Thanks to visit codestin.com
Credit goes to link.springer.com

Skip to main content
Springer Nature Link
Log in

Expression profiling of circulating tumor cells in metastatic breast cancer

  • Clinical trial
  • Published:
Breast Cancer Research and Treatment Aims and scope Submit manuscript

Abstract

Circulating tumor cells (CTCs) are prognostic in all stages of breast cancer. However, since they are extremely rare, little is known about the molecular nature of these cells. We report a novel strategy for the isolation and expression profiling of pure populations of CTCs derived from peripheral blood. We developed a method to isolate CTCs based on immunomagnetic capture followed by fluorescence-activated cell sorting (IE/FACS). After assay validation using the BT474 cell line spiked into blood samples in vitro, RNA from CTCs isolated from the blood of five metastatic breast cancer (MBC) patients was linearly amplified and subjected to gene expression profiling via cDNA microarrays. We isolated a range of 9-993 captured CTCs from five MBC patients’ blood and profiled their RNA in comparison to a diverse panel of primary breast tumors (n = 55). Unsupervised hierarchical clustering revealed that CTC profiles clustered with more aggressive subtypes of primary breast tumors and were readily distinguishable from peripheral blood (PB) and normal epithelium. Differential expression analysis revealed CTCs to have downregulated apoptosis, and they were distinguishable from PB by the relative absence of immune-related signals. As expected, CTCs from MBC had significantly higher risk of recurrence scores than primary tumors (p = 0.0073). This study demonstrates that it is feasible to isolate CTCs from PB with high purity through IE/FACS and profile them via gene expression analysis. Our approach may inform the discovery of therapeutic predictors and be useful for real-time identification of emerging resistance mechanisms in MBC patients.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+
from £29.99 /Month
  • Starting from 10 chapters or articles per month
  • Access and download chapters and articles from more than 300k books and 2,500 journals
  • Cancel anytime
View plans

Buy Now

Price includes VAT (United Kingdom)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Explore related subjects

Discover the latest articles, books and news in related subjects, suggested using machine learning.

Abbreviations

CTC:

Circulating tumor cell

IE/FACS:

Immunomagnetic enrichment followed by fluorescence-activated cell sorting

RNA:

ribonucleic acid

MBC:

Metastatic breast cancer

cDNA:

Complementary deoxyribonucleic acid

PB:

Peripheral blood

QPCR:

Quantitative real-time polymerase chain reaction

EpCAM:

epithelial cell adhesion marker

mAb:

Monoclonal antibody

HER2:

Human epidermal growth factor receptor 2

ER:

Estrogen receptor

RPMI:

Roswell Park Memorial Institute

EDTA:

Ethylenediaminetetraacetic acid

CALGB:

Cancer and Leukemia Group B

ACRIN:

American College of Radiology Imaging Network

StratRef:

Stratagene Universal Human Pooled Reference RNA

dIdC:

Poly(deoxyinosinic-deoxycytidylic) acid sodium salt

M-MLV:

Moloney Murine Leukemia Virus Reverse Transcriptase

GUS:

Beta-glucuronidase

ABI:

Applied Biosystems

CT:

Cycle threshold

HLA:

Human leukocyte antigen

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

Cy:

Cyanine dye

GSE:

Gene Expression Omnibus series format file

AU:

Approximately Unbiased

ANOVA:

Analysis of variance

BH:

Benjamini Hochberg

FDR:

False discovery rate

DAVID:

the Database for Annotation, Visualization and Integrated Discovery

PAM50:

A 50 gene intrinsic subtype classifier

BCL2:

B-cell lymphoma 2

CDC6:

Cell division cycle 6

NUF2:

Kinetochore protein Nuf2

CENPF:

Centromere protein F

CEP55:

Centrosomal protein 55 kDa

CXXC5:

CXXC finger protein 5

EGFR:

Epidermal growth factor receptor

ERBB2:

Human epidermal growth factor receptor 2

ESR1:

Estrogen receptor 1

FGFR4:

Fibroblast growth factor receptor 4

FOXC1:

Forkhead box C1

GRB7:

Growth factor receptor-bound protein 7

NDC80:

NDC80 kinetochore complex component

KRT14:

Keratin 14

MYBL2:

Myeloblastosis oncogene-like 2

PTTG1:

Pituitary tumor-transforming 1

RRM2:

Ribonucleotide reductase M2

TMEM45B:

Transmembrane protein 45B

TYMS:

