Integrated Micro Nanotechnologies for the Isolation and Single Cell Multiplex Gene Expression Analysis of Circulating Tumor Cells
Eric Lin1, Lianette Rivera1, Shamileh Fouladdel,Hyeun Joong Yoon1, Jacob Wieger1, Stephanie Guthrie1, Yadwinder S. Deol2, Evan Keller2, Vaibhav Sahai2, Diane M. Simeone2, Monika L. Burness2, Ebrahim Azizi2, Max S. Wicha2, and Sunitha Nagrath1
1Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, USA 2Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA
Introduction and Objective: Circulating tumor cells (CTCs) are shed from the primary tumor into the peripheral blood. CTCs are emerging as important biomarkers with high clinical relevance. Enumeration of CTCs may have several clinical uses, including determination of prognosis in patients with established malignancy, or even detection of previously undiagnosed cancer. However, due to the limitation of sensitivity and specificity of current technologies for CTC isolation, the full potential of CTCs has yet to be realized. Furthermore, emerging research show that a small number of cells have stem cell-like nature in various cancers and those are called cancer stem cells (CSC) which may arise from differentiated cancer cells through EMT and have the potential to self-renew and are pluripotent. Emerging microfluidic technologies are promising for isolating both CTCs and CSCs with a high yield and specificity. We present novel integrated nano microfluidic technologies that enable both functional and genomic assays beyond enumeration. We developed an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs yields the highest throughput with high CTC recovery and high blood cell removal among all the label-free technologies. The isolated CTC populations were further analyzed for single cell multiplex gene expression analysis.
Methods: The PDMS-made inertial microfluidic device has 637 mm in length with 56 corners, 500 μm in width, and 100 μm in height. The separation of is driven by two main forces: (i) inertial force that focuses the cells into streamlines, and (ii) drag force from Dean flow that migrates the focused cells to various positions based on size. Device is optimized with MCF-7 and Panc-1 cell line within PBS buffer solution and diluted blood, and is tested in patients with breast cancer on an average of 10 mLs of whole blood processed through double devices in series. CTCs isolated were analyzed for tumor specific protein markers and genomic characterization is done using singe cell analysis techniques.
Results: Samples are processed through the inertial microfluidic device and CTCs are enriched in second outlet based on size difference between CTCs and blood cells. Device is optimized to operate at an extremely high throughput of 2500 μL/min with high recovery (greater than 90%) and high white blood cells (WBCs) removal (5 log orders). In patient samples, we identified CTCs in 38 of 40 (95%) breast patients with metastatic disease (5.4±4.6 CTC/mL) with low WBC contamination (663±647 WBC/mL). We utilized multiplex PCR to interrogate expression of 96 genes in single CTC’s isolated using label free purification followed by C1 and BioMark (Fluidigm® USA) analysis. Based on the gene expression, both inter and intra patient heterogeneity of CTCs at the single cell level were discovered among the tested patient samples.
Conclusion: The molecular and genetic profiling of CTCs also presents a possible alternative to painful, costly, and invasive biopsies. – and will foster the advancement of science and engineering via the exploration of new druggable targets approaches and the further understanding of the pharmacodynamics.