A Systemic Approach to Isolate, Retrieve, and Characterize Trophoblasts from the Maternal Circulation Using a Centrifugal Microfluidic Disc and a Multiple Single-Cell Retrieval Strategy.

Rare cells in the blood often have rich clinical significance. Although their isolation is highly desirable, this goal remains elusive due to their rarity. This paper presents a systemic approach to isolate and characterize trophoblasts from the maternal circulation. A microfluidic rare cell disc assay (RaCDA) was designed to process an extremely large volume of up to 15 mL of blood in 30 min, depleting red blood cells (RBCs) and RBC-bound white blood cells (WBC) while isolating trophoblasts in the collection chip. To minimize cell loss, on-disc labeling of cells with fluorescent immuno-staining identified the trophoblasts. Retrieval of trophoblasts utilized an optimized strategy in which multiple single cells were retrieved within the same micropipette column, with each cell encapsulated in a fluid volume (50 nL) separated by an air pocket (10 nL). Further, whole-genome amplification (WGA) amplified contents from a few retrieved cells, followed by quality control (QC) on the success of WGA via housekeeping genes. For definitive confirmation of trophoblasts, short-tandem repeat (STR) of the WGA-amplified content was compared against STR from maternal WBC and amniocytes from amniocentesis. Results showed a mean recovery rate (capture efficiency) of 91.0% for spiked cells with a WBC depletion rate of 99.91%. The retrieval efficiency of single target cells of 100% was achieved for up to four single cells retrieved per micropipette column. Comparison of STR signatures revealed that the RaCDA can retrieve trophoblasts from the maternal circulation.

A spatiotemporally defined in vitro microenvironment for controllable signal delivery and drug screening.

Cancer metastasis and drug resistance are important malignant tumor phenotypes that cause roughly 90% mortality in human cancers. Current therapeutic strategies, however, face substantial challenges partially due to a lack of applicable pre-clinical models and drug-screening platforms. Notably, microscale and three-dimensional (3D) tissue culture platforms capable of mimicking in vivo microenvironments to replicate physiological conditions have become vital tools in a wide range of cellular and clinical studies. Here, we present a microfluidic device capable of mimicking a configurable tumor microenvironment to study in vivo-like cancer cell migration as well as screening of inhibitors on both parental tumors and migratory cells. In addition, a novel evaporation-based paper pump was demonstrated to achieve adaptable and sustainable concentration gradients for up to 6 days in this model. This straightforward modeling approach allows for fast patterning of a wide variety of cell types in 3D and may be further integrated into biological assays. We also demonstrated cell migration from tumor spheroids induced by an epidermal growth factor (EGF) gradient and exhibited lowered expression of an epithelial marker (EpCAM) compared with parental cells, indicative of partial epithelial-mesenchymal transition (EMT) in this process. Importantly, pseudopodia protrusions from the migratory cells - critical during cancer metastasis - were demonstrated. Insights gained from this work offer new opportunities to achieve active control of in vitro tumor microenvironments on-demand, and may be amenable towards tailored clinical applications.

Author Correction: Immunofluorescence can assess the efficacy of mTOR pathway therapeutic agent Everolimus in breast cancer models.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Immunofluorescence can assess the efficacy of mTOR pathway therapeutic agent Everolimus in breast cancer models.

When breast cancer patients start to exhibit resistance to hormonal therapy or chemotherapy, the mTOR inhibitor everolimus can be considered as an alternative therapeutic agent. Everolimus can deregulate the PI3K/AKT/mTOR pathway and affect a range of cellular functions. In some patients, the agent does not exhibit the desired efficacy and, even worse, not without the associated side effects. This study assessed the use of immunofluorescence (IF) as a modality to fill this unmet need of predicting the efficacy of everolimus prior to administration. Cell viability and MTT assays based on IF intensities of pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 on breast cancer cells (Hs578T, MCF7, BT474, MDA-MB-231) and patient-derived cell culture from metastatic sites (ABC-82T and ABC-16TX1) were interrogated. Results show that independent pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 IF expressions can classify data into different groups: everolimus sensitive and resistant. The combined IF baseline intensity of these proteins is predictive of the efficacy of everolimus, and their intensities change dynamically when cells are resistant to everolimus. Furthermore, mTOR resistance is not only consequence of the AKT/mTOR pathway but also through the LKB1 or MAPK/ERK pathway. The LKB1 and pho-GSK3ß may also be potential predictive markers for everolimus.

Modeling of cancer metastasis and drug resistance via biomimetic nano-cilia and microfluidics.

Three-dimensional (3D) tissue culture platforms that are capable of mimicking in vivo microenvironments to replicate physiological conditions are vital tools in a wide range of cellular and clinical studies. Here, learning from the nature of cilia in lungs - clearing mucus and pathogens from the airway - we develop a 3D culture approach via flexible and kinetic copolymer-based chains (nano-cilia) for diminishing cell-to-substrate adhesion. Multicellular spheroids or colonies were tested for 3-7 days in a microenvironment consisting of generated cells with properties of putative cancer stem cells (CSCs). The dynamic and reversible regulation of epithelial-mesenchymal transition (EMT) was examined in spheroids passaged and cultured in copolymer-coated dishes. The expression of CSC markers, including CD44, CD133, and ABCG2, and hypoxia signature, HIF-1α, was significantly upregulated compared to that without the nano-cilia. In addition, these spheroids exhibited chemotherapeutic resistance in vitro and acquired enhanced metastatic propensity, as verified from microfluidic chemotaxis assay designed to replicate in vivo-like metastasis. The biomimetic nano-cilia approach and microfluidic device may offer new opportunities to establish a rapid and cost-effective platform for the study of anti-cancer therapeutics and CSCs.