All-in-One digital microfluidics pipeline for proteomic sample preparation and analysis.

Highly sensitive and reproducible analysis of samples containing low amounts of protein is restricted by sample loss and the introduction of contaminants during processing. Here, we report an All-in-One digital microfluidic (DMF) pipeline for proteomic sample reduction, alkylation, digestion, isotopic labeling and analysis. The system features end-to-end automation, with integrated thermal control for digestion, optimized droplet additives for sample manipulation and analysis, and an automated interface to liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Dimethyl labeling was integrated into the pipeline to allow for relative quantification of the trace samples at the nanogram level, and the new pipeline was applied to evaluating cancer cell lines and cancer tissue samples. Several known proteins (including HSP90AB1, HSPB1, LDHA, ENO1, PGK1, KRT18, and AKR1C2) and pathways were observed between model breast cancer cell lines related to hormone response, cell metabolism, and cell morphology. Furthermore, differentially quantified proteins (such as PGS2, UGDH, ASPN, LUM, COEA1, and PRELP) were found in comparisons of healthy and cancer breast tissues, suggesting potential utility of the All-in-One pipeline for the emerging application of proteomic cancer sub-typing. In sum, the All-in-One pipeline represents a powerful new tool for automated proteome processing and analysis, with the potential to be useful for evaluating mass-limited samples for a wide range of applications.

Analysis of the effects of aryl hydrocarbon receptor expression on cancer cell invasion via three-dimensional microfluidic invasion assays.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that binds to xenobiotics and activates expression of response elements to metabolize these compounds. The AHR pathway has been associated with a long list of diseases including cancer; however, it is debated whether AHR is tumorigenic or tumour-inhibiting. In particular, there are contradictory reports in the literature regarding the effects of AHR expression level on metastatic breast cancer. Here we used a 3D invasion assay called cell invasion in digital microfluidic microgel systems (CIMMS) to study the effect of AHR expression on invasion. In this study, MDA-MB-231 cells with stable knockout of AHR (AHRko) showed enhanced invasive characteristics and reduced proliferation, and cells with transient overexpression of AHR showed reduced invasiveness. Overexpression of AHR with a mutation in the DNA binding domain showed no difference in invasiveness compared to control, which suggests that the changes in invasiveness are related to the expression of AHR. CIMMS also allowed for extraction of sub-populations of invaded cells for RNA sequencing experiments. A comparison of the transcriptomes of invaded subpopulations of wild-type and AHRko cells identified 1809 genes that were differentially expressed, with enriched pathways including cell cycle, proliferation, survival, immunoproteasome activation, and activation of matrix metalloproteases. In sum, the data reported here for MDA-MB-231 cells suggests some new interpretations of the discrepancy in the literature on the role of AHR in breast cancer. We propose that the unique combination of functional discrimination with transcriptome profiling provided by CIMMS will be valuable for a wide range of mechanistic invasion-biology studies in the future.

Cell invasion in digital microfluidic microgel systems.

Microfluidic methods for studying cell invasion can be subdivided into those in which cells invade into free space and those in which cells invade into hydrogels. The former techniques allow straightforward extraction of subpopulations of cells for RNA sequencing, while the latter preserve key aspects of cell interactions with the extracellular matrix (ECM). Here, we introduce "cell invasion in digital microfluidic microgel systems" (CIMMS), which bridges the gap between them, allowing the stratification of cells on the basis of their invasiveness into hydrogels for RNA sequencing. In initial studies with a breast cancer model, 244 genes were found to be differentially expressed between invading and noninvading cells, including genes correlating with ECM-remodeling, chemokine/cytokine receptors, and G protein transducers. These results suggest that CIMMS will be a valuable tool for probing metastasis as well as the many physiological processes that rely on invasion, such as tissue development, repair, and protection.