Wednesday, May 22, 2013

Chemosensitivity testing using CTCs from patient samples

Chemosensitivity Testing of Circulating Epithelial Tumor  Cells (CETC) in Vitro: Correlation to in Vivo Sensitivity  and Clinical Outcome
www.laborpachmann.de/pdf/Chemosensitivity_JCT_2013.pdf

interesting paper and opens up new application area for CTCs.

Method for isolation: RBC lysis followed by image analysis of all nucleated cells

Conclusion: "Thus, we are able to show that chemosensitivity testing of circulating tumor cells provides real-time information about the sensitivity of the tumor present in the patient, even at different times during therapy, and correlates with treatment success. "

Thursday, May 2, 2013

PLOS publication comparing the performance of various types of filters for CTC recovery


Filter Characteristics Influencing Circulating Tumor Cell Enrichment from Whole Blood


This is great! I have been wanting to do this myself for quite sometime. There are tons of CTC technologies based on using a filter of one type of another to recover CTCs. In fact this probably is the most crowded sub-segment of commercial CTC platforms. This is mainly due to low technical threshold needed to get to market. Thus attracting lots of market entrants.

However, not many have attempted a broader study of exactly what the correlation is between the porosity, shape and size of pores to the precisely measured cellular dimensions from cell lines as well as patient samples. This study has attempted to do it. Thanks to the authors.

Key findings:

Cell Enumeration is Easiest on a Stiff Filter with Low Porosity

>> The authors point out that there is difficulty in imaging a flexible substrate, which is intuitive, hence rigid substrates are preferred.

>> "The high porosity and low number of pores of the microsieves and the TEM grids resulted in a cell density too high to reliably distinguish adjacent cells when imaged with a 4x/NA0.13 objective" ==> this is a bit confusing. my understanding is that high porosity = high number of pores per cm2. the paper seems to suggest otherwise. Share your thoughts

>> "he ideal filter should maintain its planar form during filtration, it should not react with the sample and pores should be sufficiently separated to facilitate discrimination of cells"

Increasing Pore Size Leads to Lower Recovery and Higher Sample Purity
>> This is also intuitive as larger pores deplete WBCs better than smaller pores but also lose more CTCs

>> recoveries vary for different cell lines at different pore sizes, indicating that the filter method is not one size fits all, which also intuitive, given the vast heterogeneity among tumor cells

>> "As long as there are sufficient pores to pass the sample, recovery seems to be insensitive to the number of pores. There is almost no difference between the microsieves with 115,000 pores and the one with 26,000 pores in terms of spiked cell recovery or pressure across the filter, but the number of leukocytes retained is 3.1 fold higher on the filter with 4.4 fold more pores"

Recovery is Constant Until Approximately 2% of Pores are Occupied

The Volume that can be Filtered is Limited by the Contaminant Concentration
>> that is smaller volumes lead to better recovery rates than larger volumes of sample

Monocytes are Retained More than Other Leukocytes

EpCAM+CK+CD45− CTC (from patient samples) are Smaller than Typical Cells Derived from Tumor Cell Lines

Cell Lines with Size of CTC Typically have Low Recovery

Summary:
This paper is a highly recommended read as it clarifies several aspects of membrane-based CTC filtration devices. The key points are, a) one size does not fit all, b) there is always a recovery purity trade-off in this method, and c) CTC recovery using this method is subject to a number of factors such as geometry, number of pores, rigidity, flow rate, sample volume etc.

The authors summarize it in the manuscript "In summary, the ideal filter for CTC enrichment from 10 ml of whole blood has a pore size of about 5 µm, thickness of at least 10 µm, at least 100,000 regularly spaced pores, a porosity of 10% or less and is constructed of a stiff, flat material, which does not interact with blood cells. While cell size is an important factor in determining recovery, other factors must be involved in determining whether a cell can pass as well. To evaluate a filtration procedure, cell lines with a median size of 11–13 µm should be used to challenge the system, such as Colo-320, SW-480 and not cell lines significantly larger than CTC."