Thymidylate synthetase

UBE2C:

Ubiquitin-conjugating enzyme E2C

UBE2T:

Ubiquitin-conjugating enzyme E2

ROR ~ P:

Risk of recurrence score

PBS:

Phosphate-buffered saline

KEGG:

Kyoto Encyclopedia of Genes and Genomes

GO:

Gene ontology

UTR:

Untranslated region

UCSF:

University of California, San Francisco

USC:

University of Southern California

I-SPY1:

Investigation of Serial Studies to Predict Your Therapeutic Response with Imaging And moLecular Analysis 1 clinical trial

SWOG:

Southwest Oncology Group

NCI:

National Cancer Institute

PE:

Phycoerythrin

CI:

Confidence interval

References

  1. Allard WJ, Matera J, Miller MC, Repollet M, Connelly MC, Rao C, Tibbe AG, Uhr JW, Terstappen LW (2004) Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin Cancer Res 10(20):6897–6904. doi:10.1158/1078-0432.CCR-04-0378

    Article  PubMed  Google Scholar 

  2. Budd GT, Cristofanilli M, Ellis MJ, Stopeck A, Borden E, Miller MC, Matera J, Repollet M, Doyle GV, Terstappen LW, Hayes DF (2006) Circulating tumor cells versus imaging–predicting overall survival in metastatic breast cancer. Clin Cancer Res 12(21):6403–6409

    Article  CAS  PubMed  Google Scholar 

  3. Liu MC, Shields PG, Warren RD, Cohen P, Wilkinson M, Ottaviano YL, Rao SB, Eng-Wong J, Seillier-Moiseiwitsch F, Noone AM, Isaacs C (2009) Circulating tumor cells: a useful predictor of treatment efficacy in metastatic breast cancer. J Clin Oncol 27(31):5153–5159. doi:10.1200/JCO.2008.20.6664

    Article  PubMed  Google Scholar 

  4. Maheswaran S, Sequist LV, Nagrath S, Ulkus L, Brannigan B, Collura CV, Inserra E, Diederichs S, Iafrate AJ, Bell DW, Digumarthy S, Muzikansky A, Irimia D, Settleman J, Tompkins RG, Lynch TJ, Toner M, Haber DA (2008) Detection of mutations in EGFR in circulating lung-cancer cells. N Engl J Med 359(4):366–377. doi:10.1056/NEJMoa0800668

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Yu M, Bardia A, Wittner BS, Stott SL, Smas ME, Ting DT, Isakoff SJ, Ciciliano JC, Wells MN, Shah AM, Concannon KF, Donaldson MC, Sequist LV, Brachtel E, Sgroi D, Baselga J, Ramaswamy S, Toner M, Haber DA, Maheswaran S (2013) Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition. Science 339(6119):580–584. doi:10.1126/science.1228522

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Sieuwerts AM, Mostert B, Bolt-de Vries J, Peeters D, de Jongh FE, Stouthard JM, Dirix LY, van Dam PA, Van Galen A, de Weerd V, Kraan J, van der Spoel P, Ramirez-Moreno R, van Deurzen CH, Smid M, Yu JX, Jiang J, Wang Y, Gratama JW, Sleijfer S, Foekens JA, Martens JW (2011) mRNA and microRNA expression profiles in circulating tumor cells and primary tumors of metastatic breast cancer patients. Clin. Cancer Res 17(11):3600–3618. doi:10.1158/1078-0432.CCR-11-0255

    Article  CAS  PubMed  Google Scholar 

  7. Smirnov DA, Zweitzig DR, Foulk BW, Miller MC, Doyle GV, Pienta KJ, Meropol NJ, Weiner LM, Cohen SJ, Moreno JG, Connelly MC, Terstappen LW, O’Hara SM (2005) Global gene expression profiling of circulating tumor cells. Cancer Res 65(12):4993–4997. doi:10.1158/0008-5472.CAN-04-4330

    Article  CAS  PubMed  Google Scholar 

  8. Ring A, Smith IE, Dowsett M (2004) Circulating tumour cells in breast cancer. Lancet Oncol 5(2):79–88

    Article  PubMed  Google Scholar 

  9. Xenidis N, Perraki M, Kafousi M, Apostolaki S, Bolonaki I, Stathopoulou A, Kalbakis K, Androulakis N, Kouroussis C, Pallis T, Christophylakis C, Argyraki K, Lianidou ES, Stathopoulos S, Georgoulias V, Mavroudis D (2006) Predictive and prognostic value of peripheral blood cytokeratin-19 mRNA-positive cells detected by real-time polymerase chain reaction in node-negative breast cancer patients. J Clin Oncol 24(23):3756–3762

