Skip to main content
SpringerLink
Log in

Strategies for Isolation and Molecular Profiling of Circulating Tumor Cells

  • Chapter
  • First Online:
Isolation and Molecular Characterization of Circulating Tumor Cells

Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 994))

Abstract

Cancer is the leading cause of death by disease worldwide, and metastasis is responsible for more than 90% of the mortality of cancer patients. Metastasis occurs when tumor cells leave the primary tumor, travel through the blood stream as circulating tumor cells (CTCs), and then colonize secondary tumors at sites distant from the primary tumor. The capture, identification, and analysis of CTCs offer both scientific and clinical benefits. On the scientific side, the analysis of CTCs could help elucidate possible genetic alterations and signaling pathway aberrations during cancer progression, which could then be used to find new methods to stop cancer progression. On the clinical side, non-invasive testing of a patient’s blood for CTCs can be used for patient diagnosis and prognosis, as well as subsequent monitoring of treatment efficacy in routine clinical practice. Additionally, investigation of CTCs early in the progression of cancer may reveal targets for initial cancer detection and for anti-cancer treatment. This chapter will evaluate strategies and devices used for the isolation and identification of CTCs directly from clinical samples of blood. Recent progress in the understanding of the significance of both single CTCs and circulating tumor microemboli will be discussed. Also, advancements in the use of CTC-based liquid biopsy in clinical diagnosis and the potential of CTC-based molecular characterization for use in clinical applications will be summarized.

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

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  • Aceto N, Bardia A, Miyamoto DT, Donaldson MC, Wittner BS, Spencer JA, Yu M, Pely A, Engstrom A, Zhu H, Brannigan BW, Kapur R, Stott SL, Shioda T, Ramaswamy S, Ting DT, Lin CP, Toner M, Haber DA, Maheswaran S (2014) Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell 158(5):1110–1122. doi:10.1016/j.cell.2014.07.013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Adams JD, Thevoz P, Bruus H, Soh HT (2009) Integrated acoustic and magnetic separation in microfluidic channels. Appl Phys Lett 95(25):254103. doi:10.1063/1.3275577

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Aggarwal C, Meropol NJ, Punt CJ, Iannotti N, Saidman BH, Sabbath KD, Gabrail NY, Picus J, Morse MA, Mitchell E, Miller MC, Cohen SJ (2013) Relationship among circulating tumor cells, CEA and overall survival in patients with metastatic colorectal cancer. Ann Oncol 24(2):420–428. doi:10.1093/annonc/mds336

    Article  CAS  PubMed  Google Scholar 

  • 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 

  • Antfolk M, Magnusson C, Augustsson P, Lilja H, Laurell T (2015) Acoustofluidic, label-free separation and simultaneous concentration of rare tumor cells from white blood cells. Anal Chem 87(18):9322–9328. doi:10.1021/acs.analchem.5b02023

    Article  CAS  PubMed  Google Scholar 

  • Antonarakis ES, Lu C, Wang H, Luber B, Nakazawa M, Roeser JC, Chen Y, Mohammad TA, Chen Y, Fedor HL, Lotan TL, Zheng Q, De Marzo AM, Isaacs JT, Isaacs WB, Nadal R, Paller CJ, Denmeade SR, Carducci MA, Eisenberger MA, Luo J (2014) AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. N Engl J Med 371(11):1028–1038. doi:10.1056/NEJMoa1315815

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Antonarakis ES, Lu C, Luber B, Wang H, Chen Y, Nakazawa M, Nadal R, Paller CJ, Denmeade SR, Carducci MA, Eisenberger MA, Luo J (2015) Androgen receptor splice variant 7 and efficacy of taxane chemotherapy in patients with metastatic castration-resistant prostate cancer. JAMA Oncol 1(5):582–591. doi:10.1001/jamaoncol.2015.1341

    Article  PubMed  PubMed Central  Google Scholar 

  • Armstrong AJ, Marengo MS, Oltean S, Kemeny G, Bitting RL, Turnbull JD, Herold CI, Marcom PK, George DJ, Garcia-Blanco MA (2011) Circulating tumor cells from patients with advanced prostate and breast cancer display both epithelial and mesenchymal markers. Mol Cancer Res 9(8):997–1007. doi:10.1158/1541-7786.MCR-10-0490