Limitations:
though the authors used a polycarbonate track etched membrane, they did not use a patterned parylene membrane or other deformable membrane, so arrive at the conclusion that a hard surface is necessary may require additional experimentaiton  comparing the track etched polycarbonate versus parylene or other patterned substrate (this study may already exist, I will have to look it up)

the spiked cells used in the experiments were pre-stained. this is not the case for patient samples. typically, if staining were to be done on filter post recovery, a certain amount of cell loss can be expected due to this process

I wrote another post some time ago
The Problem with using size and deformability as the criteria to isolate Circulating tumor cells (CTC)

I found another good reference below
Pore design and engineering for filters and membranes

Tuesday, April 23, 2013

Triple positive cancer stem cells (CSCs) subtset of CTCs implicated in Metastasis


Key findings:
  • A subset of CTCs occurring at low frequency are responsible for metastasis
  • the frequency of occurrence in the reported study was 0 .6 to 33% of CTCs
  • This subset is positive for CD44, CD47, MET 
  • experimental method involved isolating CTCs from 350 breast cancer patients using cell surface markers and transplating them in mice with defective immune system
  • As the disease advances, the number of triple-positive cells increases, but the total number of CTCs does not. 
  • Patients with very high numbers of triple-positive cells had particularly high numbers of metastases and a much poorer prognosis than women in whom only few of these metastasis-inducing cells were detected
Conclusion: "On the whole, triple-positive cells seem to have a substantially higher biological relevance for disease progression than previously studied CTCs"


direct link to the nature biotechnology paper
http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.2576.html

Thursday, April 18, 2013

Lessons from On-Q-ity's failure: Reflections of a VC who invested in the company

On-Q-ity, a Cancer Diagnostic Company: R.I.P. | LifeSciVC

On-Q-ity recently announced closure of its operations. Bruce Booth, a VC who invested in On-Q-ity reflects on the lessons learnt from this unsuccessful venture.


Booth covers several key learnings, including the significance of team, product focus, getting the market timing right.
Many of these are routinely covered in venture advice articles. Its still worth reading from the point of view of someone who went through the drill. 
I highly recommend giving it a read as its relevant to any CTC company and there are many startup companies trying to commercialize CTC technologies. You can find the updated list of CTC companies in this post.
http://circulating-tumor-cells.blogspot.sg/2010/12/commercial-circulating-tumor-cell-ctc.html

Saturday, March 30, 2013

High throughput capture of circulating tumor cells using an integrated microfluidic system

ScienceDirect.com - Biosensors and Bioelectronics - High throughput capture of circulating tumor cells using an integrated microfluidic system


"we achieved an enrichment factor of 1500X, and a high processing throughput of 9.6 mL/min with
90% capture yield and more than 50% capture purity at cell concentration 102 cells/mL"

I believe this is the highest reported throughput with 90% capture efficiency. Impressive!

The principle used in isolation is a combination of Deterministic lateral displacement (DLD) and EpCAM-based capture.

Most of you know about EpCAM based capture on microposts. Below is a video on how DLD works



some related prior art:

Isolation of rare circulating tumour cells in cancer patients by microchip technology



Tuesday, March 19, 2013

A focus review on affinity-based, dielectrophoresis and hydrophoresis

Microfluidic devices for the isolation of circulating rare cells: a focus on affinity-based, dielectrophoresis and hydrophoresis - Hyun - ELECTROPHORESIS - Wiley Online Library

