Microfluidic Technologies for Isolating
and Studying Circulating Tumor Cells
Sunitha Nagrath
Assistant Professor of Chemical Engineering & Biomedical
Engineering
Biointerface Institute, Translational Oncology Program
University of Michigan
Learning Objectives

Circulating Tumor Cells (CTCs)
fundamentals, understanding their role
in cancer diagnosis, monitoring and
prognosis, significance of CTCs as liquid
biopsy in personalized medicine,
Isolation Technologies, state of the art
using microfluidics, emerging
applications of CTCs.
Circulating Tumor Cells: The Liquid Biopsy
“For people with cancer or suspected cancer, the biopsy
is a necessary evil — an uncomfortable and somewhat
risky procedure to extract tissue for diagnosis or
analysis.”
-The New York Times
April 7, 2014
Kaiser. Science, 2010
The term “liquid biopsy”
originally introduced for
the analysis of CTCs is
also used now for ctDNA
analysis. The current
definition of a biopsy is,
however, ‘the removal of
cells or tissues for
examination by a
pathologist’ (National
Cancer Institute at the
National Institutes of
Health). While this term is
suitable for CTCs, it
appears somewhat
misleading when applied
to ctDNA, a fragmented
cell component released
mainly by dying tumor
cells.
Alix-Panabières et. al. Transl
Med. 2013 Jul;1(2):18.
Luis A. Diaz Jr, and Alberto Bardelli JCO 2014;32:579-586
Bednarz-Knoll,
etc.
Metastasis Rev, 2012
all.,
Cancer
Cell Separation Techniques
Integrated
Magsorter
Labyrinth
GO Chip
Evolution of CTC Isolation
Technologies
Yoon, et al., Nagrath, ACS Nano, 2014
Microfluidic Technologies
CTC-Chip 1 mL/hr
Nagrath et al, Nature (2007)
HTMSU 1.6 mL/hr
Adams et al., JACS (2008)
Aptamer chip 2.16 mL/hr Filtration 7.5mL/hr
Sheng et al., Analytical Chemistry (2012)
S. Y. Zheng et al., Biomed. Microdevices, (2011)
HB Chip 1.2 mL/hr
Stott et al., PNAS (2010)
I-chip 3-9 mL/hr
Ozkumur et al., Sci. Transl Med (2013)
GO Chip 3mL/hr
Yoon et al, Nature
Nanotechnology (2014)
Muralidhar et al, Small (2014)
Nano Velcro 1mL/hr
Radial Flow 10mL/hr
CTC Chip: First Microfluidic Device
CTC-Chip Design Goals:
• Minimal handling
– single-step, no pre-labeling, no pre-processing of the sample
• Gentle
• Uniform processing conditions
• >85% of all cancers are epithelial in origin
• Microposts coated with Ab for EpCAM
Longitudinal Correlation of CTCs with Disease
Course
NAGRATH ET AL., NATURE 2007
CTC Biopsy
Maheswaran ET AL., NEJM, 2008
Yoon, et al., Nature Nanotechnology, 2013
CTC-Chip, Nature 2007,
NEJM 2008, STM 2009
HB-Chip, PNAS 2010
GO-Chip, Nature
Nanotech, 2013
Graphene Oxide (GO)
• GO is a derivative of graphene consisting of
oxygen functional groups on their basal plane and edges.
• GO is a single-layer form of graphite oxide.
- Strong paper-like material
- Substantial material for
the manufacture of graphene
- Useful material for biological
applications
• GO layers are about
1.1 ± 0.2nm in thickness
HARP Engineering, LLC
Graphene Oxide (GO) Chip
Yoon, et al., Nature Nanotechnology, 2013
Self-assembly of Graphene Oxide
SEM image of a cancer cell captured on nano post
 High Sensitivity
 High Purity
 Live cells
 Whole blood
 Ability to expand
 RNA/DNA analysis
Graphene Oxide CTC Chip (GO-Chip)
• Cell Capture Experiment (Cells spiked into whole blood)
Cell Capture Efficiency (3~100 cells in Blood)
Mean: 94.2
Mean: 87.3
Mean: 73
Mean: 48
Mean: 13
Mean: 48
Zhang, et al., Oncotarget, 2014
Expansion of CTCs
Expansion of CTCs
Expansion of CTCs from Early Stage
Lung Cancer Patients
Capture yield of early lung CTCs on day 0
Expansion of CTCs from Early Stage
Lung Cancer Patients
Functional and Genomic Assays
Next Generation Sequencing of Cultured Lung
CTCs