Genesis of the CTC-iChip

 

When cancer cells detach from a primary tumor, the cells enter and exit the blood vessels.  Some of these tumor cells shedding into the vasculature will go on to form metastases at sites distant from the primary cancer site.  Tumor cells that are identified in transit within the blood stream are known as circulating tumor cells (CTCs).  CTCs are extraordinarily rare, with only one tumor cell per billion normal blood cells in the circulation of patients with advanced cancer.   An innovation team of MGH bioengineers, molecular biologists, and cancer clinicians has developed novel microfluidic-based CTC capture platforms that can sort and isolate pure populations of CTCs – one tumor cell at a time – in sufficient numbers and under conditions that are compatible with detailed molecular and functional analysis after capture.  This rare cell sorting technology enables real-time CTC probing for noninvasive screening of tumor evolution and for predictive biomarkers to guide cancer therapy for the millions of new cancer patients diagnosed worldwide each year. Read More

There are a variety of CTC isolation approaches that rely on the physical or chemical properties, expression of biomarkers, or functional characteristics of the cells. Since the early 2000’s, bioengineers from the MGH Center for Engineering in Medicine have designed, developed, and tested new microfluidic-based methodologies and systems to isolate common and rare bioparticles, single cells, and homogeneous populations of living cells from various bodily fluids. Read more information about using inertial focusing to separate bioparticles in microchannels.  In exploring potential applications for their novel rare cell sorting technologies, the group recognized how important CTCs were becoming in the cancer field.  Together with cancer clinicians from the MGH Cancer Center, the team recognized that CTC-based diagnostics and therapeutics could be realized only if the CTCs were captured in high numbers with high purity.  In one of their most clinically relevant applications, the team has developed microfluidics-based technologies to provide very high accuracy, sensitivity, and specificity in detecting CTCs.  The CTC-iChip is capturing CTCs from many different types of cancers to help investigators further characterize the molecular and cellular features of different CTCs, the factors underlying their shedding into the bloodstream, and their role as key drivers of human blood-borne metastasis, the condition directly responsible for nearly all cancer-related deaths.  Read more about the clinical testing results from the CTC-iChip.

CTC detection

Fluorescent micrographs of CTCs

Patient CTCs come in different sizes and shapes

Ultrasensitive techniques to isolate CTCs have advanced the emerging field of CTC biology to identify, enumerate, and characterize CTCs.  Distinguishing CTCs from normal blood cells is the first step in being able to capture these rare cells in patient blood samples.  The most widely used CTC isolation techniques rely on antibody-based capture of CTCs, which express epithelial cell surface markers that are not present in normal leukocytes (white blood cells, WBCs).  EpCAM, epithelial cell adhesion molecule, is most commonly used because its expression is virtually universal in cells of epithelial origin (including epithelial cancers like lung, breast, and prostate), but it is absent in blood cells.  Immunomagnetic bead capture is a commercially available methodology in which an EpCAM antibody is used to capture CTCs, which are then visualized by staining with a mixture of antibodies against the cytoplasmic epithelial cytokeratins to eliminate cells of epithelial origin. These and the other current CTC isolation methods relying on the chemical and physical properties of CTCs, which traditionally involve multiple batch processing steps, have unique advantages and limitations.

The micropost CTC-Chip

&copy Nagrath et al. Originally published in Nature. 450: 1235-1239. doi: 10.1038/nature06385

Scanning electron micrograph of the microposts array showing pillars coated with an antibody to bind CTCs

The MGH bioengineers designed and developed a microfluidic system that could capture large numbers of viable CTCs in a single step from whole blood without pre-dilution, pre-labeling, pre-fixation, or any other processing steps.  The first microfluidic device or chip was an array consisting of 78,000 microposts coated with EpCAM antibodies. Using deterministic lateral displacement separation principles, the bioengineers optimized the laminar flow kinetics and determined a velocity that would maximize the duration of cell-micropost contact and minimize the shear forces for maximal contact, so that the EpCAM-expressing CTCs could bind to the microposts as they moved within the system, and white blood cells and red blood cells could be washed from the chip.  The captured CTCs attached to the microposts were visualized and confirmed as CTCs through staining with antibodies, which differentiates nonspecifically bound white blood cells from epithelial CTCs.  For CTC enumeration, the entire capture device was imaged using a semi-automated imaging system while on-chip lysis allowed for DNA and RNA extraction and subsequent molecular analysis of the CTCs.  Initial proof-of-principle studies testing blood samples from patients with metastatic lung, prostate, pancreas, breast, and colon cancer showed a high sensitivity (1 target cell in 1 billion blood cells), more than 47% purity, and 99% yield across all five cancers.   And the capture system sorted these rare cells directly from whole blood in a single step. Performance testing confirmed that the high capture rates and viable condition of the CTCs (98% were viable) isolated via the micropost CTC-Chip allowed the captured CTCs to be used for high accuracy cell enumeration and molecular characterization, including mutational analysis on DNA recovered from the CTCs (Nagrath et al, 2007).

