Integrated Analysis of Multiple Biomarkers from Circulating Tumor Cells Enabled by Exclusion-Based Analyte Isolation.

Purpose: There is a critical clinical need for new predictive and pharmacodynamic biomarkers that evaluate pathway activity in patients treated with targeted therapies. A microscale platform known as VERSA (versatile exclusion-based rare sample analysis) was developed to integrate readouts across protein, mRNA, and DNA in circulating tumor cells (CTC) for a comprehensive analysis of the androgen receptor (AR) signaling pathway.

Experimental Design: Utilizing exclusion-based sample preparation principles, a handheld chip was developed to perform CTC capture, enumeration, quantification, and subcellular localization of proteins and extraction of mRNA and DNA.

Efficient sample preparation from complex biological samples using a sliding lid for immobilized droplet extractions.

Sample preparation is a major bottleneck in many biological processes. Paramagnetic particles (PMPs) are a ubiquitous method for isolating analytes of interest from biological samples and are used for their ability to thoroughly sample a solution and be easily collected with a magnet. There are three main methods by which PMPs are used for sample preparation: (1) removal of fluid from the analyte-bound PMPs, (2) removal of analyte-bound PMPs from the solution, and (3) removal of the substrate (with immobilized analyte-bound PMPs).

A microfluidic coculture and multiphoton FAD analysis assay provides insight into the influence of the bone microenvironment on prostate cancer cells.

In prostate cancer, bone is a frequent site of metastasis; however, the molecular mechanisms of this tumor tropism remain unclear. Here, we integrate a microfluidic coculture platform with multi-photon imaging based techniques to assess both phenotypic cell behavior and FAD fluorescence intensity and fluorescence lifetime in the same cell. This platform combines two independent assays normally performed with two different cell populations into a single device, allowing us to simultaneously assess both phenotypic cell behavior and enzyme activity.

Rapid translation of circulating tumor cell biomarkers into clinical practice: technology development, clinical needs and regulatory requirements.

The great hope in circulating tumor cell (CTC) research lies in the use of these rare cells as an accessible "fluid biopsy" that would permit frequent, minimally invasive sampling of tumor cells for similar molecular assays that are performed on traditional biopsies. Given the rarity of CTCs in peripheral circulation, microscale methods show great promise and superiority to capture and analyze these cells from patients with solid tumors. Novel technologies that produce validated CTC biomarkers may finally provide medical oncologists the tools needed to provide precise, personalized medical care for patients with advanced cancer.

Paired diagnostic and pharmacodynamic analysis of rare non-small cell lung cancer cells enabled by the VerIFAST platform.

Lung cancer is the leading cause of cancer-related deaths in the United States and worldwide. This has led to major research initiatives focusing on improving early diagnosis rate, as well as the development of pharmacodynamic biomarkers. However, broad clinical integration of these approaches is limited due to the invasive nature of lung biopsies, needle aspirates and resections.

Selective nucleic acid removal via exclusion (SNARE): capturing mRNA and DNA from a single sample.

The path from gene (DNA) to gene product (RNA or protein) is the foundation of genotype giving rise to phenotype. Comparison of genomic analyses (DNA) with paired transcriptomic studies (mRNA) is critical to evaluating the pathogenic processes that give rise to human disease. The ability to analyze both DNA and mRNA from the same sample is not only important for biologic interrogation but also to minimize variance (e.

A negative selection methodology using a microfluidic platform for the isolation and enumeration of circulating tumor cells.

Circulating tumor cells (CTCs) exist in the peripheral blood stream of metastatic cancer patients at rates of approximately 1 CTC per billion background cells. In order to capture and analyze this rare cell population, various techniques exist that range from antibody-based surface marker positive selection to methods that use physical properties of CTCs to negatively exclude background cells from a CTC population. However, methods to capture cells for functional downstream analyses are limited due to inaccessibility of the captured sample or labeling techniques that may be prohibitive to cell function.

Suspended microfluidics.

Although the field of microfluidics has made significant progress in bringing new tools to address biological questions, the accessibility and adoption of microfluidics within the life sciences are still limited. Open microfluidic systems have the potential to lower the barriers to adoption, but the absence of robust design rules has hindered their use. Here, we present an open microfluidic platform, suspended microfluidics, that uses surface tension to fill and maintain a fluid in microscale structures devoid of a ceiling and floor.

The VerIFAST: an integrated method for cell isolation and extracellular/intracellular staining.

Isolation and characterization of a specific subset of cells from a large heterogeneous population is necessary for studying rare subpopulations of cells. Existing methods require transfer or wash steps that risk causing loss of the rare cell population of interest. Integrated methods reduce loss, making these methods especially useful for reliable isolation of rare cell populations.

Automated operation of immiscible filtration assisted by surface tension (IFAST) arrays for streamlined analyte isolation.

The purification of analytes is an important prerequisite for many analytical processes. Although automated infrastructure has dramatically increased throughput for many of these processes, the upstream analyte purification throughput has lagged behind, partially due to the complexity of conventional isolation processes. Here, we demonstrate automated operation of arrays of a new sample preparation technology--immiscible filtration assisted by surface tension (IFAST).

Circulating tumor cells: getting more from less.

Recent insights into circulating tumor cells (CTCs) have been driven by numerous technological innovations aimed at isolating, purifying, and analyzing these rare cells. However, the information density within these cells has yet to be truly accessed and exploited for patient benefit. A device reported by Issadore et al.