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

无标记、多参数台式平台，用于功能保留、活的癌症干细胞分离和生物标志物发现，以探测膀胱尿路上皮癌

• 批准号: 10086817
• 负责人: Karthik Ratna Balakrishnan
• 金额: $ 5.5万
• 依托单位: NODEXUS, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-20 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10086817
• 关键词: Address; Benchmarking; Biological Assay; Biological Markers; Bladder Neoplasm; CD44 gene; Calibration; Cell Line; Cell Separation; Cell Survival; Cell Therapy; Cells; Cellular Assay; Cessation of life; Cisplatin; Clinical; Detection; Development; Early Diagnosis; Flow Cytometry; Fluorescence; Fluorescence-Activated Cell Sorting; Generations; Genes; Growth; Heterogeneity; Label; Lead; Maintenance; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of urinary bladder; Marker Discovery; Masks; Microfluidics; Molecular; Monitor; Outcome; Pain; Patient-Focused Outcomes; Patients; Phase; Population; Population Control; Property; Quantitative Reverse Transcriptase PCR; Regulation; Resistance; Sampling; Screening for cancer; Sorting - Cell Movement; System; Technology; Testing; Time; Transitional Cell Carcinoma; Transurethral Resection; United States; Universities; Urinary tract; Urine; Urothelium; Validation; Walking; Xenograft procedure; base; biomarker discovery; biomarker identification; biomarker panel; bladder Carcinoma; bladder transitional cell carcinoma; cancer cell; cancer stem cell; cancer subtypes; chemotherapeutic agent; early detection biomarkers; improved; insight; instrument; medical schools; novel; novel therapeutics; preservation; screening; stem-like cell; targeted treatment; therapy development; tool; trait; treatment optimization; tumor

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.

Nodexus先前开发的NX One系统利用节点孔传感电检测（非 基于标记的）和基于荧光的标记检测 用于活细胞分选和单细胞分离的阀门。在本提案中，我们强调了一项新的文书， 将我们证明的节点-孔感测（“Node-pore sensing”）单细胞分离与无标记的多标记物 筛选快速出现的癌症干细胞（CSC）空间，有效的生物标志物发现， 靶向治疗开发的功能研究将是变革性的，但容易获得可行的， 使用任何单一的商业仪器不可能获得功能性保存的活单细胞。而 该平台最终将适用于更多的癌症亚型，这是我们进入这一领域的最初起点。 膀胱尿路上皮癌（UCB）膀胱尿路上皮癌最常见 泌尿道恶性肿瘤，超过600，000人患有膀胱癌，约79，030例新发病例和16，870例 美国每年死亡人数。通过与该领域专家的广泛交谈，我们发现 Nodexus平台可以解决的与UCB相关的关键痛点。 CSC是癌细胞的异质混合物中的亚群，其具有增强的促增殖活性。 恶性性质，但特异性标志物对干细胞样性状的贡献及其临床效用， 生物标志物尚未最终确定，使得针对这些细胞的靶向治疗非常困难。 挑战性更好地理解尿路上皮CSC调控的分子机制， 鉴定与CSC生成和维持相关的关键分子对于研究CSC的生物学特性是至关重要的。 确定普遍接受的、临床上准确的生物标志物，用于早期癌症检测和监测 经尿道膀胱肿瘤电切术（TURBT）后，以及有效的靶向治疗的发展， 治疗 由于肿瘤内的异质性，研究CSC的复杂性大大增加， 不同癌症亚型中CSC样特征和功能重要性的差异。结论性确认 标志物相关功能的开发将为靶向治疗的开发打开大门。进一步研究 同时进行的全面的标志物小组将为下游提供重要价值， 用于确定生物标志物鉴定和优化治疗的研究。关键是， 必须评估和理解这些标记;这需要能够全面筛查 许多标记，分离单细胞群体以揭示被掩盖的异质性，并在下游进行 对这些存活的， 功能保存的分离的单细胞。虽然现有的技术，如FACS、MACS和CyTOF， 在更广阔的空间内提供了巨大的价值，没有一个提供了广泛的无标签轻松访问 以Nodexus平台将提供的方式鉴定、活的、功能性保存的单个CSC。