Lindsay N. Strotman

Purification of cell subpopulations via immiscible filtration assisted by surface tension (IFAST)

The selective isolation of a sub-population of cells from a larger, mixed population is a critical preparatory process to many biomedical assays. Here, we present a new cell isolation platform with a unique set of advantages over existing devices. Our technology, termed Immiscible Filtration Assisted by Surface Tension, exploits physical phenomena associated with the microscale to establish fluidic barriers composed of immiscible liquids. By attaching magnetically-responsive particles to a target cell population via immunocapture, we can selectively transport this population across the immiscible barrier and into a separate aqueous solution. The high interfacial energy associated with the immiscible phase / aqueous phase boundaries prevents unwanted cells or other contaminants from inadvertently crossing the immiscible phase. We have demonstrated, using fluorescent particles, stromal cells, and whole blood as “background”, that we can successfully isolate ~70% of a target breast cancer cell population with an average purity of >80%. Increased purity was obtained by coupling two immiscible barriers in series, a modification that only slightly increases operational complexity. Furthermore, several samples can be processed in parallel batches in a near-instantaneous manner without the requirement of any washing, which can cause dilution (negative selection) or significant uncontrolled loss (positive selection) of target cells. Finally, cells were observed to remain viable and proliferative following traverse through the immiscible phase, indicating that this process is suitable for a variety of downstream assays, including those requiring intact living cells.

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.

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.

Characterization of Molecules Binding to the 70K N-terminal Region of Fibronectin by IFAST Purification Coupled with Mass Spectrometry

Fibronectin (Fn) is a large glycoprotein present in plasma and extracellular matrix and is important for many processes. Within Fn the 70 kDa N-terminal region (70k-Fn) is involved in cell-mediated Fn assembly, a process that contributes to embryogenesis, development, and platelet thrombus formation. In addition, major human pathogens including Staphlycoccus aureus and Streptococcus pyogenes bind the 70k-Fn region by a novel form of protein-protein interaction called β-zipper formation, facilitating bacterial spread and colonization. Knowledge of blood plasma and platelet proteins that interact with 70k-Fn by β-zipper formation is incomplete. In the current study, we aimed to characterize these proteins through affinity purification. For this affinity purification, we used a novel purification technique termed immiscible filtration assisted by surface tension (IFAST). The foundation of this technology is immiscible phase filtration, using a magnet to draw paramagnetic particle (PMP)-bound analyte through an immiscible barrier (oil or organic solvent) that separates an aqueous sample from an aqueous eluting buffer. The immiscible barrier functions to remove unbound proteins via exclusion rather than dilutive washing used in traditional isolation methods. We identified 31 interactors from plasma, of which only seven were previously known to interact with Fn. Furthermore, five proteins were identified to interact with 70k-Fn from platelet lysate, of which one was previously known. These results demonstrate that IFAST offers advantages for proteomic studies of interacting molecules in that the technique requires small sample volumes, can be done with high enough throughput to sample multiple interaction conditions, and is amenable to exploratory mass spectrometric and confirmatory immuno-blotting read-outs.

Streamlining Gene Expression Analysis: Integration of Co-Culture and mRNA Purification

Co-culture of multiple cell types within a single device enables the study of paracrine signaling events. However, extracting gene expression endpoints from co-culture experiments is laborious, due in part to pre-PCR processing of the sample (i.e., post-culture cell sorting and nucleic acid purification). Also, a significant loss of nucleic acid may occur during these steps, especially with microfluidic cell culture where lysate volumes are small and difficult to access. Here, we describe an integrated platform for performing microfluidic cell culture and extraction of mRNA for gene expression analysis. This platform was able to recover 30-fold more mRNA than a similar, non-integrated system. Additionally, using a breast cancer/bone marrow stroma co-culture, we recapitulated stromal-dependent, estrogen-independent growth of the breast cancer cells, coincident with transcriptional changes. We anticipate that this platform will be used for streamlined analysis of paracrine signaling events as well as for screening potential drugs and/or patient samples.

Cellular Microenvironment Dictates Androgen Production by Murine Fetal Leydig Cells in Primary Culture

ABSTRACT Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3–5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cell-cell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture.

Nucleic Acid Sample Preparation using Spontaneous Biphasic Plug Flow

Nucleic acid (NA) extraction and purification has become a common technique in both research and clinical laboratories. Current methods require repetitive wash steps with a pipet that are laborious and time-consuming, making the procedure inefficient for clinical settings. We present here a simple technique that relies on spontaneous biphasic plug flow inside a capillary to achieve sample preparation. By filling the sample with oil, aqueous contaminants were displaced from the captured NA without pipetting wash buffers or use of external force and equipment. mRNA from mammalian cell culture was purified, and polymerase chain reaction (PCR) amplification showed similar threshold cycle values as those obtained from a commercially available kit. Human immunodeficiency virus (HIV) viral-like particles were spiked into serum and a 5-fold increase in viral RNA extraction yield was achieved compared to the conventional wash method. In addition, viral RNA was successfully purified from human whole blood, and a limit of detection of approximately 14 copies of RNA extracted per sample was determined. The results demonstrate the utility of the current technique for nucleic acid purification for clinical purposes, and the overall approach provides a potential method to implement nucleic acid testing in low-resource settings.

Weak protein-protein interactions revealed by immiscible filtration assisted by surface tension.

