Functional profiling of circulating tumor cells with an integrated vortex capture and single-cell protease activity assay

Significance The current paradigm in liquid biopsies focuses on circulating tumor cell (CTC) count and genomics to provide clinically actionable information. We push beyond current capabilities to introduce a functional assay that quantifies protease secretion from live CTCs with single-cell resolution. In this assay, an integrated microfluidic device captures CTCs from blood and washes and encapsulates them into nanoliter-scale droplets with fluorogenic substrates within minutes, maintaining physiologic conditions. Our data support the presence of outlier CTCs with high protease activity that may drive metastasis or immune evasion. We reveal an intriguing correlation between CTC protease activity and metastatic progression. Such functional liquid biopsy approaches provide avenues to understand the metastatic process and potential for companion diagnostics. Tumor cells are hypothesized to use proteolytic enzymes to facilitate invasion. Whether circulating tumor cells (CTCs) secrete these enzymes to aid metastasis is unknown. A quantitative and high-throughput approach to assay CTC secretion is needed to address this question. We developed an integrated microfluidic system that concentrates rare cancer cells >100,000-fold from 1 mL of whole blood into ∼50,000 2-nL drops composed of assay reagents within 15 min. The system isolates CTCs by size, exchanges fluid around CTCs to remove contaminants, introduces a matrix metalloprotease (MMP) substrate, and encapsulates CTCs into microdroplets. We found CTCs from prostate cancer patients possessed above baseline levels of MMP activity (1.7- to 200-fold). Activity of CTCs was generally higher than leukocytes from the same patient (average CTC/leukocyte MMP activity ratio, 2.6 ± 1.5). Higher MMP activity of CTCs suggests active proteolytic processes that may facilitate invasion or immune evasion and be relevant phenotypic biomarkers enabling companion diagnostics for anti-MMP therapies.

[1]  P. Gresele,et al.  Platelets release active matrix metalloproteinase‐2 in vivo in humans at a site of vascular injury: lack of inhibition by aspirin , 2007, British journal of haematology.

[2]  Jongyoon Han,et al.  Single Cell Analysis of Leukocyte Protease Activity Using Integrated Continuous-Flow Microfluidics. , 2016, Analytical chemistry.

[3]  L. Portella,et al.  Circulating Tumor Cells in Prostate Cancer Diagnosis and Monitoring: An Appraisal of Clinical Potential , 2014, Molecular Diagnosis & Therapy.

[4]  Hamed Haddadi,et al.  Inertial flow of a dilute suspension over cavities in a microchannel , 2016, Journal of Fluid Mechanics.

[5]  Klaus Pantel,et al.  Clinical Applications of Circulating Tumor Cells and Circulating Tumor DNA as Liquid Biopsy. , 2016, Cancer discovery.

[6]  O. Matsushita,et al.  Purification and characterization of Clostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the corresponding gene , 1994, Journal of bacteriology.

[7]  D. Grignon,et al.  Membrane type 1-matrix metalloproteinase (MT1-MMP) and MMP-2 immunolocalization in human prostate: change in cellular localization associated with high-grade prostatic intraepithelial neoplasia. , 1999, Clinical cancer research : an official journal of the American Association for Cancer Research.

[8]  C. Lin,et al.  Matrix metalloproteinase‐9 cooperates with transcription factor Snail to induce epithelial–mesenchymal transition , 2011, Cancer science.

[9]  M. G. Christiansen,et al.  Magnetically Actuated Protease Sensors for in Vivo Tumor Profiling. , 2016, Nano letters.

[10]  S. Jeffrey,et al.  Fast and Label-Free Isolation of Circulating Tumor Cells from Blood: From a Research Microfluidic Platform to an Automated Fluidic Instrument, VTX-1 Liquid Biopsy System , 2018, SLAS technology.

[11]  Jennifer L. Doyle,et al.  Differential role of β‐catenin in VEGF and histamine‐induced MMP‐2 production in microvascular endothelial cells , 2009, Journal of cellular biochemistry.

[12]  S. Bhatia,et al.  Ultrasensitive tumour-penetrating nanosensors of protease activity , 2017, Nature Biomedical Engineering.

[13]  Miles A. Miller,et al.  Single cell multiplexed assay for proteolytic activity using droplet microfluidics. , 2016, Biosensors & bioelectronics.

[14]  Yo Sup Moon,et al.  Quantitative Diagnosis of Malignant Pleural Effusions by Single-Cell Mechanophenotyping , 2013, Science Translational Medicine.

[15]  Guang Yao.  Modelling mammalian cellular quiescence , 2014, Interface Focus.

[16]  Rajan P Kulkarni,et al.  High efficiency vortex trapping of circulating tumor cells. , 2015, Biomicrofluidics.

[17]  Anne Vincent-Salomon,et al.  Circulating Tumor Cell Detection Predicts Early Metastatic Relapse After Neoadjuvant Chemotherapy in Large Operable and Locally Advanced Breast Cancer in a Phase II Randomized Trial , 2008, Clinical Cancer Research.

[18]  Alison Stopeck,et al.  Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[19]  D. Weitz,et al.  Throughput enhancement of parallel step emulsifier devices by shear-free and efficient nozzle clearance. , 2019, Lab on a chip.

[20]  Guocheng Yuan,et al.  GiniClust: detecting rare cell types from single-cell gene expression data with Gini index , 2016, Genome Biology.

[21]  D. Weitz,et al.  Single-cell analysis and sorting using droplet-based microfluidics , 2013, Nature Protocols.

[22]  Charles H. Yoon,et al.  Antibody-mediated inhibition of MICA and MICB shedding promotes NK cell–driven tumor immunity , 2018, Science.

[23]  B. Mroczko,et al.  Serum levels and tissue expression of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinases 2 (TIMP-2) in colorectal cancer patients , 2014, Tumor Biology.

[24]  Fei Zhang,et al.  Tumor-derived Matrix Metalloproteinase-13 (MMP-13) correlates with poor prognoses of invasive breast cancer , 2008, BMC Cancer.

[25]  Z. Werb,et al.  New functions for the matrix metalloproteinases in cancer progression , 2002, Nature Reviews Cancer.

[26]  J. C. Love,et al.  Functional analysis of single cells identifies a rare subset of circulating tumor cells with malignant traits. , 2014, Integrative biology : quantitative biosciences from nano to macro.

[27]  J. Reynolds,et al.  Overexpression of MMP-9 Contributes to Invasiveness of Prostate Cancer Cell Line LNCaP , 2011, Immunological investigations.

[28]  Glenn Heller,et al.  Circulating Tumor Cell Number and Prognosis in Progressive Castration-Resistant Prostate Cancer , 2007, Clinical Cancer Research.

[29]  Yong-Ak Song,et al.  Enhancing protease activity assay in droplet-based microfluidics using a biomolecule concentrator. , 2011, Journal of the American Chemical Society.

[30]  P. Loadman,et al.  Membrane type matrix metalloproteinases (MMPs) show differential expression in non-small cell lung cancer (NSCLC) compared to normal lung: correlation of MMP-14 mRNA expression and proteolytic activity. , 2007, European journal of cancer.

[31]  Miles A. Miller,et al.  Multiplexed protease activity assay for low-volume clinical samples using droplet-based microfluidics and its application to endometriosis. , 2013, Journal of the American Chemical Society.

[32]  Rajan P Kulkarni,et al.  Classification of large circulating tumor cells isolated with ultra-high throughput microfluidic Vortex technology , 2016, Oncotarget.

[33]  Jongyoon Han,et al.  Jetting microfluidics with size-sorting capability for single-cell protease detection. , 2015, Biosensors & bioelectronics.