Wow, its review season, quite a number of reviews already in 2013
Focus of review: Microfluidic- affinity-based (antibody-antigen), dielectrophoresis and Hydrophoresis- (aka inertial microfluidics)

some perspectives expressed by the authors

"Several isolation methodologies based on affinity-based positive enrichment using epitopes
expressed on the cell surface have been developed. However, all circulating rare cells do not express the same specific antigens, as they are heterogeneous by nature. Although it is controversial whether positive or negative enrichment is more efficient, it is clear that the latter is more advantageous than the former because the target cells can be captured in intact form. Novel approaches for negative enrichment (affinity-based methods using antibodies to isolate hematologic cells or non-affinity-based methods such as dielectrophoresis and hydrophoresis) should be developed, because a negative enrichment microfluidic chip enables the simultaneous isolation of various types of circulating rare cells, such as circulating endothelial cells (CECs), cancer stem cells (CSCs), circulating progenitor cells (CPCs), and circulating tumor cells (CTCs), including nucleated red blood cells (nRBCs). The number of intact and heterogeneous circulating rare cells collected continuously by such a device will provide researchers with many opportunities to investigate the molecular nature of these rare cells."

Wednesday, March 13, 2013

CTC user survey results 2013

Here is a survey conducted by World CTC organization to gather feedback on the top priorities of CTC community in 2013 and beyond

http://worldctc-berlin.com/uploads/ffiles/2013/03/145979.pdf

Key questions covered in the survey

How highly would you rate the priority of CTCs in your organisation?

What do you think is the biggest challenge in current CTC research?

What is your current research priority?

In your opinion, what needs to be done to validate the use of CTCs in the clinic?

What do you think is the timeline for CTCs to become an important part of the clinical decision
making process?

What do you think is stopping you from making CTCs your top priority?

Which companies/technologies do you think hold the most promise for improving our ability to study and measure CTCs?


Highlights:
  • 74.3% of respondents in the survey highlighted CTCs as either a "very high" or "high priority" for their organisation 
  • When questioned what was the biggest challenges faced in this field the top challenge was identified as “Current technology is neither sensitive or specific enough” (33%) with the second biggest challenge being that “There is no agreed golden standard for technology” (25.5%) 
  • A resounding 48.8% thought that to validate CTCs in the clinic there needs to be “more clinical trials validating CTCs as a diagnostic/prognostic prospective”

Thanks to world CTC for conducting the survey and publishing the results. 
please view full report here
http://worldctc-berlin.com/uploads/ffiles/2013/03/145979.pdf



Circulating Tumor DNA Detects Metastatic Breast Cancer

Circulating Tumor DNA Detects Metastatic Breast Cancer (original link)

link with my annotations is here
https://diigo.com/0xl25

link for NJEM paper is http://www.nejm.org/doi/full/10.1056/NEJMoa1213261

The battle between cell-free DNA versus cells was won by cf DNA in non-invasive prenatal diagnostics (at least for now)

It will be interesting to watch this unfold for cancer

Saturday, February 23, 2013

Microfluidics and cancer: are we there yet?

Microfluidics and cancer: are we there yet? [Biomed Microdevices. 2013] - PubMed - NCBI

A review by Sunitha Nagrath on the applications of Microfluidic technology to cancer biology and cancer diagnostics, including, CTC isolation, molecular diagnosis, tumor biology and high throughput screening

Sunday, February 17, 2013

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.

Thursday, February 7, 2013

An Automated High-Throughput Counting Method for Screening Circulating Tumor Cells in Peripheral Blood - Analytical Chemistry (ACS Publications)

An Automated High-Throughput Counting Method for Screening Circulating Tumor Cells in Peripheral Blood - Analytical Chemistry (ACS Publications)

Summary:
This is a very unique paper that presents direct labelling of cells in whole blood. besides the traditional EpCAM, CD4-, CK markers, the authors also used CD44+ marker for stemness.

methods:
1 ml of blood directly stained with markers, centrifuged to remove excess labelling antibody and resuspended and flown through the microfluidic chip. A custom built line-confocal imaging system captures images of cells flowing past the microfluidic channel.
authors had previously published a variation of this technique here http://www.ncbi.nlm.nih.gov/pubmed/22389033

performance:
  • 94% recovery 
  • 1 ml of blood processed in ~ 30 mins
  • no isolation/enrichment needed
interestingly, the performance of this system was compared with cellsearch in 90 clinical samples with the following findings

91% positive samples vs 44% positive using cellsearch in 7.5 ml of blood
range of cells 15 to 3375 per 7.5ml vs 1 to 846 by cellsearch
average 305 cells/ml vs 36 cells/ml by cellsearch

very impressive performance improvement over cellsearch.

