Pairing Microwell Arrays with an Affordable, Semiautomated Single-Cell Aspirator for the Interrogation of Circulating Tumor Cell Heterogeneity.

Comprehensive analysis of tumor heterogeneity requires robust methods for the isolation and analysis of single cells from patient samples. An ideal approach would be fully compatible with downstream analytic methods, such as advanced genomic testing. These endpoints necessitate the use of live cells at high purity. A multitude of microfluidic circulating tumor cell (CTC) enrichment technologies exist, but many of those perform bulk sample enrichment and are not, on their own, capable of single-cell interrogation. To address this, we developed an affordable semiautomated single-cell aspirator (SASCA) to further enrich rare-cell populations from a specialized microwell array, per their phenotypic markers. Immobilization of cells within microwells, integrated with a real-time image processing software, facilitates the detection and precise isolation of targeted cells that have been optimally seeded into the microwells. Here, we demonstrate the platform capabilities through the aspiration of target cells from an impure background population, where we obtain purity levels of 90%-100% and demonstrate the enrichment of the target population with high-quality RNA extraction. A range of low cell numbers were aspirated using SASCA before undergoing whole transcriptome and genome analysis, exhibiting the ability to obtain endpoints from low-template inputs. Lastly, CTCs from patients with castration-resistant prostate cancer were isolated with this platform and the utility of this method was confirmed for rare-cell isolation. SASCA satisfies a need for an affordable option to isolate single cells or highly purified subpopulations of cells to probe complex mechanisms driving disease progression and resistance in patients with cancer.

User-defined morphogen patterning for directing human cell fate stratification.

Concentration gradients of biochemical stimuli such as morphogens play a critical role in directing cell fate patterning across species and throughout development but are not commonly recapitulated in vitro. While in vitro biomolecule gradients have been generated using customized microfluidic platforms, broad implementation has been limited because these platforms introduce new variables to cell culture such as externally driven flow, culture in a specialized matrix, or extended time for in situ long range diffusion. Here we introduce a method that enables preforming and then transferring user-controlled gradients to cells in standard "open" cultures. Our gradient patterning devices are modular and decoupled from the culture substrate. We find that gradient generation and transfer are predictable by finite element modeling and that device and loading parameters can be used to tune the stimulus pattern. Furthermore, we demonstrate use of these devices to spatially define morphogen signal gradients and direct peri-gastrulation fate stratification of human pluripotent stem cells. This method for extrinsic application of biochemical signal gradients can thus be used to spatially influence cellular fate decisions in a user-controlled manner.

Interrogating Bronchoalveolar Lavage Samples via Exclusion-Based Analyte Extraction.

Although average survival rates for lung cancer have improved, earlier and better diagnosis remains a priority. One promising approach to assisting earlier and safer diagnosis of lung lesions is bronchoalveolar lavage (BAL), which provides a sample of lung tissue as well as proteins and immune cells from the vicinity of the lesion, yet diagnostic sensitivity remains a challenge. Reproducible isolation of lung epithelia and multianalyte extraction have the potential to improve diagnostic sensitivity and provide new information for developing personalized therapeutic approaches. We present the use of a recently developed exclusion-based, solid-phase-extraction technique called SLIDE (Sliding Lid for Immobilized Droplet Extraction) to facilitate analysis of BAL samples. We developed a SLIDE protocol for lung epithelial cell extraction and biomarker staining of patient BALs, testing both EpCAM and Trop2 as capture antigens. We characterized captured cells using TTF1 and p40 as immunostaining biomarkers of adenocarcinoma and squamous cell carcinoma, respectively. We achieved up to 90% (EpCAM) and 84% (Trop2) extraction efficiency of representative tumor cell lines. We then used the platform to process two patient BAL samples in parallel within the same sample plate to demonstrate feasibility and observed that Trop2-based extraction potentially extracts more target cells than EpCAM-based extraction.

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. Recently, an advance for sampling suspicious lung nodules to collect mini-bronchoalveolar lavage (mBAL) samples was shown to be diagnostically relevant but limited by standard cytology techniques leading to low sensitivity and specificity. In addition, a second non-invasive method that holds great promise is the collection of circulating tumor cells, a rare population of tumor cells that have shed into peripheral circulation from primary or metastatic tumor sites, from blood. Here, we utilize a recently published platform, VerIFAST, for the capture and proteomic analysis of rare cells, to isolate cells of interest from lung cancer patients using both mBAL and blood samples. The VerIFAST platform leverages surface tension at the microscale to pin aqueous and oil fluids in adjacent chambers to create a virtual filter between two aqueous fluids. In this manuscript, the VerIFAST was further enhanced to include oil pinning, which allowed on-device tumbling, further eliminating a laborious and time consuming step that could result in increased sample loss. Finally, we further developed the base assays used in standard histopathologic assays for diagnostic and pharmacodynamic analysis of these rare lung cancer cells. Specifically, we examined thyroid transcription factor-1 (TTF-1) signal intensity, in which loss is associated with more aggressive disease, and epidermal growth factor receptor (EGFR) signal intensity, which is a high value therapeutic target in lung cancer.