Automating the image analysis platform

The micropost CTC-Chip represented landmark technology and provided improved yield and purity of captured CTCs isolated by commercial platforms.  However, the micropost CTC-Chip isolation strategy required highly skilled sample handling and the hand counting of thousands of images for each chip. The investigators recognized that manual image analysis would limit its use in high-throughput applications. To develop a robust and automated platform capable of high-throughput complex analysis of rare cells captured in a three-dimensional matrix, the investigators developed image processing algorithms and scoring criteria to quantify the number of captured CTCs, using available unique prostate cancer markers as a test.  The digital imaging system with integration of the complete CTC-Chip footprint provided multiplane scanning capacity, a 75% reduction in scanning time, increased image quality, and reduced interoperator variability (Stott et al, STM, 2010).

Second generation design improvements in the herringbone CTC-Chip

&copy Stott et al. Originally published in Proceedings of the National Academy of Sciences U S A. 107:18392-18397. doi: 10.1073/pnas.1012539107

Micrograph of the herringbone grooved surface

The micropost CTC-Chip relied on laminar flow, thereby limiting the interactions of target cells with surfaces.  In addition, the complex micropost geometry proved challenging to scale up for high-throughput production and larger-scale clinical applications.  In the next phase of the design process, the bioengineers created surface ridges or herringbones in the wall of the device wherein herringbone-induced microvortices disrupted the laminar flow streamlines that cells traveled, causing them to ‘shift’, increasing the number of cell-surface interactions in the antibody-coated device.  In proof-of-principle studies, the cell capture efficiency of the herringbone CTC-Chip (HB Chip) was greater than that of the traditional flat-walled microfluidic device.  A larger version of the HB-chip was created to compare the cell capture efficiency between the HB-Chip and the micropost CTC-Chip for CTCs captured from patients with metastatic prostate cancer and lung cancer and again, the HB-Chip captured more CTCs with greater purity (Stott et al, PNAS, 2010).

The high-throughput microvortex mixing device ensured effective contact of cells with antibody-coated surfaces, while designed with simple geometry amenable for large-scale manufacturing. The use of transparent materials allowed for imaging of the captured CTCs using standard clinical histopathological stains, in addition to immunofluorescence-conjugated antibodies.  The low shear design of the HB-Chip revealed a surprising finding on the chips – the capture of rare multicellular CTC clusters in the bloodstream of patients with metastatic prostate or lung cancer . CTC microclusters had never been identified using the micropost CTC-Chip, which was designed to allow flow of single cells between the microposts.  The clinical importance of this most unexpected finding is that circulating CTC clusters have the potential to lodge in distal capillary beds and initiate the formation of metastatic lesions.

Substantial performance enhancements with the CTC-iChip

&copy Karabacak et al. Originally published in Nature Protocols. 9:694-710. doi:10.1038/nprot.2014.044

Two separate microfluidic devices house three different microfluidic components engineered for inline operation

On both the micropost CTC-Chip and HB-Chip, the CTCs were bound by tumor antigens and yielded cells that were physically attached to a microfluidic surface. CTC sorting as cells in suspension and not bound to a surface allows for high-quality clinically standardized morphological and immunohistochemical analyses and RNA-based, single cell micromanipulation.  To overcome the critical shortcoming of the cells being bound to the microposts, the bioengineers incorporated the strength of microfluidics for rare cell handling with the benefits of magnetic-based cell sorting.

Three sequential microfluidic technologies within a single, integrated, and automated microfluidic system were designed and optimized in the microfluidic CTC capture platform now called the “CTC-iChip.”  The CTC-iChip isolation strategy uses two operational modes of immunomagnetic sorting to isolate CTCs: a positive selection mode (tumor antigen-dependent) whereby CTCs are identified and sorted on the basis of their expression of EpCAM and a negative depletion mode (tumor antigen-independent) in which the blood sample is depleted of normal white blood cells with antibodies like CD45 and CD14 directed against them.

&copy Ozkumur et al. Originally published in Science Translational Medicine. 5: 179ra47. doi: 10.1126/scitranslmed.3005616

Magnetopheretic depletion of leukocytes. (2) Cells align in a single stream after passing through asymmetric focusing units. (3) Magnetically tagged cells are then deflected using an external magnetic field. (4) CTCs and WBCs are separated by the end of the channel.

After target cell labeling, the first stage within the CTC-iChip uses hydrodynamic sized-based sorting to achieve low shear microfluidic debulking of whole blood.  The technology created in the micropost CTC-Chip without the antibody is used to separate and discard the red blood cells, platelets, plasma proteins, and free magnetic beads.  The nucleated cells (white blood cells and CTCs) are retained and presented to the second stage for inertial focusing.  These nucleated cells are aligned within the microfluidic channel using inertial focusing to order the cells both laterally and longitudinally, so that the cells can be precisely deflected into a collection channel with minimal magnetic moment. In the third and final component, magnetically-labeled cells are separated from unlabeled cells within a deflection channel.  In essence, the three integrated microfluidic technologies replace bulk red blood cell lysis and/or centrifugation, hydrodynamic sheath flow in flow cytometry, and magnetic-activated cell sorting.

