Benjamin P. Casavant

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. We developed a simple and ubiquitous model predicting fluid flow in suspended microfluidic systems and show that it encompasses many known capillary phenomena. Suspended microfluidics was used to create arrays of collagen membranes, mico Dots (μDots), in a horizontal plane separating two fluidic chambers, demonstrating a transwell platform able to discern collective or individual cellular invasion. Further, we demonstrated that μDots can also be used as a simple multiplexed 3D cellular growth platform. Using the μDot array, we probed the combined effects of soluble factors and matrix components, finding that laminin mitigates the growth suppression properties of the matrix metalloproteinase inhibitor GM6001. Based on the same fluidic principles, we created a suspended microfluidic metabolite extraction platform using a multilayer biphasic system that leverages the accessibility of open microchannels to retrieve steroids and other metabolites readily from cell culture. Suspended microfluidics brings the high degree of fluidic control and unique functionality of closed microfluidics into the highly accessible and robust platform of open microfluidics.

Circulating Tumor Cells: Getting More from Less

Continued technologic innovation may soon permit the interrogation of rare circulating tumor cells to understand tumor biology, guide drug development, and personalize treatment for cancer patients. 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. in this issue of Science Translational Medicine proposes a highly sensitive methodology that may both extend CTC capture to a broader patient population and provide greater understanding of biological targets for personalized medical therapies.

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. Here, we present a negative selection method that leverages a Microfluidic Cell Concentrator (MCC) to allow collection and analysis of this rare cell population without needing cell adhesion or other labeling techniques to keep the cells within the chamber. Because the MCC is designed to allow collection and analysis of non-adherent cell populations, multiple staining steps can be applied in parallel to a given CTC population without losing any of the population. The ability of the MCC for patient sample processing of CTCs for enumeration was demonstrated with five patient samples, revealing an average of 0.31 CTCs/mL. The technique was compared to a previously published method - the ELISPOT - that showed similar CTC levels among the five patient samples tested. Because the MCC method does not use positive selection, the method can be applied across a variety of tumor types with no changes to the process.

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.g., sample loss) unrelated to the biology. Existing methods for RNA and DNA purification from a single sample are typically time-consuming and labor intensive or require large sample sizes to split for separate RNA and DNA extraction procedures. Thus, there is a need for more efficient and cost-effective methods to purify both RNA and DNA from a single sample. To address this need, we have developed a technique, termed SNARE (Selective Nucleic Acid Removal via Exclusion), that uses pinned oil interfaces to simultaneous purify mRNA and DNA from a single sample. A unique advantage of SNARE is the elimination of dilutive wash and centrifugation processes that are fundamental to conventional methods where sample is typically discarded. This minimizes loss and maximizes recovery by allowing nondilutive reinterrogation of the sample. We demonstrate that SNARE is more sensitive than commercially available kits, robustly and repeatably achieving mRNA and DNA purification from extremely low numbers of cells for downstream analyses. In addition to sensitivity, SNARE is fast, easy to use, and cost-effective and requires no laboratory infrastructure or hazardous chemicals. We demonstrate the clinical utility of the SNARE with prostate cancer circulating tumor cells to demonstrate its ability to perform both genomic and transcriptomic interrogation on rare cell populations that would be difficult to achieve with any current method.

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). IFAST uses surface tension to position an immiscible liquid barrier between a biological sample and downstream buffer. Paramagnetic particles are used to capture analytes of interest and draw them across the immiscible barrier, thus resulting in purification in a single step. Furthermore, the planarity of the IFAST design enables facile and simultaneous operation of multiple IFAST devices. To demonstrate the application of automation to IFAST, we successfully perform an array of 48 IFAST-based assays to detect the presence of a specific antibody. This assay array uses only a commercial automated liquid handler to load the devices and a custom-built magnet actuator to operate the assays. Automated operation of the IFAST devices resulted in more repeatable results relative to manual operation.

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. We observed that the osteotropic prostate cancer cell line (C4-2B), when in a coculture with bone marrow stromal cells (MC3T3-E1), has increased protrusive phenotype and increased total and protein-bound FAD compared to its parent cell line (LNCaP). We hypothesized that an increase in ROS-generating APAO activity may be responsible for these effects, and found that the effects were decreased in the presence of the antioxidant N-Acetyl Cysteine (NAC). This suggests that an ROS-related signaling mechanism at the bone metastatic site may be correlated with and play a role in increased invasion of metastasizing prostate cancer cells. The studies performed using this combined platform will lead to new insights into the mechanisms that drive prostate cancer metastasis.

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. This technology was validated across integrated endpoints in cell lines and a cohort of patients with castrate-resistant prostate cancer (CRPC) treated with AR-targeted therapies and chemotherapies. Results: The VERSA was validated in cell lines to analyze AR protein expression, nuclear localization, and gene expression targets. When applied to a cohort of patients, radiographic progression was predicted by the presence of multiple AR splice variants and activity in the canonical AR signaling pathway. AR protein expression and nuclear localization identified phenotypic heterogeneity. Next-generation sequencing with the FoundationOne panel detected copy number changes and point mutations. Longitudinal analysis of CTCs identified acquisition of multiple AR variants during targeted treatments and chemotherapy. Conclusions: Complex mechanisms of resistance to AR-targeted therapies, across RNA, DNA, and protein endpoints, exist in patients with CRPC and can be quantified in CTCs. Interrogation of the AR signaling pathway revealed distinct patterns relevant to tumor progression and can serve as pharmacodynamic biomarkers for targeted therapies. Clin Cancer Res; 23(3); 746–56. ©2016 AACR.

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). In this paper, we explore the third and least studied method for PMP-based sample preparation using a platform termed Sliding Lid for Immobilized Droplet Extractions (SLIDE). SLIDE leverages principles of surface tension and patterned hydrophobicity to create a simple-to-operate platform for sample isolation (cells, DNA, RNA, protein) and preparation (cell staining) without the need for time-intensive wash steps, use of immiscible fluids, or precise pinning geometries. Compared to other standard isolation protocols using PMPs, SLIDE is able to perform rapid sample preparation with low (0.6%) carryover of contaminants from the original sample. The natural recirculation occurring within the pinned droplets of SLIDE make possible the performance of multistep cell staining protocols within the SLIDE by simply resting the lid over the various sample droplets. SLIDE demonstrates a simple easy to use platform for sample preparation on a range of complex biological samples.

Cell-based Microfluidic Assays in Translational Medicine

Microfluidics is the study and use of fluid flow at small volumes (typically microliter and below). Microfluidic principles have great potential utility in many engineering and medical arenas, including point-of-care diagnostics tests. The home pregnancy test is an early example of a “microfluidic” diagnostic assay that utilized a colorimetric readout to detect human chorionic gonadotropin. More recently, microfluidics has been used to study and quantify cellular characteristics. Two areas where microfluidic cell-based assays have been used for clinical applications are chemotaxis (gradient-dependent cell migration) and the isolation and analysis of rare cells such as circulating tumor cells. This chapter will review current translational research studies, integrating microfluidics with chemotaxis and rare cell analysis, as well as future research directions in these emerging fields.

OCT-based angiography of human dermal microvascular reactions to local stimuli: Implications for increasing capillary blood collection volumes: OCTA OF DERMAL MICROVASCULAR REACTIONS TO STIMULI

To measure and compare microvascular responses within the skin of the upper arm to local stimuli, such as heating or rubbing, through the use of optical coherence tomography angiography (OCTA), and to investigate its impact on blood volume collection.