    Article  CAS  PubMed  Google Scholar 

  10. Apostolaki S, Perraki M, Kallergi G, Kafousi M, Papadopoulos S, Kotsakis A, Pallis A, Xenidis N, Kalmanti L, Kalbakis K, Agelaki S, Kalykaki A, Stournaras C, Stathopoulos E, Georgoulias V, Mavroudis D (2009) Detection of occult HER2 mRNA-positive tumor cells in the peripheral blood of patients with operable breast cancer: evaluation of their prognostic relevance. Breast Cancer Res Treat 117(3):525–534. doi:10.1007/s10549-008-0239-3

    Article  PubMed  Google Scholar 

  11. Sieuwerts AM, Kraan J, Bolt-de Vries J, van der Spoel P, Mostert B, Martens JW, Gratama JW, Sleijfer S, Foekens JA (2009) Molecular characterization of circulating tumor cells in large quantities of contaminating leukocytes by a multiplex real-time PCR. Breast Cancer Res Treat 118(3):455–468. doi:10.1007/s10549-008-0290-0

    Article  CAS  PubMed  Google Scholar 

  12. Powell AA, Talasaz AH, Zhang H, Coram MA, Reddy A, Deng G, Telli ML, Advani RH, Carlson RW, Mollick JA, Sheth S, Kurian AW, Ford JM, Stockdale FE, Quake SR, Pease RF, Mindrinos MN, Bhanot G, Dairkee SH, Davis RW, Jeffrey SS (2012) Single cell profiling of circulating tumor cells: transcriptional heterogeneity and diversity from breast cancer cell lines. PLoS One 7(5):e33788. doi:10.1371/journal.pone.0033788

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Magbanua MJ, Park JW (2013) Isolation of circulating tumor cells by immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS) for molecular profiling. Methods. doi:10.1016/j.ymeth.2013.07.029

    PubMed  Google Scholar 

  14. Magbanua MJ, Sosa EV, Roy R, Eisenbud LE, Scott JH, Olshen A, Pinkel D, Rugo HS, Park JW (2013) Genomic profiling of isolated circulating tumor cells from metastatic breast cancer patients. Cancer Res 73(1):30–40. doi:10.1158/0008-5472.CAN-11-3017

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Lang JE, Magbanua MJ, Scott JH, Makrigiorgos GM, Wang G, Federman S, Esserman LJ, Park JW, Haqq CM (2009) A comparison of RNA amplification techniques at sub-nanogram input concentration. BMC Genom 10:326. doi:10.1186/1471-2164-10-326

    Article  Google Scholar 

  16. Lasfargues EY, Coutinho WG, Redfield ES (1978) Isolation of two human tumor epithelial cell lines from solid breast carcinomas. J Natl Cancer Inst 61(4):967–978

    CAS  PubMed  Google Scholar 

  17. Baugh LR, Hill AA, Brown EL, Hunter CP (2001) Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29(5):E29

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25(4):402–408. doi:10.1006/meth.2001.1262

    Article  CAS  PubMed  Google Scholar 

  19. Haqq C, Nosrati M, Sudilovsky D, Crothers J, Khodabakhsh D, Pulliam BL, Federman S, Miller JR 3rd, Allen RE, Singer MI, Leong SP, Ljung BM, Sagebiel RW, Kashani-Sabet M (2005) The gene expression signatures of melanoma progression. Proc Natl Acad Sci USA 102(17):6092–6097

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Hughes TR, Mao M, Jones AR, Burchard J, Marton MJ, Shannon KW, Lefkowitz SM, Ziman M, Schelter JM, Meyer MR, Kobayashi S, Davis C, Dai H, He YD, Stephaniants SB, Cavet G, Walker WL, West A, Coffey E, Shoemaker DD, Stoughton R, Blanchard AP, Friend SH, Linsley PS (2001) Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer. Nat Biotechnol 19(4):342–347

    Article  CAS  PubMed  Google Scholar 

  21. DeRisi J, Penland L, Brown PO, Bittner ML, Meltzer PS, Ray M, Chen Y, Su YA, Trent JM (1996) Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nat Genet 14(4):457–460