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ashworth TR (1869) A case of cancer in which cells similar to those in the tumours were seen in the blood after death. Aust Med J 14:146

    Google Scholar 

  • Au SH, Storey BD, Moore JC, Tang Q, Chen YL, Javaid S, Sarioglu AF, Sullivan R, Madden MW, O’Keefe R, Haber DA, Maheswaran S, Langenau DM, Stott SL, Toner M (2016) Clusters of circulating tumor cells traverse capillary-sized vessels. Proc Natl Acad Sci U S A 113(18):4947–4952. doi:10.1073/pnas.1524448113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bayarri-Lara C, Ortega FG, Cueto Ladron de Guevara A, Puche JL, Ruiz Zafra J, de Miguel-Perez D, Ramos AS, Giraldo-Ospina CF, Navajas Gomez JA, Delgado-Rodriguez M, Lorente JA, Serrano MJ (2016) Circulating tumor cells identify early recurrence in patients with non-small cell lung cancer undergoing radical resection. PLoS One 11(2):e0148659. doi:10.1371/journal.pone.0148659

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Bidard FC, Huguet F, Louvet C, Mineur L, Bouche O, Chibaudel B, Artru P, Desseigne F, Bachet JB, Mathiot C, Pierga JY, Hammel P (2013) Circulating tumor cells in locally advanced pancreatic adenocarcinoma: the ancillary CirCe 07 study to the LAP 07 trial. Ann Oncol 24(8):2057–2061. doi:10.1093/annonc/mdt176

    Article  CAS  PubMed  Google Scholar 

  • Bidard FC, Peeters DJ, Fehm T, Nole F, Gisbert-Criado R, Mavroudis D, Grisanti S, Generali D, Garcia-Saenz JA, Stebbing J, Caldas C, Gazzaniga P, Manso L, Zamarchi R, de Lascoiti AF, De Mattos-Arruda L, Ignatiadis M, Lebofsky R, van Laere SJ, Meier-Stiegen F, Sandri MT, Vidal-Martinez J, Politaki E, Consoli F, Bottini A, Diaz-Rubio E, Krell J, Dawson SJ, Raimondi C, Rutten A, Janni W, Munzone E, Caranana V, Agelaki S, Almici C, Dirix L, Solomayer EF, Zorzino L, Johannes H, Reis-Filho JS, Pantel K, Pierga JY, Michiels S (2014) Clinical validity of circulating tumour cells in patients with metastatic breast cancer: a pooled analysis of individual patient data. Lancet Oncol 15(4):406–414. doi:10.1016/S1470-2045(14)70069-5

    Article  PubMed  Google Scholar 

  • 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. doi:10.1158/1078-0432.CCR-05-1769

    Article  CAS  PubMed  Google Scholar 

  • Chaffer CL, Weinberg RA (2011) A perspective on cancer cell metastasis. Science 331(6024):1559–1564. doi:10.1126/science.1203543

    Article  CAS  PubMed  Google Scholar 

  • Chang MC, Chang YT, Chen JY, Jeng YM, Yang CY, Tien YW, Yang SH, Chen HL, Liang TY, Wang CF, Lee EY, Chang YC, Lee WH (2016) Clinical significance of circulating tumor microemboli as a prognostic marker in patients with pancreatic ductal adenocarcinoma. Clin Chem 62(3):505–513. doi:10.1373/clinchem.2015.248260

    Article  CAS  PubMed  Google Scholar 

  • Chen L, Han X (2015) Anti-PD-1/PD-L1 therapy of human cancer: past, present, and future. J Clin Invest 125(9):3384–3391. doi:10.1172/JCI80011

    Article  PubMed  PubMed Central  Google Scholar 

  • Chen Q, Zhang XH, Massague J (2011) Macrophage binding to receptor VCAM-1 transmits survival signals in breast cancer cells that invade the lungs. Cancer Cell 20(4):538–549. doi:10.1016/j.ccr.2011.08.025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen JY, Tsai WS, Shao HJ, Wu JC, Lai JM, Lu SH, Hung TF, Yang CT, Wu LC, Chen JS, Lee WH, Chang YC (2016) Sensitive and specific biomimetic lipid coated microfluidics to isolate viable circulating tumor cells and microemboli for cancer detection. PLoS One 11(3):e0149633. doi:10.1371/journal.pone.0149633

