Versatile exclusion-based sample preparation platform for integrated rare cell isolation and analyte extraction

Abstract

Rare cell populations provide a patient-centric tool to monitor disease treatment, response, and resistance. However, understanding rare cells is a complex problem, which requires cell isolation/purification and downstream molecular interrogation – processes challenged by non-target populations, which vary patient-to-patient and change with disease. As such, cell isolation platforms must be amenable to a range of sample types while maintaining high efficiency and purity. The Multiplexed Technology for Automated Extraction (mTAE) is a versatile magnetic bead-based isolation platform that facilitates positive, negative, and combinatorial selection with integrated protein staining and nucleic acid isolation. mTAE is validated by isolating circulating tumor cells (CTCs) – a model rare cell population – from breast and prostate cancer patient samples. Negative selection yielded high efficiency capture of CTCs while positive selection yielded higher purity with an average of only 95 contaminant cells captured per milliliter of processed whole blood. With combinatorial selection, an overall increase in capture efficiency was observed, highlighting the potential significance of integrating multiple capture approaches on a single platform. Following capture (and staining), on platform nucleic acid extraction enabled the detection of androgen receptor-related transcripts from CTCs isolated from prostate cancer patients. The flexibility (e.g. negative, positive, combinatorial selection) and capabilities (e.g. isolation, protein staining, and nucleic acid extraction) of mTAE enable users to freely interrogate specific cell populations, a capability required to understand the potential of emerging rare cell populations and readily adapt to the heterogeneity presented across clinical samples

Live Cell Molecular Analysis of Primary Prostate Cancer Organoids Identifies Persistent Androgen Receptor Signaling

“…To con rm cancer-cell content in our low-volume primary tissue specimen, we have developed a multiplex epigenetic biomarker assay to detect promoter methylation of PC-associated genes including GSTP1, RASSF1, RARb and APC [27][28][29][30][31]. We collected matched samples of ~100-1500 cells from single cell suspensions generated from native biopsies and subsequent prostate PDCO specimens, isolated total DNA and performed and MBDbased enrichment of methylated DNA using the SEEMLIS method on the semi-automated VERSA micro uidic platform [32]. We then performed quantitative PCR analysis to interrogate the promotermethylation of selected PC-associated genes (Figure 5).…”

Development and initial clinical testing of a multiplexed circulating tumor cell assay in patients with clear cell renal cell carcinoma

“…Analytes were bound to antibody‐functionalized paramagnetic particles (PMPs) and magnetically drawn across phase boundaries (aqueous/oil interface) using an external magnetic force to isolate the PMP‐bound analyte from the sample. Antibody concentration was tested on live and fixed RCC cell lines to optimize capture efficiency using the ExtractMAX, an automated ESP platform [37].…”

Live cell molecular analysis of primary prostate cancer organoids identifies persistent androgen receptor signaling

“…To confirm cancer-cell content in our low-volume primary tissue specimen, we have developed a multiplex epigenetic biomarker assay to detect promoter methylation of PC-associated genes including GSTP1, RASSF1, RARb and APC [27][28][29][30][31]. We collected matched samples of ~ 100-1500 cells from single cell suspensions generated from native biopsies and subsequent prostate PDCO specimens, isolated total DNA and performed and MBD-based enrichment of methylated DNA using the SEEMLIS method on the semi-automated VERSA microfluidic platform [32]. We then performed quantitative PCR analysis to interrogate the promoter-methylation of selected PC-associated genes (Fig.…”