Saturday, December 29, 2012

Microfluidics separation reveals the stem-cell–like deformability of tumor-initiating cells

Microfluidics separation reveals the stem-cell–like deformability of tumor-initiating cells

Key findings:
Here we report a microfluidics method to enrich physically deformable cells by mechanical manipulation through artificial microbarriers. Driven by hydrodynamic forces, flexible cells or cells with high metastatic propensity change shape to pass through the microbarriers and exit the separation device, whereas stiff cells remain trapped. We demonstrate the separation of (i) a mixture of two breast cancer cell types (MDA-MB-436 and MCF-7) with distinct deformabilities and metastatic potentials, and (ii) a heterogeneous breast cancer cell line (SUM149), into enriched flexible and stiff subpopulations. We show that the flexible phenotype is associated with overexpression of multiple genes involved in cancer cell motility and metastasis, and greater mammosphere formation efficiency. Our observations support the relationship between tumor-initiating capacity and cell deformability, and demonstrate that tumor-initiating cells are less differentiated in terms of cell biomechanics.

Significance: This is a key finding, which raises the following question; are technologies that use size and deformability as a criteria to enrich Circulating Tumor Cells (CTCs) are inherently biased towards missing cells with high metastatic propensity?


Saturday, December 22, 2012

Published CTC patents and patent applications

A list of all relevant patents for "rare cells from fluid" can be found here

http://www.patentfish.com/rare-cells-from-fluid

=======================================================================


http://www.google.com/patents/US7863012
http://www.google.com/patents/EP1425294B1?cl=en
http://www.google.com.sg/patents/EP1597353A2?cl=en
http://www.google.com.sg/patents/US8329422

Technology: EpCAM based enrichment using magnetic beads
Abstract:
The methods and reagents described in this invention are used to analyze circulating tumor cells, clusters, fragments, and debris. Analysis is performed with a number of platforms, including flow cytometry and the CellSpotter® fluorescent microscopy imaging system. Analyzing damaged cells has shown to be important. However, there are two sources of damage: in vivo and in vitro. Damage in vivo occurs by apoptosis, necrosis, or immune response. Damage in vitro occurs during sample acquisition, handling, transport, processing, or analysis. It is therefore desirable to confine, reduce, eliminate, or at least qualify in vitro damage to prevent it from interfering in analysis. Described herein are methods to diagnose, monitor, and screen disease based on circulating rare cells, including malignancy as determined by CTC, clusters, fragments, and debris. Also provided are kits for assaying biological specimens using these methods.
(Company:  Veridex LLC)
========================================================================

http://www.google.com.sg/patents/EP2229441A2?cl=en

Technology: EpCAM based moagentic beads
Abstract:
Described here is an automated robotic device that isolates circulating tumor cells (CTCs) or other biological structures with extremely high purity. The device uses powerful magnetic rods covered in removable plastic sleeves. These rods sweep through blood samples, capturing, e.g., cancer cells labeled with antibodies linked to magnetically responsive particles such as superparamagnetic beads. Upon completion of the capturing protocol, the magnetic rods undergo several rounds of washing, thereby removing all contaminating blood cells. The captured target cells are released into a final capture solution by removing the magnetic rods from the sleeves. Additionally, cells captured by this device show no reduced viability when cultured after capture. Cells are captured in a state suitable for genetic analysis. Also disclosed are methods for single cell analysis. Being robotic allows the device to be operated with high throughput.
(Company:  Magsweeper)
========================================================================

http://www.google.com.sg/patents/EP1984030A4?cl=en

Technology: HD CTC technology
Abstract:
Methods are provided for detecting circulating tumor cells in a mammalian subject. Methods of diagnosing cancer in a mammalian subject are provided. The methods of detection or diagnosis indicate the presence of metastatic cancer or early stage cancer.
(Company:  Scripps institute (EPIC Sciences))
========================================================================
http://www.google.com.sg/patents/EP1874920A2?cl=en


