Developing Tools for Interrogation of Clinically Significant Rare Cell Populations

摘要

Rare cell populations are a potential tool for assessing disease progression, treatment, and monitoring while also providing the opportunity for new diagnostic and prognostic biomarkers. Hidden within large, diverse contaminating populations, rare cell populations present challenges in assessing the information they contain. Physically or analytically, rare cell populations must be identified and distinguished from the contaminant populations they reside in to obtain and utilize the information they hold. Despite their clinical potential, utilization of rare cell populations requires assays able to overcome the challenges presented by each rare cell population, challenges that change and emerge with each population. By evaluating and optimizing magnetic bead isolation approaches, a rare cell isolation platform is developed and validated through the targeting of a rare cell population, circulating tumor cells. This platform leverages Exclusion-based Sample Preparation (ESP) to facilitate positive, negative, and combinatorial selection of cell populations. ESP is then used to combine cell isolation with downstream immunostaining and nucleic acid extraction capabilities. Expanding on the targeting of rare cell populations, an alternative rare cell population, the HIV viral reservoir, is assessed. Lacking differential external protein markers, the HIV viral reservoir requires alternative approaches (often intracellular analytes following activation) to identify and quantify this rare cell population. Herein, ESP is used to facilitate multiplexed RNA extractions, which increases sensitivity in detecting rare RNA transcripts from large cellular inputs. Following targeting of rare RNA transcripts, an alternative approach to reservoir quantification is pursued, specifically detection of reservoir cells capable of producing infectious virus. Here, a dual fluorescent reporter cell line is used to provide a sensitive readout enabling the live visualization of a single infection event. Building on the reporter readout, an alternative culture approach was shown to enhance infection rates, further enhancing sensitivity of the readout. The presented tools and approaches aim to advance understanding of rare, clinically valuable rare cell populations as despite their value, rare cell populations present significant challenges in transitioning into clinical care.