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Chu PG, Weiss LM (2002) Keratin expression in human tissues and neoplasms. Histopathology 40(5):403–439

    Article  CAS  PubMed  Google Scholar 

  • Coumans FA, Ligthart ST, Uhr JW, Terstappen LW (2012) Challenges in the enumeration and phenotyping of CTC. Clin Cancer Res 18(20):5711–5718. doi:10.1158/1078-0432.CCR-12-1585

    Article  PubMed  Google Scholar 

  • Court CM, Ankeny JS, Hou S, Tseng HR, Tomlinson JS (2015) Improving pancreatic cancer diagnosis using circulating tumor cells: prospects for staging and single-cell analysis. Expert Rev Mol Diagn 15(11):1491–1504. doi:10.1586/14737159.2015.1091311

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J, Miller MC, Reuben JM, Doyle GV, Allard WJ, Terstappen LW, Hayes DF (2004) Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 351(8):781–791. doi:10.1056/NEJMoa040766

    Article  CAS  PubMed  Google Scholar 

  • de Bono JS, Scher HI, Montgomery RB, Parker C, Miller MC, Tissing H, Doyle GV, Terstappen LW, Pienta KJ, Raghavan D (2008) Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer. Clin Cancer Res 14(19):6302–6309. doi:10.1158/1078-0432.CCR-08-0872

    Article  PubMed  CAS  Google Scholar 

  • Deng G, Krishnakumar S, Powell AA, Zhang H, Mindrinos MN, Telli ML, Davis RW, Jeffrey SS (2014) Single cell mutational analysis of PIK3CA in circulating tumor cells and metastases in breast cancer reveals heterogeneity, discordance, and mutation persistence in cultured disseminated tumor cells from bone marrow. BMC Cancer 14:456. doi:10.1186/1471-2407-14-456

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Duda DG, Duyverman AM, Kohno M, Snuderl M, Steller EJ, Fukumura D, Jain RK (2010) Malignant cells facilitate lung metastasis by bringing their own soil. Proc Natl Acad Sci U S A 107(50):21677–21682. doi:10.1073/pnas.1016234107

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Esselen KM, Cronin AM, Bixel K, Bookman MA, Burger RA, Cohn DE, Cristea M, Griggs JJ, Levenback CF, Mantia-Smaldone G, Meyer LA, Matulonis UA, Niland JC, Sun C, O’Malley DM, Wright AA (2016) Use of CA-125 tests and computed tomographic scans for surveillance in ovarian cancer. JAMA Oncol. doi:10.1001/jamaoncol.2016.1842

    PubMed  PubMed Central  Google Scholar 

  • Faugeroux V, Pailler E, Auger N, Taylor M, Farace F (2014) Clinical utility of circulating tumor cells in alk-positive non-small-cell lung cancer. Front Oncol 4:281. doi:10.3389/fonc.2014.00281

    Article  PubMed  PubMed Central  Google Scholar 

  • Fischer KR, Durrans A, Lee S, Sheng J, Li F, Wong ST, Choi H, El Rayes T, Ryu S, Troeger J, Schwabe RF, Vahdat LT, Altorki NK, Mittal V, Gao D (2015) Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance. Nature 527(7579):472–476. doi:10.1038/nature15748

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gerhard R, Schmitt FC (2014) Liquid-based cytology in fine-needle aspiration of breast lesions: a review. Acta Cytol 58(6):533–542. doi:10.1159/000362805

    CAS  PubMed  Google Scholar 

  • Gonsalves WI, Morice WG, Rajkumar V, Gupta V, Timm MM, Dispenzieri A, Buadi FK, Lacy MQ, Singh PP, Kapoor P, Gertz MA, Kumar SK (2014) Quantification of clonal circulating plasma cells in relapsed multiple myeloma. Br J Haematol 167(4):500–505. doi:10.1111/bjh.13067

    Article  PubMed  PubMed Central  Google Scholar 

  • Grisanti S, Almici C, Consoli F, Buglione M, Verardi R, Bolzoni-Villaret A, Bianchetti A, Ciccarese C, Mangoni M, Ferrari L, Biti G, Marini M, Ferrari VD, Nicolai P, Magrini SM, Berruti A (2014) Circulating tumor cells in patients with recurrent or metastatic head and neck carcinoma: prognostic and predictive significance. PLoS One 9(8):e103918. doi:10.1371/journal.pone.0103918