Technology:
Abstract:
The invention features devices and methods for detecting, enriching, and analyzing circulating tumor cells and other particles. The invention further features methods of diagnosing a condition, e.g., cancer, in a subject by analyzing a cellular sample from the subject.
(Company:  CellPoint diagnostics)
========================================================================
http://www.google.com.sg/patents/US20110294186
Technology:
Abstract:
The invention features devices and methods for detecting, enriching, and analyzing circulating tumor cells and other particles. The invention further features methods of diagnosing a condition, e.g., cancer, in a subject by analyzing a cellular sample from the subject.
(Company:  On-Q-ity)
========================================================================

http://www.google.com.sg/patents/US7993821
http://www.google.com.sg/patents/US20120178097

Technology:
Abstract:
Embodiments in accordance with the present invention relate to methods and apparatuses for concentrating and isolating Circulating Tumor Cells (CTCs) from body fluids. One embodiment of the present invention includes a micro-fabricated or nano-fabricated device having channels configured for separating and excluding. Embodiments in accordance with the present invention utilize features that reduce the hydrodynamic pressure experienced by the cells during the separation, isolation and concentration processes, and therefore reduce the likelihood of cell lysis or other damage to the cells..
(Company:  Daniel Chiu, U Washington)
========================================================================
http://www.google.com.sg/patents/US20120100521

Technology:
Abstract:
Microdevices are disclosed to efficiently, accurately, and rapidly isolate and enumerate rare cells, such as circulating tumor cells, from liquids such as whole blood. The system employs multiple parallel meandering channels having a width on the order of 1-2 cell diameters. The microdevices can be produced at low-cost, may readily be automated, and in many instances may be used without pre-processing of the sample. They may be used to isolate and enumerate rare cells, including for example the detection and diagnosis of cancers, cancer staging, or evaluating the effectiveness of a therapeutic intervention, or detecting pathogenic bacteria. The device may optionally be used to nondestructively capture and later to release target cells.
(Company:  Steven Soper, Biofluidica)
========================================================================
http://www.google.com/patents/EP2201361B1?cl=en

Technology: multiple antibodies on magnetic beads
Abstract:The present invention relates to the detection of tumor stem cells and tumor cells in epithelial-mesenchymal transition and uses of such methods. According to the present invention said method comprises a selecting step for selection or enrichment of said predetermined cells from the sample wherein the sample is contacted with the solid surface for preferential binding of said predetermined cells to the solid surface and then the sample is removed from the solid surface in a washing step. The inventive method is characterized in that the sample contains a polyol at least during one of contacting the sample with the solid surface and the washing step and in a detection step detecting in said cells, preferentially selected or enriched by said selecting step, the presence or absence of expression of at least one marker associated with at least one of the group comprising tumor stem cells and tumor cells in epithelial-mesenchymal transition.
(Company:  Adnagen)
========================================================================

http://www.google.com/patents/US7846393

Technology: 3D parylene membrane filter for isolation of CTC
Abstract:
The present invention provides a parylene-based membrane filter device for capturing of circulating tumor cells (CTC). The membrane filter has an array of holes having a predetermined geometric design with precisely controlled size, shape and density. In one aspect, the device has a stack of substantially parallel membrane filters with uniformly-spaced and/or monodispersed holes.
(Company:  Filtini)
========================================================================

http://www.google.com.sg/patents/US8088715

Technology: automated microscope
Abstract:
An automated, highly sensitive, specific and potentially quantitative detection method using an automated microscope for identifying and enumerating rare cancer cells in blood and other fluids.
(Company:  Ikonisys)

========================================================================
http://www.google.com/patents/US6949355
http://www.google.com.sg/patents/EP2041299A2?cl=en

Technology: negative enrichment of CTCs
Abstract:
The invention provides methods and compositions for isolating and detecting rare cells from a sample containing other types of cells. In particular, the invention includes a debulking step that can use a microfabricated filter or a cell lysis step or density-based methods to remove certain types of cells, and immobilization methods to remove other types of cells. The invention also includes a method for determining the number or proportion of cancer cells in a biological sample by detecting the presence or amount of telomerase activity or telomerase nucleic acid or telomerase expression after enrichment of rare cells. This invention further provides an efficient and rapid method to specifically remove red blood cells as well as white blood cells from a biological sample, resulting in the enrichment of rare target cells. The presence or level of target cells provides information about various physiological conditions, such as cancer
(Company:  Aviva Biosciences)


