Microtube Device for Selectin- Mediated Capture of Viable Circulating Tumor Cells from Blood A.D. Hughes, J. Mattison, L.T. Western, J.D. Powderly, B.T.

Microtube Device for Selectin-Mediated Capture of Viable Circulating Tumor Cells from Blood

A.D. Hughes, J. Mattison, L.T. Western, J.D. Powderly, B.T. Greene, and M.R. King

May 2012

www.clinchem.org/cgi/content/article/58/5/846

© Copyright 2012 by the American Association for Clinical Chemistry

© Copyright 2009 by the American Association for Clinical Chemistry

IntroductionIntroduction

Metastasis> Cancer morbidity is congruent with the dissemination of malignant cells throughout the body> Responsible for 90% of deaths

Circulating tumor cells (CTCs)> Malignant cells travel through the circulatory system> Prevalence of CTCs in blood correlates with disease severity> CTCs are easily accessible, making them promising agents for cancer study, diagnosis, and personalized treatment development

© Copyright 2009 by the American Association for Clinical Chemistry

IntroductionIntroduction Circulating tumor cells (CTCs)

> Typically ~106-fold less abundant than leukocytes> No known universal marker

- Epithelial cellular adhesion molecule (EpCAM) is most widely used in research but not present on 100% of CTCs

CTC Detection> CTC count is used to track disease> Two major classes of techniques:

- Ficoll density centrifugation followed by ID with antibodies- PCR

> CellSearch®

- Sole FDA-approved technique for CTC enumeration- Ficoll centrifugation followed by EpCAM-based immunomagnetic

separation> CellSearch® and PCR necessitate cell death

© Copyright 2009 by the American Association for Clinical Chemistry

IntroductionIntroduction CTC isolation techniques

> Microfluidic devices- Capture cells using antibodies (eg. anti-EpCAM)- Cell viability is not compromised- Slow nature of Ab binding requires slower flow rates (~1 mL/h)

Selectin-based CTC isolation>Normal function of selectins is to recruit fast-flowing leukocytes to endothelium during inflammation

- CTC may use the same process to bind to the endothelium> Selectin-bound cells are afforded time to bind to antibodies

- Samples can therefore be processed at higher flow rates> This natural ‘biomimetic’ process does not compromise cell viability

Nanoparticle/nanotube coatings have been used to alter cell behavior> Halloysite nanotube coating has been used to enhance capture of model cell lines

© Copyright 2009 by the American Association for Clinical Chemistry

Study AimsStudy Aims Construct a device for selectin-based CTC capture

Evaluate the impact of a halloysite nanotube coating

Evaluate utility of device in clinical setting

Compare device to gold standard CellSearch®

© Copyright 2009 by the American Association for Clinical Chemistry

QuestionsQuestions What effect, if any, would increased surface roughness have on cell

capture?

What other schema could increase sample throughput?

What are the limitations to current state of knowledge on CTC? How may this be influenced by existing isolation techniques?

© Copyright 2009 by the American Association for Clinical Chemistry

Methods – Device PreparationMethods – Device Preparation Coat microtube surface in successive steps to achieve a surface

coated with both E-selectin and anti-epithelial antibodies (EpCAM or PSMA). This slide contains animation.

© Copyright 2009 by the American Association for Clinical Chemistry

Methods – CTC isolationMethods – CTC isolation Peripheral blood collected from a patient diagnosed with stage IV

metastatic carcinoma

Buffy coat (leukocytes + CTC) isolated by Ficoll centrifugation

Washed buffy coat perfused through device

Non-adherent and loosely adherent cells washed out of the device

Adherent cells released from the device and cultured for up to 5 days

Cells still viable after 5 days in culture were stained with DAPI and anti-EpCAM or anti-PSMA for enumeration.

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Validation: Capture of model cells from whole blood Validation: Capture of model cells from whole blood

Figure 1. Relationship between numbers of KG1a acute myeloid leukemia cells supplemented into 4 mL diluted blood and the number of cells recovered. Error bars represent the SEM determined from calibration experiments. p < 0.001.

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Primary CTC capture – Cumulative results and comparison to Primary CTC capture – Cumulative results and comparison to CellSearchCellSearch

Figure 2. Left: The number of CTC captured from the blood of all patients is compiled along with the results of samples collected from healthy participants. CTC counts from CellSearch® reported here were derived from 7.5 mL samples and renormalized for comparison. Participants Norm-1 through -5 were processed in tubes coated with EpCAM in addition to E-selectin. The symbol indicates samples processed through smooth tubes only. Right: Representative micrographs of CTC in culture following isolation from the smooth device (top) and nanotube-coated device (bottom), stained for EpCAM (green) and DAPI (blue). Scale bar 50 μm.

Smooth

Nanotube-Coating

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Figure 3. The purity values of samples analyzed on both surfaces were compared and purity was significantly greater on the nanotube-coated surface. O’s indicate breast cancer patients, Δ’s indicate prostate cancer, X’s indicate lung cancer, and +’s indicate ovarian cancer samples. p < 0.001 using paired nondirectional t-test.

Capture purity is enhanced by the nanotube coating Capture purity is enhanced by the nanotube coating

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Figure 4. The contact area between adherent leukocytes and planar E-selectin-functionalized surfaces was measured on smooth and nanotube-coated surfaces. (A) Comparison of cell area on smooth versus nanotube-coated surfaces by two-tailed paired t test yielded p = 0.0038. (Insert) Cell spreading was also quantified based on cell perimeter on either surface. Paired t test yielded p = 0.0175. Error bars represent standard deviation. (B) Representative micrograph of adherent leukocytes on the smooth surface. (C) Representative micrograph of adherent leukocytes on the nanotube-coated surface. Cell membranes were stained with octadecyl rhodamine B (green) and nuclei stained with DAPI (blue).

Nanotube coating resists leukocyte Nanotube coating resists leukocyte adhesionadhesion

A

B

C

© Copyright 2009 by the American Association for Clinical Chemistry

ResultsResults Viable cancer cells were successfully isolated from each sample

processed> Significant numbers of cells were detected in culture following isolation

Capture purity was improved using the nanotube coating> Nanotube coating resisted adhesion of contaminating leukocytes

Utility was demonstrated in use of either blood or buffy coat

Device performance was a significant improvement over the gold standard CellSearch®

© Copyright 2009 by the American Association for Clinical Chemistry

QuestionsQuestions Cell spiking experiments are usually used in determining capture

efficiency of isolation devices. How relevant is this?

What is the most appropriate use for this new approach (eg. diagnosis, research, etc.)?

How could the device be further optimized?

By what mechanism(s) does the nanotube coating enhance cell capture?

What may we conclude when comparing the different types of cancers addressed in this study?

© Copyright 2009 by the American Association for Clinical Chemistry

Thank you for participating in this month’sClinical Chemistry Journal Club.

Additional Journal Clubs are available atwww.clinchem.org

Follow us