Performance tests of both the positive selection and negative depletion modes has been conducted in epithelial cancers (lung, prostate, pancreas, breast, and brain) and non-epithelial cancers (melanoma). The CTC-iChip has the ability to process large volumes of whole blood (8 ml per hour), with high throughput (10 million cells per second), and at high efficiency, in either positive selection or negative depletion mode (Ozkumur et al, 2013).  This unique approach (described in Karabacak et al, 2014) enables cytopathological and molecular characterization of both epithelial and non-epithelial cancers .  In cancers that have undergone endothelial-mesenchymal transition or do not express established epithelial cell surface markers (triple-negative breast cancer, pancreatic cancer, and melanoma as examples), the CTC-iChip can capture these CTCs.  In addition, the CTC-iChip displays increased sensitivity for cancer patients with low counts of CTCs, which may also have relatively low expression that may not be captured using existing immunoaffinity techniques.

Support for the design, development, and testing of the microfluidic-based CTC capture platforms has come from National Institutes of Health grants, an award from the Prostate Cancer Foundation, a “dream team award” from Stand Up To Cancer, and Johnson & Johnson.

Transforming the CTC-iChip from bench top to bedside

The CTC-iChip is an unbiased, broadly applicable, high throughput, and operator-independent rare cell sorting technology compatible with standardized molecular assays, cytopathology, and single-cell expression profiling.  Two distinct features of the CTC-iChip enable a variety of applications for research and clinical diagnostics of CTCs: the cells are in suspension rather than mobilized on a chip, and the mode of CTC isolation is tumor antigen-independent.  Clinical applications that are becoming routine in cancer treatment include initial genotyping of cancer, molecular characterization of cancer, and repeated noninvasive sampling of tumors during treatment.  The CTC-iChip is an excellent tool to provide detailed molecular analyses and reliable information for clinical monitoring of individual patients undergoing cancer therapy, and  could offer a novel approach for early detection of invasive cancer before the establishment of metastatic disease.  The MGH team is working in partnership with Johnson & Johnson scientists to expand the clinical testing and availability of the CTC-iChip technology.

Relevant publications

Nagrath S, Sequist LV, Maheswaran S, Bell DW, Irimia D, Ulkus L, et al.  Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature. 2007 Dec 20;450(7173):1235-9. PubMed PMID: 18097410; PubMed Central PMCID: PMC3090667

Maheswaran S, Sequist LV, Nagrath S, Ulkus L, Brannigan B, Collura CV, et al.   Detection of mutations in EGFR in circulating lung-cancer cells. N Engl J Med. 2008 Jul 24;359(4):366-77. PubMed PMID: 18596266; PubMed Central PMCID: PMC3551471

Sequist LV, Nagrath S, Toner M, Haber DA, Lynch TJ. The CTC-chip: an exciting new tool to detect circulating tumor cells in lung cancer patients. J Thorac Oncol. 2009 Mar;4(3):281-3. PubMed PMID: 19247082

Stott SL, Lee RJ, Nagrath S, Yu M, Miyamoto DT, Ulkus L, et al. Isolation and characterization of circulating tumor cells from patients with localized and  metastatic prostate cancer. Sci Transl Med. 2010 Mar 31;2(25):25ra23. PubMed PMID: 20424012; PubMed Central PMCID: PMC3141292

Stott SL, Hsu CH, Tsukrov DI, Yu M, Miyamoto DT, Waltman BA, et al. Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. Proc Natl Acad Sci U S A. 2010 Oct 26;107(43):18392-7. PubMed PMID: 20930119; PubMed Central PMCID: PMC2972993

Yu M, Stott S, Toner M, Maheswaran S, Haber DA. Circulating tumor cells: approaches to isolation and characterization. J Cell Biol. 2011 Feb 7;192(3):373-82. Review. PubMed PMID: 21300848; PubMed Central PMCID: PMC3101098

Ozkumur E, Shah AM, Ciciliano JC, Emmink BL, Miyamoto DT, Brachtel E, et al.  Inertial focusing for tumor antigen-dependent and -independent sorting of rare circulating tumor cells. Sci Transl Med. 2013 Apr 3;5(179):179ra47. PubMed PMID: 23552373; PubMed Central PMCID: PMC3760275

Karabacak NM, Spuhler PS, Fachin F, Lim EJ, Pai V, Ozkumur E, et al. Microfluidic, marker-free isolation of circulating tumor cells from blood samples. Nat Protoc. 2014 Mar;9(3):694-710. PubMed PMID: 24577360; PubMed Central PMCID: PMC4179254

Contact

Mehmet Toner, PhD Daniel A. Haber, MD Shannon L. Stott, PhD Nezihi Karabacak, PhD
mtoner@hms.harvard.edu dhaber@mgh.harvard.edu sstott@mgh.harvard.edu kmurat@gmail.com/a>
617-724-5336 617-726-7805 617-726-7805 617-726-3474

See Less

Grace McDonald-SmithGenesis of the CTC-iChip