========================================================================
http://www.google.com.sg/patents/US20070026413

Technology: microposts coated with EpCAM
Abstract:
The invention features devices and methods for detecting, enriching, and analyzing circulating tumor cells and other particles. The invention further features methods of diagnosing a condition, e.g., cancer, in a subject by analyzing a cellular sample from the subject.
(Company:  Mehmet Toner, Harvard)
========================================================================

http://www.google.com.sg/patents/US20070026419

Technology: Materal displacement obstacle chip
Abstract:
The invention features devices and methods for detecting, enriching, and analyzing circulating tumor cells and other particles. The invention further features methods of diagnosing a condition, e.g., cancer, in a subject by analyzing a cellular sample from the subject.
(Company:  Martin Fuchs, Cellective Dx)
========================================================================

http://www.google.com/patents/US8263404
Technology: RBC agglutination followed by filtration
Abstract:
An antigen-dependent negative selection blood cell separation method is described. Rare circulating epithelial cells can be separated from blood by depleting erythrocytes from a blood sample. Erythrocytes are depleted by agglutination. The new method comprises the use of an agglutinating agent, such as an anti-glycophorin A or glycophorin B antibody, as glycophorin A or B are present on erythrocytes and not on the desired epithelial cells. With regular mixing, desired rare circulating epithelial cells do not become entrapped in the red cell agglutinate.

(Company:  Medical Discovery partners)

========================================================================

http://www.google.com.sg/patents/US20120100560
Technology: anti-body coated microfluidic chip and quantum dots-based imagingi
Abstract:
Applications in nanomedicine, such as diagnostics and targeted therapeutics, rely on the detection and targeting of membrane biomarkers. The present invention, in one embodiment, utilizes quantitative profiling, spatial mapping, and multiplexing of cancer biomarkers using functionalized quantum dots. This approach provides highly selective targeting molecular markers for pancreatic cancer with extremely low levels of non-specific binding and provides quantitative spatial information of biomarker distribution on a single cell, which is important since tumors cell populations are inherently heterogeneous. The quantitative measurements (number of molecules per square micron) is validated using flow cytometry and demonstrated using multiplexed quantitative profiling using color-coded quantum dots.


(Company:  johns Hopkins university)

========================================================================

http://www.google.com/patents/US8263404
Technology: RBC agglutination followed by filtration
Abstract:
An antigen-dependent negative selection blood cell separation method is described. Rare circulating epithelial cells can be separated from blood by depleting erythrocytes from a blood sample. Erythrocytes are depleted by agglutination. The new method comprises the use of an agglutinating agent, such as an anti-glycophorin A or glycophorin B antibody, as glycophorin A or B are present on erythrocytes and not on the desired epithelial cells. With regular mixing, desired rare circulating epithelial cells do not become entrapped in the red cell agglutinate.
(Company:  Medical Discovery partners)
========================================================================

WO2010/144745 A2
http://www.sumobrain.com/patents/wipo/Sheath-flow-devices-methods/WO2010144745.html
Technology: Microfluidic cheath flow for cell isolation
Abstract: The invention relates generally to fluid processing and, in particular aspects, processing fluids for detection, selection, trapping and/or sorting of particulate moieties. Sheath flow devices described allow isolation of target species from fluid samples while avoiding non-specific binding of unwanted species to the surfaces of the separation device. Biological fluid processing, detection, sorting or selection of cells, proteins, and nucleic acids is described. The invention finds particular use in diagnostic settings, analyzing a patient's medical condition, monitoring and/or adjusting a therapeutic regimen and producing cell based products
 (Company: Cynvenio Biosystems)
========================================================================

   US2011/0070642 A1
http://www.patentlens.net/patentlens/patents.html?patnums=US_2011_0070642_A1&language=&

Abstract: A device for isolating and cultivating live cells on a filter or for extracting the genetic material thereof includes: a filter holder (108) connected to a filter; a compartment (102) having an upper opening and a lower opening; and an element (110) that is mobile relative to the compartment for applying a force on the holder and releasing the holder. According to the embodiments, the filter holder is mechanically connected to the compartment or to the mobile element until the application of the force. Preferably, the device further includes a removable end piece (104) tightly and removably attached and adapted for preventing the relative movement of the mobile element and the compartment for applying the force and releasing the holder.
(Assignee: ScreenCell)
========================================================================

