Isolation and retrieval of circulating tumor cells using centrifugal forces : Scientific Reports : Nature Publishing Group

Isolation and retrieval of circulating tumor cells using centrifugal forces : Scientific Reports : Nature Publishing Group

This paper is from Clearbridge Biomedics, manufacturers of a commercial CTC system (CTC chip), which    is used to isolate CTCs based on physical properties (size and deformability)

The present paper uses inertial microfluidics (explained here and here ), which uses a spiral channel and the centrifugal forces generated within the channel to separate cells based on their physical properties.

http://www.nature.com/srep/2013/130212/srep01259/carousel/srep01259-f1.jpg

Performance Summary:
> 85% recovery
> positive CTCs enumeration in all samples from patients with metastatic lung cancer (n = 20; 5–88 CTCs per mL
> throughput: 3ml/hr.

Commentary:
This work is concurrently ongoing at Clearbridge and University of Cincinnati (now seemingly independently, though the source of origin is university of cincinnati). Prof. Ian Papautsky from the University of Cincinnati has pioneered inertial microfluidics-based cell sorting for a number of years, leading to many publications.

Epithelial to Mesenchymal Transition in CTCs from breast cancer patients: MGH paper published in Science

Circulating tumor cells get wanderlust

Science 1 February 2013: 
Vol. 339 no. 6119 pp. 580-584DOI:10.1126/science.1228522
Key highlights of the paper:

• Both mesenchymal & epithelial markers were expressed  in rare primary tumor cells but mesenchymal cells were highly enriched in CTCs. 
• An association of mesenchymal CTCs with disease progression was observed by serial monitoring of 11 patients
• Reversible shifts between these epithelial & mesenchymal cell fates accompanied each cycle of response to therapy and disease progression in an index patient
• Provides evidence of EMT in human breast cancer specimens, both in rare cells within primary tumors and more abundantly in CTCs. 
• The researchers found a striking association between expression of mesenchymal markers and clusters of CTCs, rather than single migratory cells.
• The proposal that mesenchymal transformation of epithelial cells is mediated by TGF-β released from platelets is supported by the researchers who note of strong TGF-β signatures in mesenchymal CTC clusters, many of which carry attached platelets
• human primary breast tumors contain rare cancer cells that coexpress mesenchymal and epithelial markers

Methods used:
Herringbone chip using an antibody cocktail (EpCAM, EGFR and Her2). Dual-colorimetric RNA–in situ hybridization (ISH) assay was used to examine tumor cells for expression of  epithelial (E) transcripts: [keratins (KRT) 5, 7, 8, 18, and 19; EpCAM (epithelial cell adhesion molecule); and CDH1 (cadherin 1)] and mesenchymal (M) transcripts: [FN1 (fibronectin 1), CDH2 (cadherin 2), and SERPINE1/PAI1 (serpin peptidase inhibitor, clade E)].

Focus review 2013: Label free microfluidic technologies for isolation of circulating tumor cells (CTCs)

Label-free isolation of circulating tumor cells in microfluidic devices: Current research and perspectives

This paper is quite comprehensive in covering the many label free approaches to CTC isolation reported so far, including, filters, accoustophoresis, magnetophoresis, etc.

The general recommendation of the paper is towards adoption of these technologies for clinical use.

undoubtedly, there are several advantages to label-free isolation of CTCs, specially filtration techniques, which are simple, inexpensive, fast and easy to use. However, there are some important limitations that need to be considered,  which I was hoping the paper would shed light upon, but didnt.

the general limitations of filtration techniques are listed here.

It was also recently reported that the deformability of tumor initiating cells is less differentiated from normal blood cells. This makes the sized and deformability based techniques vulnerable to missing these important subtypes of CTCs. This is covered here 

High-purity and label-free isolation of circulating tumor cells (CTCs) in a microfluidic platform by using optically-induced-dielectrophoretic (ODEP) force

pubs.rsc.org/en/content/articlepdf/2013/lc/c3lc41256c

Probing circulating tumor cells in microfluidics - Latest review paper on Microfluidic CTC technologies

Probing circulating tumor cells in microfluidics - Lab on a Chip (RSC Publishing) DOI:10.1039/C2LC90148J

this is a review paper detailing the latest developments in microfluidics based circulating tumor cell isolation and analysis.

Microfluidics and Circulating Tumor Cells. by On-Q-ity

Microfluidics and Circulating Tumor Cells. [J Mol Diagn. 2012] - PubMed - NCBI

Paper Commentary: SSA-MOA: a novel CTC isolation platform using selective size amplification (SSA) and a multi-obstacle architecture (MOA) filter - Lab on a Chip (RSC Publishing)

SSA-MOA: a novel CTC isolation platform using selective size amplification (SSA) and a multi-obstacle architecture (MOA) filter - Lab on a Chip (RSC Publishing)

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Update (10 Feb, 2013): A follow up improvement of this technology has been published here
performance: 95% isolation efficiency, 59% purity from 3 ml of blood (total isolation time ~ 30 mins)
no results from clinical samples were reported.

another recent publication from Samsung in Biomicrofluidics 
an older publication from Samsung advanced Institute of Technology can be found here
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Summary:

This paper presents a novel method of enriching CTCs by combining EpCAM based bead tagging followed by size filtration. The basic premise of this paper is that purely size-based techniques suffer from size overlap between CTCs and WBCs. To overcome this, CTCs are selectively tagged with 3 micron polymer beads to enhance their size, followed by filtration through a lateral filter surface. recoveries of upto 99.1% are reported in spiked cells.

Advantages:

• Results show that the size enhancement seems to work in appropriately discriminating between WBCs and MCF-7 cells

Limitations:

• Fundamentally, this is an antigen dependent technology, hence belongs to positive enrichment family of techniques. Its inherently limited by antigen expression and its variability as well as the efficiency of binding interaction.
• There are other known cancer cell lines which are smaller than the MCF-7 cells, how does this technique work with smaller cancer cells? smaller cells will require larger beads for effective differentiation, however, steric hindrance will become a factor between adjoining binding sites
• silicon manufacturing technology is expensive as it is. it is unclear how expensive is the silicon on glass technology
• throughput is 20ul/min, which is considerably lower than size filtration systems, which have reported as fast as 2 mls in 5 minutes. the overall assay time is also increased due to the need to pre-conjugate beads to cells
• the effect of occupancy of antigen binding sites by microbeads on downstream molecular characterization of tumor cells
• lack of clinical data
• lateral flow filtration is inherently limited by the requirement of scaling for higher throughput versus large area staining and imaging requirement

other considerations:

1. http://www.clearbridgebiomedics.com/ --> has a platform for size and deformability based CTC isolation
2. cell size enhancement product is available here  http://pluriselect.com/home.html, 
3. how is the performance of this paper in comparison to item 2 above, which should be relatively inexpensive and has a higher thorughput