    Article  CAS  PubMed  Google Scholar 

  22. Yang YH, Paquet A, Dudoit S (2009) marray: Exploratory analysis for two-color spotted microarray data.. R package version 1.36.0. edn.,

  23. R_Core_Team (2012) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria

  24. Smyth GK (2005) Limma: linear models for microarray data. Bioinformatics and Computational Biology Solutions using R and Bioconductor. Springer, New York

    Google Scholar 

  25. Suzuki R, Hidetoshi S (2011) pvclust: Hierarchical clustering with P-values via multiscale bootstrap resampling. R package version 1.2–2. edn.,

  26. Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA (2003) DAVID: database for annotation, visualization, and integrated discovery. Genome Biol 4(5):P3

    Article  PubMed  Google Scholar 

  27. Parker JS, Mullins M, Cheang MC, Leung S, Voduc D, Vickery T, Davies S, Fauron C, He X, Hu Z, Quackenbush JF, Stijleman IJ, Palazzo J, Marron JS, Nobel AB, Mardis E, Nielsen TO, Ellis MJ, Perou CM, Bernard PS (2009) Supervised risk predictor of breast cancer based on intrinsic subtypes. J Clin Oncol 27(8):1160–1167. doi:10.1200/JCO.2008.18.1370

    Article  PubMed Central  PubMed  Google Scholar 

  28. McShane LM, Hayes DF (2012) Publication of tumor marker research results: the necessity for complete and transparent reporting. J Clin Oncol 30(34):4223–4232. doi:10.1200/JCO.2012.42.6858

    Article  PubMed Central  PubMed  Google Scholar 

  29. Esserman LJ, Berry DA, DeMichele A, Carey L, Davis SE, Buxton M, Hudis C, Gray JW, Perou C, Yau C, Livasy C, Krontiras H, Montgomery L, Tripathy D, Lehman C, Liu MC, Olopade OI, Rugo HS, Carpenter JT, Dressler L, Chhieng D, Singh B, Mies C, Rabban J, Chen YY, Giri D, van ‘t Veer L, Hylton N (2012) Pathologic complete response predicts recurrence-free survival more effectively by cancer subset: results from the I-SPY 1 TRIAL–CALGB 150007/150012, ACRIN 6657. J Clin Oncol 30(26):3242–3249. doi:10.1200/JCO.2011.39.2779

    Article  PubMed Central  PubMed  Google Scholar 

  30. van ’t Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, Mao M, Peterse HL, van der Kooy K, Marton MJ, Witteveen AT, Schreiber GJ, Kerkhoven RM, Roberts C, Linsley PS, Bernards R, Friend SH (2002) Gene expression profiling predicts clinical outcome of breast cancer. Nature 415(6871):530–536. doi:10.1038/415530a

    Article  Google Scholar 

  31. van de Vijver MJ, He YD, van’t Veer LJ, Dai H, Hart AA, Voskuil DW, Schreiber GJ, Peterse JL, Roberts C, Marton MJ, Parrish M, Atsma D, Witteveen A, Glas A, Delahaye L, van der Velde T, Bartelink H, Rodenhuis S, Rutgers ET, Friend SH, Bernards R (2002) A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med 347(25):1999–2009. doi:10.1056/NEJMoa021967347/25/1999

    Article  PubMed  Google Scholar 

  32. Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, Deng S, Johnsen H, Pesich R, Geisler S, Demeter J, Perou CM, Lonning PE, Brown PO, Borresen-Dale AL, Botstein D (2003) Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci USA 100(14):8418–8423

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Meng S, Tripathy D, Frenkel EP, Shete S, Naftalis EZ, Huth JF, Beitsch PD, Leitch M, Hoover S, Euhus D, Haley B, Morrison L, Fleming TP, Herlyn D, Terstappen LW, Fehm T, Tucker TF, Lane N, Wang J, Uhr JW (2004) Circulating tumor cells in patients with breast cancer dormancy. Clin. Cancer Res 10(24):8152–8162

    Article  PubMed  Google Scholar 

  34. Smerage JB, Barlow WE, Hortobagyi GN, Winer EP, Leyland-Jones B, Srkalovic G, Tejwani S, Schott AF, O’Rourke MA, Lew DL, Doyle GV, Gralow JR, Livingston RB, Hayes DF (2014) Circulating tumor cells and response to chemotherapy in metastatic breast cancer: SWOG S0500. J Clin Oncol. doi:10.1200/JCO.2014.56.2561

    PubMed  Google Scholar 

Download references

Acknowledgments

We would like to thank Daisuke Nonaka, Daniel Khodabakhsh, Kavitha Krishnan, Hema Parmar, Alfred Au, Scot Federman, Pamela Derish, David Ginzinger, Joe Gray, and Eduardo Sosa for their contributions to this work. We acknowledge William Hyun and the UCSF Comprehensive Cancer Center Molecular Cytometry Core. We acknowledge Emily Park and Tom Frey of BD Biosciences for contributing reagents used for these experiments.