20120258475 

Abstract: A microflow apparatus for separating or isolating cells from a bodily fluid or other liquid sample uses a flow path where straight-line flow is interrupted by a pattern of transverse posts. The posts are spaced across the width of a collection region in the flow path, extending between the upper and lower surfaces thereof; they have rectilinear surfaces, have arcuate cross-sections, and are randomly arranged so as to disrupt streamlined flow. Sequestering agents, such as Abs, are attached to all surfaces in the collection region via a hydrophilic coating, preferably a hydrogel containing isocyanate moieties or a PEG or polyglycine of substantial length, and are highly effective in capturing cells or other targeted biomolecules as a result of such streamlined flow disruption.
(Assignee: Biocept)
========================================================================

Sunday, December 16, 2012

Circulating tumor cells as surrogate biomarkers of epithelial mesenchymal transition and metastatic phenotype in prostate cancer patients

Prognostic impact of circulating tumor cells assessed with the CellSearch AssayTM and AdnaTest BreastTM in metastatic breast cancer patients: the DETECT study

Study Helps Resolve Debate About How Tumors Spread

IsoFlux System for Circulating Tumor Cell Analysis

Thursday, December 13, 2012

Useful CTC resources and links




CTC e-learning module with CME credits

"Learning Objectives
To understand the role of circulating tumour cells (CTCs) and disseminating cancer cells (DCCs) in metastatic process
To learn about basic techniques for CTCs isolation, their molecular profile analysis, and predictive and prognostic characteristics
To discuss most important reports regarding implementation of CTCs in biomarker studies and to correlate their molecular profiles with clinical data"
====================================================

Link to CTC research at Scripps Institute. Lots of useful info
http://cancer.scripps.edu/default.aspx
=====================================================

A broad collection of staining, molecular analysis and other protocols relating to CTCs can be found here Circulating Tumor Cell protocols
=====================================================

useful summary and commentary on CTCs The Arts, Sciences and Medicine: CTCs ( CIRCULATING TUMOR CELLS )
=======================================================

The Grand Challenge in Circulating Tumor Cell Isolation: The need for a Gold Standard Platform