Biological mechanisms are often mediated by transient interactions between multiple proteins. The isolation of intact protein complexes is essential to understanding biochemical processes and an important prerequisite for identifying new drug targets and biomarkers. However, low-affinity interactions are often difficult to detect. Here, we use a newly described method called immiscible filtration assisted by surface tension (IFAST) to isolate proteins under defined binding conditions. This method, which gives a near-instantaneous isolation, enables significantly higher recovery of transient complexes compared to current wash-based protocols, which require reequilibration at each of several wash steps, resulting in protein loss. The method moves proteins, or protein complexes, captured on a solid phase through one or more immiscible-phase barriers that efficiently exclude the passage of nonspecific material in a single operation. We use a previously described polyol-responsive monoclonal antibody to investigate the potential of this new method to study protein binding. In addition, difficult-to-isolate complexes involving the biologically and clinically important Wnt signaling pathway were isolated. We anticipate that this simple, rapid method to isolate intact, transient complexes will enable the discoveries of new signaling pathways, biomarkers, and drug targets.

Abstract 1602: Development and validation of an NGS-based assay to detect all classes of genomic alterations in circulating tumor cells (CTCs) from patients with solid tumors

Purpose With increased use of targeted therapeutics, the ability to perform a ‘liquid biopsy’ on circulating tumor cells (CTCs) holds potential to address the need for a more representative and longitudinal means to monitor tumor burden in patients. However, the analytic and clinical validity of such an assay has proved difficult to establish, as the cell population in circulation is complex, and the CTC portion is heterogeneous, evolving, and highly dependent on clinical factors as well as CTC isolation methods. No studies to date have characterized CTCs for the full range of relevant genomic alterations in the context of patient-matched primary and metastatic tissue biopsies. We set out to develop a method to perform comprehensive NGS-based genomic profiling on phenotypically-isolated CTCs, and to compare CTC analysis with a comprehensive tissue-based assessment of paired primary and metastatic tissue in patients. Methods An integrated platform (VERSA) was employed for capture of EpCAM CTCs, enumeration and DNA extraction. Extracted DNA was amplified using a modified version of MDA-based whole genome amplification (WGA), followed by standard adaptor-ligated library construction and solution hybridization using a custom DNA baitset before sequencing on Illumina HiSeq. Data was analyzed using a computational workflow developed to detect base substitutions, indels, high-level copy number alterations, and select gene rearrangements. We performed extensive analytic validation of the assay using over 65 cell line mixtures, constructed to contain all classes of known variants across all allele frequencies (0-100%). We used the assay to analyze CTC DNA from 20 lung cancer patients, paired with analysis of temporally-matched primary and/or metastatic FFPE samples analyzed by FoundationOne, an FFPE tissue-based assay. Results Validation experiments showed that for samples meeting minimum acceptance criteria (50 cells at 50% purity), assay sensitivity exceeded 95% for detections of base substitutions, 85% for indels and rearrangements, and 80% for high-level copy number alterations, while maintaining tight specificity control. WGA-induced coverage bias limited sensitivity to low-level copy number alterations. Comparison of driver alterations across CTC samples and paired primary/metastatic tissue for 20 patients with lung cancer is ongoing. Conclusions A comprehensive NGS-based approach to genomic profiling is feasible on CTCs isolated from lung cancer patients, and demonstrated accuracy sufficient to characterize actionable targets for clinical decision-making. However, more extensive tumor-type specific studies with a similar matched-tissue approach must be performed in order to establish full analytic and clinical validation, and assay specifications will need to be optimized to enable use on broader patient populations. Citation Format: Allison Welsh, Garrett M. Frampton, Zachary R. Chalmers, Jamie Sperger, Roman Yelensky, Doron Lipson, Geoff Otto, Lindsay Strotman, Scott Berry, Hannah Pezzi, Anne Traynor, David J. Beebe, Vincent A. Miller, Joshua M. Lang, Philip J. Stephens. Development and validation of an NGS-based assay to detect all classes of genomic alterations in circulating tumor cells (CTCs) from patients with solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1602. doi:10.1158/1538-7445.AM2015-1602

Abstract 4814: Molecular interrogation of the androgen receptor in circulating tumor cells to identify therapeutic resistance to androgen targeting therapies

There is a critical need to evaluate and target mechanisms of resistance to Androgen Receptor (AR) targeting agents, such as Enzalutamide and Abiraterone Acetate. Preclinical models have identified resistance mechanisms that include AR gene mutations and rearrangements, expression of AR splice variants and AR overexpression. Circulating tumor cells (CTCs) represent a potential source of tumor samples for further molecular interrogation into resistance mechanisms of AR directed therapy. Here we present, an integrated CTC capture and analysis technology known as VERSA (Vertical Exclusion-based Rare Sample Analysis) to allow evaluation of the AR at the protein, DNA and mRNA level. Using the VERSA platform we have captured EpCAM+ CTCs from castrate-resistance prostate cancer patients and quantified nuclear localization of the AR in CTCs as a potential predictive therapeutic protein biomarker. Using quantitative immunocytochemistry, results have shown patients that are responding to various AR-targeting and chemotherapy treatments have lower percentages of AR nuclear localization (38.3±7.4%) as compared to patients that have progressed on AR-targeting therapies (60.4±4.9%). We have also identified significant heterogeneity in both AR localization and total AR protein expression that correlated with treatment history. Furthermore, CTCs have been identified in having 100 % AR nuclear localization in patients undergoing AR targeted therapies, indicating a possible subpopulation of CTCs that exhibit resistance phenotypes. mRNA was also extracted from CTCs of men with CRPC for analysis of AR splice variant expression. Results showed various levels of splice variant expression from men previously on AR targeting therapies. These studies indicate that a comprehensive, real-time analysis of the AR can be serially assessed for early signs of tumor resistance to AR targeting agents. Citation Format: Lindsay N. Strotman, Jamie Sperger, Benjamin Casavant, Stephanie Theide, David Beebe, Joshua Lang. Molecular interrogation of the androgen receptor in circulating tumor cells to identify therapeutic resistance to androgen targeting therapies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4814. doi:10.1158/1538-7445.AM2014-4814