1. 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

2. © Copyright 2009 by the American Association for Clinical Chemistry Introduction  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

3. © Copyright 2009 by the American Association for Clinical Chemistry Introduction  Circulating tumor cells (CTCs) > Typically ~10 6 -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

4. © Copyright 2009 by the American Association for Clinical Chemistry Introduction  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

5. © Copyright 2009 by the American Association for Clinical Chemistry Study 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 ®

6. © Copyright 2009 by the American Association for Clinical Chemistry Questions  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?

7. © Copyright 2009 by the American Association for Clinical Chemistry Methods – 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.

8. © Copyright 2009 by the American Association for Clinical Chemistry Methods – 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.

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

10. © Copyright 2009 by the American Association for Clinical Chemistry Primary CTC capture – Cumulative results and comparison to CellSearch 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

11. © 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

12. © 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 adhesion A B C

13. © Copyright 2009 by the American Association for Clinical Chemistry Results  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 ®

14. © Copyright 2009 by the American Association for Clinical Chemistry Questions  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?

15. © Copyright 2009 by the American Association for Clinical Chemistry Thank you for participating in this month’s Clinical Chemistry Journal Club. Additional Journal Clubs are available at www.clinchem.org Follow us