The urgent need for a Standardized Technology for Tumor Cell Isolation
In recent years, a number of technologies have claimed the ability to isolate tumor cells for clinical cancer management. These technologies can be broadly classified as falling into the following categories with regards to their value proposition,
a)      Using specific antibodies (either single-EpCAM or combination) in a conventional magnetic isolation assay or a microfluidics assay (Eg., Veridex, Biocept, OnQity, Biofluidica, Adnagen)
b)      Using size and biomechanical properties as a differentiator between tumor cells and normal blood cells (Eg., Clearbridge, CellSeivo, Rare cells, Screen Cell, Creative Microtech, Filtini, Parsortix)
c)       Technologies whose value proposition is in automated enumeration and identification such as automated microscopy  (Eg., Bioview, Ikonisys, eDAR, Epic Sciences)
d)      Technologies that use electrical properties (Silicon biosystems, Apocell)
The advantages and disadvantages of various techniques are tabulated extensively in various reviews [1 -8]. It’s also widely noted that most of these technologies remain to be independently validated and not considered optimal for tumor cell isolation [9-15]. Some criterion were recently (2012) suggested by a group of KOLs, titled “Considerations in the development of circulating tumor cell technology for clinical use” [16]. A list of technological requirements of an ideal CTC platform were previously suggested in a Lab on a chip paper in 2011 titled “Circulating Tumor Cells: The Grand Challenge” [17].
The pain due to lack of robust and standardized tumor cell isolation technology:
So far CTCs have only been FDA approved for cancer prognosis, which is of limited clinical utility. The real clinical impact of CTCs will be realized when their relevance is shown for personalized therapy & monitoring and early diagnosis. The clinical community is now investigating this relevance. However, the present technology platforms are inherently biased and may lead to poor clinical interest. For example, “A recent study revealed that “normal-like” breast cancer cells, which usually display an aggressive phenotype, express low expression of EpCAM and are not detected by the CellSearch® test [65]. Moreover, a retrospective study that involved 292 patients with metastatic breast cancer has shown that 36% of them showed an undetectable CTC status, which could be due, at least in part, to an underestimation of CTCs by the Cell-Search® test due to CTC undergoing epithelial-mesenchymal transition (EMT) ” [1]
Another paper noted that, “ The challenge of CTC detection is related to the requirement of both high sensitivity and specificity. A wrong labeling of ‘‘non-tumor cells’’ (epithelial non tumor cells or normal hepatocytes, for instance) as ‘‘tumor cells’ could generate poor clinical interest” [8]
This problem is well summarized by the following passage in recent literature “The true potential of CTCs has yet to be realized because of limits in technology used to capture these cells and our lack of a complete understanding of metastasis. This is complicated by the fact that our understanding of CTCs is subject to the techniques available to identify and isolate CTCs, and the biases inherent to them.” [6]
From the above passage it is very clear that what is urgently needed now in CTC field is not “cheap” or “fast” technology but the “most reliable” platform to accurately reflect the patient physiology. 
The problem with existing technologies:
“Despite the recent technological advances, the development of a single device capable of simultaneously achieving high throughput, high target cancer cell recovery, high purity, and high cell viability remains challenging.” [5]
As pointed out by the numerous review papers, all technology platforms existing today suffer from one trade-off or another. For example, antibody-based technologies are limited by the expression of specific antibodies. A universal CTC marker is yet unknown and given the heterogeneity of cancer [18], it is doubtful that a single universal cancer marker will be found.
The problem with Size and biomechanical properties based isolation  “The size range of different tumor cells is highly variable and does overlap with that of normal blood cells” [19]. Also, “CTC may not be always .8 mm making the sensitivity of these assays questionable.” [20]. Moreover, “ these methods suffer from low cell viability resulting from potential damage incurred as the cells pass through narrow filter pores, which renders the use of microfilters less compatible for live cell interrogations (e.g., cell suspensions were partially fixed before being passed through a membrane micro-filter)” [4]
The assessment of technologies based on size is well summed up in this review “Unfortunately, large leukocytes can be trapped by the filter as well, therefore contaminating the CTC fraction or small CTC can pass through the pores therefore depleting the CTC population. This is why this technique is generally considered not highly sensitive and poorly specific” [8]
There are several companies based on the principle of size and biomechanical properties and this is the most crowded sub-space within CTC. A reason for this is because of low technological barrier to entry and wide availability of hole making technology in various materials such as polycarbonate, silicon nitride and other polymers. As it can be readily inferred by-passability is easy and patentability is low for technologies operating on this principle. 
The Solution:
The clinical need and technology requirements are well documented [16, 17]. Briefly, the assay must be highly sensitive, well characterized with analytical and clinical samples, standardized for cross reference studies at various clinical sites, should be free of operator bias, should use standard blood collection and processing techniques and should be time stable over a period to allow for stable sample transportability. Above all, the assay should isolate viable, pure tumor cells with high efficiency in a simple and cost effective manner. 
Neegative depletion approach has been recommended over the positive selection method [2, 21]. In negative depletion approach, all normal cells are eliminated, leaving behind “abnormal cells”. These cells are then identified by immunohistochemical staining or molecular methods.
Conclusion: Despite the presence of several technologies, the need for a gold standard tumor cell isolation platform remains unmet. 