Financial support

Dr. Park’s laboratory was supported by the National Cancer Institute (NCI) Interdisciplinary Research Teams for Molecular Target Assessment (U54 CA90788). Dr. Lang’s laboratory was supported by a Ginny Clements Research Award, a Better than Ever Award (University of Arizona), a Society of Surgical Oncology Clinical Investigator Award, a California Breast Cancer Research Program Idea Award, and a STOP Cancer Award. The project described was supported in part by award number P30CA014089 from the National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health. Southwest Environmental Health Sciences Center Grant ES006694 supported Petr Novak. We acknowledge the I-SPY Program for additional statistical support.

Conflict of interest

Christopher M. Haqq owns stock in Johnson & Johnson. The other authors have no relevant financial disclosures.

Author information

Authors and Affiliations

  1. Division of Breast and Soft Tissue Surgery, Department of Surgery, Norris Comprehensive Cancer Center (NCCC), University of Southern California (USC), 1510 San Pablo Street Suite 412, Los Angeles, CA, 90033-0800, USA

    Julie E. Lang, Weizhu Zhu & Neal Mineyev

  2. Division of Hematology and Medical Oncology, Department of Medicine, University of California San Francisco (UCSF), San Francisco, CA, USA

    Janet H. Scott, Mark Jesus M. Magbanua, Julia R. Crothers & John W. Park

  3. UCSF Department of Laboratory Medicine, San Francisco, CA, USA

    Denise M. Wolf & Laura van ’t Veer

  4. Biology Centre ASCR, Institute of Plant Molecular Biology, 37001, Ceske Budejovice, Czech Republic

    Petr Novak

  5. NCCC Bioinformatics Core and Division of Hematology, Keck School of Medicine, NCCC, University of Southern California (USC), Los Angeles, CA, USA

    Vasu Punj

  6. Atara Biotherapeutics, Westlake Village, Brisbane, CA, USA

    Christopher M. Haqq

  7. Section of Breast Care Surgery, UCSF Department of Surgery, San Francisco, CA, USA

    Laura J. Esserman

  8. Division of Oncology, USC Department of Medicine, NCCC, Los Angeles, CA, USA

    Debasish Tripathy

Authors
  1. Julie E. Lang
    View author publications

    Search author on:PubMed Google Scholar

  2. Janet H. Scott
    View author publications

    Search author on:PubMed Google Scholar

  3. Denise M. Wolf
    View author publications

    Search author on:PubMed Google Scholar

  4. Petr Novak
    View author publications

    Search author on:PubMed Google Scholar

  5. Vasu Punj
    View author publications

    Search author on:PubMed Google Scholar

  6. Mark Jesus M. Magbanua
    View author publications

    Search author on:PubMed Google Scholar

  7. Weizhu Zhu
    View author publications

    Search author on:PubMed Google Scholar

  8. Neal Mineyev
    View author publications

    Search author on:PubMed Google Scholar

  9. Christopher M. Haqq
    View author publications

    Search author on:PubMed Google Scholar

  10. Julia R. Crothers
    View author publications

    Search author on:PubMed Google Scholar

  11. Laura J. Esserman
    View author publications

    Search author on:PubMed Google Scholar

  12. Debasish Tripathy
    View author publications

    Search author on:PubMed Google Scholar

  13. Laura van ’t Veer
    View author publications

    Search author on:PubMed Google Scholar

  14. John W. Park
    View author publications

    Search author on:PubMed Google Scholar

Corresponding author

Correspondence to Julie E. Lang.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lang, J.E., Scott, J.H., Wolf, D.M. et al. Expression profiling of circulating tumor cells in metastatic breast cancer. Breast Cancer Res Treat 149, 121–131 (2015). https://doi.org/10.1007/s10549-014-3215-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1007/s10549-014-3215-0

Keywords

Profiles

  1. Julie E. Lang View author profile
  2. Petr Novak View author profile