 尿路恶性肿瘤，有60万人患有膀胱癌，约79,030例新病例和16,870例 美国每年的死亡人数。从与该领域专家的广泛对话中，我们发现 Nodexus平台可以解决与UCB相关的关键痛点。 CSCs是异质混合癌细胞中的一个亚群，它增强了细胞的促进性。 恶性特征，但特定标记物对干细胞样特征的贡献及其临床应用 生物标志物尚未确定，这使得对这些细胞的靶向治疗变得极其重要 很有挑战性。更好地理解尿路上皮CSC调控的分子机制 识别与CSC生成和维持相关的关键分子对于 用于癌症早期检测和监测的普遍接受的、临床上准确的生物标志物的测定 经尿道膀胱肿瘤电切术(TURBT)术后有效靶向的研究进展 治疗。 由于肿瘤内的异质性，研究肿瘤干细胞的复杂性大大增加。 不同癌症亚型中CSC样性状和功能重要性的差异。确凿无疑 标记物相关功能的发展将为靶向治疗的开发打开大门。关于的进一步研究 同时进行的综合标记面板将为下游提供重要价值 确凿的生物标记物识别和优化治疗的研究。关键是，功能相关性 必须对这些标记进行评估和理解；这需要能够全面筛选 众多的标记，分离单细胞群体以揭示掩盖的异质性，并在下游执行 对这些活体进行功能研究(例如生长、侵袭和对化疗药物的耐药性)， 功能保存的分离的单细胞。而现有技术，如FACS、Mac和CyTOF 在更广阔的空间内提供了巨大的价值，但没有一家公司提供了广泛的免标签轻松访问 以Nodexus平台提供的方式表征、实时、功能保存的单个CSC。

Nodexus’ previously developed NX One system leverages node-pore sensing electrical detection (non- marker-based) and fluorescence-based marker detection of cells in combination with low-shear microfluidic valving for viable cell sorting and single-cell isolation. In this proposal, we highlight a new instrument that integrates our demonstrated node-pore sensing (“NPS”) single-cell isolation with label-free multi-marker screening for the quickly emerging cancer stem cell (CSC) space, efficient biomarker discovery and functional studies for targeted therapy development would be transformative, but easy access to viable, functionally-preserved live single-cells is not possible using any single commercial instrument. While the platform will eventually be applicable across more cancer subtypes, our initial point of entry into this sector focuses on urothelial carcinoma of the bladder (UCB). Urothelial carcinoma of the bladder is the most common malignancy of the urinary tract, with >600,000 living with bladder cancer and ~79,030 new cases and 16,870 deaths per year in the United States. From extensive conversations with experts in the field, we have found critical pain points related to UCB that the Nodexus platform can address. CSCs are a subpopulation within a heterogeneous mixture of cancer cells that have enhanced pro- malignant properties, but the contribution of specific markers to stem cell-like traits and their clinical utility as biomarkers have not been conclusively determined, making targeted therapy for these cells extremely challenging. A better understanding of the molecular mechanisms underlying urothelial CSC regulation and identification of key molecules associated with CSC generation and maintenance are pivotal for the determination of universally accepted, clinically-accurate biomarkers for early cancer detection and monitoring following transurethral resection of bladder tumor (TURBT) as well as the development of effective targeted therapies. The complexity with studying CSCs is immensely increased due to the heterogeneity within tumors and the variance in CSC-like traits and functional importance in different cancer subtypes. Conclusive confirmation of marker-associated functionality will open the door for targeted therapy development. Further studies of comprehensive panels of markers performed simultaneously will provide significant value for downstream studies for conclusive biomarker identification and optimized therapies. Critically, the functional relevance of such markers must be evaluated and understood; this requires being able to comprehensively screen for numerous markers, isolate single-cell populations to unveil masked heterogeneity, and perform downstream functional studies (e.g. growth, invasion, and resistance to chemotherapeutic agents) on these viable, functionally-preserved isolated single-cells. While existing technologies, such as FACS, MACS, and CyTOF have provided tremendous value within the broader space, none provide label-free easy access to extensively characterized, live, functionally preserved single CSCs in a manner that the Nodexus platform will offer.