References:
1.       Am. J Cancer Res 2011;1(6):740-751
2.       Nature reviews, clinical oncology, December, 2010
3.       Clinical Chemistry 58:5 (2012)
4.       Lab Chip, 2012, 12, 1753–1767
5.       Review article, Frontiers in oncology, vol 2, article 69, july 2012
6.       J Cancer Res Clin Oncol DOI 10.1007/s00432-011-0988-y
7.       International Journal of Hepatology, Volume 2012 (2012),
8.       Methods 50 (2010) 289–297
9.       Circulating tumor cells: approaches to isolation, and characterization, JCB review, 2011
10.   New technologies for the detection of circulating tumour cells, British Medical Bulletin 2010; 1–16
11.   Let Me Do More Than Count the Ways: What Circulating Tumor Cells Can Tell Us about the Biology of Cancer  VOL. 6, NO. 5, 1307–1310 MOLECULAR PHARMACEUTICS, 2009
12.   Disseminated Tumor Cells in Bone Marrow and Circulating Tumor Cells in Blood of Breast Cancer Patients: Current State of Detection and Characterization
 Pathobiology 2008;75:140–148
13.   Circulating tumor cells (CTC) detection: Clinical impact and future directions Patrizia Paterlini-Brechot *, Naoual Linda Benali  Cancer Letters 253 (2007) 180–204
14.   Clin Cancer Res; 16(20) October 15, 2010
15.   Cancer Letters 253 (2007) 180–204
16.   Journal of Translational Medicine 2012, 10:138 
17.   Lab Chip, 2011,11, 375-377
18.   PLoS ONE 7(5): e33788
19.   Current Opinion in Genetics & Development 2010, 20:96–99
20.   British Medical Bulletin 2010; 1–16
21.   Cancer Res; 71(18), 2011

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)

============================================================
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
=============================================================
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

Paper Commentary: Microsieve lab-chip device for rapid enumeration and fluorescence in situ hybridization of circulating tumor cells - Lab on a Chip (RSC Publishing)

Microsieve lab-chip device for rapid enumeration and fluorescence in situ hybridization of circulating tumor cells - Lab on a Chip (RSC Publishing)


Summary:
This paper is among several that use the perforated membranes to capture CTCs. The premise is that there is a clear size and deformability difference between tumor and non-tumor cells. This premise is contested.

The main issue with this approach is that its always a tradeoff between capture efficiency and purity. Quoted from manuscript "Physical size separations could potentially undercount a small portion of CTCs
"
The size filtration is combined with a tweaked metamorph image classification algorithm for automated image analysis.  
similar to automated microscopes and algorithms offered by Bioview and Ikonisys.


Advantages:
  • A generic advantage of filtration schemes is "fast" and "cheap". 
Limitations:
    • In comparison with polymer based filters, silicon filters are much more expensive.
    • the key limitation of filtration schemes is their inherent trade-off between recovery and purity
    • A wide variety of cell types and tumor types should be used to demonstrate broader utility of this device
    • cells undergoing EMT also undergo structural and biomechanical adaption to allow passage through blood vessels thus may be missed by physical property based approaches

Considerations in the development of circulating tumor cell technology for clinical use


Journal of Translational Medicine 2012, 10:138 doi:10.1186/1479-5876-10-138

Abstract
This manuscript summarizes current thinking on the value and promise of evolving
circulating tumor cell (CTC) technologies for cancer patient diagnosis, prognosis, and
response to therapy, as well as accelerating oncologic drug development. Moving forward
requires the application of the classic steps in biomarker development―analytical and
clinical validation and clinical qualification for specific contexts of use. To that end, this
review describes methods for interactive comparisons of proprietary new technologies,
clinical trial designs, a clinical validation qualification strategy, and an approach for
effectively carrying out this work through a public-private partnership that includes test
developers, drug developers, clinical trialists, the US Food & Drug Administration (FDA)
and the US National Cancer Institute (NCI).

click here for the full article

A Brief Comparison Of The Various Techniques And Methods For Isolation Of Circulating Tumor Cells (CTC’s)


The link below has a brief but informative comparison of various technologies available for Circulating Tumor cell isolation
www.rgccusa.com/index.php/download_file/view/38/1/

The Problem with using size and deformability as the criteria to isolate Circulating tumor cells (CTC)



