Tiffany Marshall

Saint Mary-of-the-Woods College

Tim McKnight

Measurement Science and SystemsEngineering Division

Dr. Kristina Thiagarajan

Computing and ComputationalSciences Directorate

Research Alliance in Math and Science

August 7, 2012

Improved Methods for Capturing and Analyzing Circulating Tumor Cells

Managed by UT-Battellefor the U.S. Department of Energy

Outline

• Introduction• Methods• Results• Future work• Conclusion• Questions

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Circulating tumor cells (CTCs)

• Found in bloodstream • Important to isolate and enrich

– Facilitates early detection in malignant disease patients – More effective treatment

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Commercial systems for CTCenrichment

• CTCs are rare in bloodstream• Magnetic beads used to capture CTCs from a vast

majority of non-CTC cells• Bead capture is harsh and results in phenotypic disturbances or

loss of viability

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Veridex CellSearch system is used to isolate CTCs

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Emerging microfluidic systemsfor CTC enrichment

• Requires large processing volumes (7ml of blood)• Low flow throughout requires long processing times• Shear stresses still cause phenotypic shift or even loss of

viability

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Demonstrated effective, but

with very limited throughput

From Stott et al (2010) PNAS

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ORNL designed microfluidiccapture and analysis platform

• Vertically-aligned carbon nanofibers (VACNF)– Cellular isolation– Direct gene delivery

• Fabricated at the Center for Nanophase Materials Sciences (CNMS)

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McKnight et al, CRC Nanotechnology

in Biology and Medicine, 2007.

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ORNL methods should improve capture efficiency and cell viability

enabling post enrichment assay

• Maintain cell viability due to lower shear stress• Higher throughput (due to smaller flow restriction)• Faster processing times

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vs.

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Development of post enrichmentlive cell assay methods

• Cell culture

• Plasmid preparation

• Rapid transgene assay

• Analysis of transgene expression

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Cell culture

• MCF-7 cell line used as a surrogate of metastatic breast cancer• Cultured in Dulbecco’s Modified Eagles Medium (DMEM)

– 10% fetal bovine serum– Penicillin– Non-essential amino acids– L-glutamine– Sodium pyruvate

• Feed cells every 2-3 days– Aspirate old media – Add 5 new mls

• Harvest cells approximately twice a week– Experimental use – Room to grow

• Place in incubator to grow with CO2 environment

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Plasmid preparation

• Use pd2eYFP-N1 plasmid that encodes destabilized yellow fluorescent protein (YFP) used as a marker for rapid transgene expression

• Heat shock plasmid into competent e. coli bacterial cells

• Transformed bacteria resist antibiotic selection and produce high copies of plasmid DNA

• Grow bacterial cultures overnight

• Chemically lyse cultures so high copy DNA plasmid can be extracted and purified using affinity-based kits.

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Rapid transgene assay

• Prepared VACNF chips using ambient air plasma• Applied 100 ng of yellow fluorescent protein plasmid (pd2eYFP)

to chip and dried• Centrifuged cells into pellet• Impaled pelleted cells onto chip• Used time lapse fluorescence

microscopy to observe onset of transgene expression

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Analysis of transgene expression

• Impaled cells receiving DNA are tracked usingtimelapse fluorescence microscopy

• Acquired images 1/min at 1500 msec exposure• Analyzed and processed image data

– Used image J software– MatLab routine to

determinefluorescencetrajectories anddata binning

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Results

• Maintain cell viability following rapid transgene technique• YFP rapidly expressed and observed • Earliest transgene expression observed within 50 min.• Average transgene expression

observed ~ 130 min.• Timeframes are consistent with

nuclear delivery

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Next step - delivery of TRF2-GFP transgene

• Telomere binding protein (TRF2) mislocalizes in metastatic breast cancer cells

• Delivery and expression of TRF2-GFP provides visualization of TRF2 localization within cells

• Rapid transgene studies– Assess metastatic potential of cells– In-vitro studies of cellular response

to chemotherapy

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Fluorescence in situ hybridization (FISH) probing

• Nuclear delivery of fluorescent probe

• Use to measure HER2 gene amplification

• Test positive for HER2 amplification

– Higher mortality rate

– Higher rate of cancer reoccurring

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Conclusion

• Deliver genes into nucleus of MCF-7 cells is possible while maintaining viability

• Plasmid DNA delivery results in rapid transgene expression– Nuclear delivery of probes– Continued viability of interfaced MCF-7 cells

• Establishes rapid transgene assay as a potential means of tailoring therapeutic intervention using enriched CTCs from clinical samples

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Acknowledgments

• A special thanks to Tim McKnight, Morten Madsen, and

Dr. Kristina Thiagarajan for their mentoring. • The Research Alliance in Math and Science program is

sponsored by the Office of Advanced Scientific Computing Research, U.S. Department of Energy.

• The work was performed at the Oak Ridge National Laboratory, which is managed by UT-Battelle, LLC under Contract

No. De-AC05-00OR22725. This work has been authored by a contractor of the U.S. Government, accordingly, the U.S. Government retains a non-exclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do so, for U.S. Government purposes.

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Sources• Melechko, Anatoli V, R. D. (2009). Synthesis of vertically aligned carbon nanofibres for interfacing

with live systems. Journal of Physics D: Applied Physics .

• How many women get breast cancer? (2012, March 12). Retrieved June 1, 2012, from American Cancer Society: http://www.cancer.org/Cancer/BreastCancer/OverviewGuide/breast-cancer-overview-key-statistics

• Lui, M., Griffin, G., & McKnight, T. (n.d.). Hybrid Nanostructured/Microstructured Platforms for the Enrichment of Circulating Tumor Cells. Seed Proposal to the Georgetown/ORNL CTSA .

• Markman, Maurie B. (2012, June 13). Breast Cancer and HER2 Overview of HER2 Breast Cancer. Retrieved July 12, 2012, from Medscape Reference: http://emedicine.medscape.com/article/1689966-overview#aw2aab6b4

• Liu, Minetta C., P. G. (2009). Circulating Tumor Cells: A Useful Predictor of Treatment. Journal of Clinical Oncology , 5153-5158.

• Nijjar, T. (2004). Accumulation and altered localization of telomere-associated protein TRF2 in immortally. Lawrence Berkeley National Laboratory .

• Stott, Shannon L. b. C.-H. (2010). Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. PNAS , 18392-18397.

• Nagrath, Sunitha L. V. (2007). Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature , 1235-1238.

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Questions?

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