Tuesday, February 5, 2013

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)].

Wednesday, January 30, 2013

Capturing and release of circulating tumor cells (CTCs) from blood using nanopillars or other nanostructures

Capturing cancer tumor cells from the blood - MaterialsViews

This paper describes the use is 3D silicon nanopillar array coated with specific antibodies to capture and release CTCs using thermal means.

A similar approach is reported here

Scientists develop new nanotechnology to capture, release tumor cells that circulate in bloodstream


(using nano-velcro)

Nanostructured Surfaces to Target and Kill Circulating Tumor Cells While Repelling Leukocytes

(using Halloysite nanotubes)

the approach reported here is also in a similar category
(using DNA tentacles)

Specific Capture and Release of Circulating Tumor Cells Using Aptamer-Modified Nanosubstrates


High-Purity Prostate Circulating Tumor Cell Isolation by a Polymer Nanofiber-Embedded Microchip for Whole Exome Sequencing



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 

Saturday, January 12, 2013

Circulating Tumor Cell ( CTC ) market info


Below is a compilation of publicly available market info from various sources

Market Potential

A recent market report was published by Transparency Market Research (http://www.transparencymarketresearch.com) titled "Circulating Tumor Cells (CTCs) and Cancer Stem Cells (CSCs) Market - Global Scenario, Trends, Size, Growth and Industry Analysis, 2011 - 2018." Globally, CTCs and CSCs Market is estimated to reach USD 7.9 billion by 2018. The global market for cancer diagnostics based on Circulating Cancer Cells (CTCs) was estimated at USD 1.0 billion in 2011. Majority of the share is most likely held by Veridex LLC (Johnson and Johnson)

CTC market estimates used by other CTC companies
company
Price/test
Market size
comment
Creatv Microtech [1]
USD 600
10-20 Bln
20-30 mln tests in US.
1 test/year/patient
Ikonisys [2]
USD 400
35 Bln
Total Addressable Market: 4 Bln (monitoring), 35 Bln (early detection)
Parsortix (listed) [3]
na
4 Bln
Est. 20,000 separations from single pharma site p.a. 25 Mln p.a. early stage rev.
Rheoblast [4]
na
5.2 Bln
10 Mln US patients= 25 M tests/year. 2-3 tests/yr/pat.
6000 clinical labs in US (2-3% growth)
1400 cancer treatment centers in US (6-7% growth)
150 biomarker companies and research labs

Figure 4:  source [4]

CTC application segment
CAGR
Prognosis
-
Therapy monitoring
26.54%
Diagnostics
26.48%
Prognostic functions hold 58.28% of the total CTC Technologies market



Figure 5 is the estimated market share by a CTC company, Silicon Biosystems, which provides solution for cell sorting.

Figure 6 is the estimated market share by a CTC company, Filtini, which provides microfilter based CTC isolation device

The demand for cellular analysis will also expand significantly during the next five years. This sector was worth $10 million in 2010 and is expected to increase at a 95.4% compound annual growth rate (CAGR) to reach $296 million in 2015.

List of all active commercial circulating tumor cell ( CTC ) technologies is here
http://circulating-tumor-cells.blogspot.com/2010/12/commercial-circulating-tumor-cell-ctc.html

other documents of interest
https://docs.google.com/file/d/0B6AYySNe0mJCTTd0Sll4NVhZMGs/edit?usp=sharing