  • “The size range of different tumor cells is highly variable and does overlap with that of normal blood cells” [1]
  • However, CTC may not be always .8 mm making the sensitivity of this assay questionable. [2]
  • However, these methods suffer from low cell viability resulting from potential damage incurred as the cells pass through narrow filter pores, which renders the use of microfilters less compatible for live cell interrogations (e.g., cell suspensions were partially fixed before being passed through a membrane micro-filter72) [3]
  • Due to the force (centrifugation or pumping) many of the cells are destroyed (viability decreased upto 70% of CTCs). Many CTCs will pass through the narrow passage due to EMT as they become more flexible and bendable. Many blood cells still remain that produce contamination and noiseHence it is not considered to be the best method[4]
  • isolation of circulating tumor cells (CTCs) by size exclusion can yield poor purity and low recovery rates, due to large variations in size of CTCs, which may overlap with leukocytes and render size-based filtration methods unreliable [5].
  • Physical size separations could potentially undercount a small portion of CTCs  [6]
  • Our observations support the relationship between tumor-initiating capacity and cell deformability, and demonstrate that tumor-initiating cells are less differentiated in terms of cell biomechanics (from normal cells). Thus deformability-based techniques may miss tumor initiating cells. [7]
  • Metastatic cells are more deformable and pass through capillaries faster than non metastatic cells [8]



[1] Current Opinion in Genetics & Development 2010, 20:96–99
[2] British Medical Bulletin 2010; 1–16
[3] Lab Chip, 2012, 12, 1753
[5] Anal Chem. 2012 Sep 4;84(17):7400-7
[6] Lab Chip, 2012,12, 4388-4396
[7] PNAS November 13, 2012 vol. 109no. 46 18707-18712
[8] http://www.pnas.org/content/early/2013/04/19/1218806110

Relationship among circulating tumor cells, CEA and overall survival in patients with metastatic colorectal cancer


doi:10.1093/annonc/mds336

Background: We previously reported results of a prospective trial evaluating the significance of circulating tumor cells
(CTCs) in patients with metastatic colorectal cancer (mCRC). This secondary analysis assessed the relationship of the
CTC number with carcinoembryonic antigen (CEA) and overall survival.
Patients and methods: Patients with mCRC had CTCs measured at baseline and specific time points after the
initiation of new therapy. Patients with a baseline CEA value ≥10 ng/ml and CEA measurements within ±30 days of the
CTC collection were included.
Results: We included 217 patients with mCRC who had a CEA value of ≥10 ng/ml. Increased baseline CEA was
associated with shorter survival (15.8 versus 20.7 months, P = 0.012). Among all patients with a baseline CEA value of
≥25 ng/ml, patients with low baseline CTCs (<3, n = 99) had longer survival than those with high CTCs (≥3, n = 58;
20.8 versus 11.7 months, P = 0.001). CTCs added prognostic information at the 3–5- and 6–12-week time points
regardless of CEA. In a multivariate analysis, CTCs at baseline but not CEA independently predicted survival and both
CTCs and CEA independently predicted survival at 6–12 weeks.
Conclusions: This study demonstrates that both CEA and CTCs contribute prognostic information for patients with
mCRC.

Paper commentary: A simple packed bed device for antibody labelled rare cell capture from whole blood





Abstract:
We have developed a system to isolate rare cells from whole blood using commercially available components and simple microfluidics. We characterized the capture of MCF-7 cells spiked into whole human blood using this system to demonstrate that enrichment and enumeration studies give results similar to in situ surface-modified devices while reducing fabrication and operation complexity.

Comments:

>>EpCAM-based technique

>> very low throughput 
"This suspension of whole blood was pumped from 3 mL syringes (ten-syringe infusion/withdraw pump, Cole-Parmer) at 0.2 mL h−1 for 1 h through the microfluidic packed bed to allow immobilization of the cancer cells. "

at this rate it will take 5 hours to process 1 mL of blood.

>> seems like an improvement over the Harvard CTC-chip, but still does not solve some fundamental issues with EpCAM based capture. The throughput is impractical

multiple channels can be used, but that will dramatically increase the imaging and staining area, making it difficult to perform high resolution cytomorphological analysis

Paper commentary: Microfluidic flow fractionation device for label-free isolation of circulating tumor cells (CTCs) from breast cancer patients

Biosensors and BioelectronicsVolume 40, Issue 1, 15 February 2013, Pages 206–212

Principle of operation: Based on hydrodynamic forces and cell size differences

steps: 7.5 ml of blood subject to RBC lysis followed by centrifugation followed by resuspension followed by processing through chip

cell yield: 0 to 21 cells in 7.5 ml of blood.

purity: NA