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Grobe A, Blessmann M, Hanken H, Friedrich RE, Schon G, Wikner J, Effenberger KE, Kluwe L, Heiland M, Pantel K, Riethdorf S (2014) Prognostic relevance of circulating tumor cells in blood and disseminated tumor cells in bone marrow of patients with squamous cell carcinoma of the oral cavity. Clin Cancer Res 20(2):425–433. doi:10.1158/1078-0432.CCR-13-1101

    Article  PubMed  CAS  Google Scholar 

  • Hanash SM, Baik CS, Kallioniemi O (2011) Emerging molecular biomarkers–blood-based strategies to detect and monitor cancer. Nat Rev Clin Oncol 8(3):142–150. doi:10.1038/nrclinonc.2010.220

    Article  PubMed  Google Scholar 

  • Hayes DF, Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Miller MC, Matera J, Allard WJ, Doyle GV, Terstappen LW (2006) Circulating tumor cells at each follow-up time point during therapy of metastatic breast cancer patients predict progression-free and overall survival. Clin Cancer Res 12(14 Pt 1):4218–4224. doi:10.1158/1078-0432.CCR-05-2821

    Article  CAS  PubMed  Google Scholar 

  • He W, Xu D, Wang Z, Xiang X, Tang B, Li S, Hou M, Zhang Y, Chen JF, Lin M, Wang L, Hou S, Tseng HR, Kuang M, Ke ZF (2016) Detecting ALK-rearrangement of CTC enriched by nanovelcro chip in advanced NSCLC patients. Oncotarget. doi:10.18632/oncotarget.8305

    Google Scholar 

  • Hegerfeldt Y, Tusch M, Brocker EB, Friedl P (2002) Collective cell movement in primary melanoma explants: plasticity of cell-cell interaction, beta1-integrin function, and migration strategies. Cancer Res 62(7):2125–2130

    CAS  PubMed  Google Scholar 

  • Hodgkinson CL, Morrow CJ, Li Y, Metcalf RL, Rothwell DG, Trapani F, Polanski R, Burt DJ, Simpson KL, Morris K, Pepper SD, Nonaka D, Greystoke A, Kelly P, Bola B, Krebs MG, Antonello J, Ayub M, Faulkner S, Priest L, Carter L, Tate C, Miller CJ, Blackhall F, Brady G, Dive C (2014) Tumorigenicity and genetic profiling of circulating tumor cells in small-cell lung cancer. Nat Med 20(8):897–903. doi:10.1038/nm.3600

    Article  CAS  PubMed  Google Scholar 

  • Hou JM, Krebs MG, Lancashire L, Sloane R, Backen A, Swain RK, Priest LJ, Greystoke A, Zhou C, Morris K, Ward T, Blackhall FH, Dive C (2012) Clinical significance and molecular characteristics of circulating tumor cells and circulating tumor microemboli in patients with small-cell lung cancer. J Clin Oncol 30(5):525–532. doi:10.1200/JCO.2010.33.3716

    Article  PubMed  Google Scholar 

  • Husemann Y, Geigl JB, Schubert F, Musiani P, Meyer M, Burghart E, Forni G, Eils R, Fehm T, Riethmuller G, Klein CA (2008) Systemic spread is an early step in breast cancer. Cancer Cell 13(1):58–68. doi:10.1016/j.ccr.2007.12.003

    Article  PubMed  CAS  Google Scholar 

  • Iinuma H, Watanabe T, Mimori K, Adachi M, Hayashi N, Tamura J, Matsuda K, Fukushima R, Okinaga K, Sasako M, Mori M (2011) Clinical significance of circulating tumor cells, including cancer stem-like cells, in peripheral blood for recurrence and prognosis in patients with Dukes’ stage B and C colorectal cancer. J Clin Oncol 29(12):1547–1555. doi:10.1200/JCO.2010.30.5151

    Article  PubMed  Google Scholar 

  • Ilie M, Long E, Butori C, Hofman V, Coelle C, Mauro V, Zahaf K, Marquette CH, Mouroux J, Paterlini-Brechot P, Hofman P (2012) ALK-gene rearrangement: a comparative analysis on circulating tumour cells and tumour tissue from patients with lung adenocarcinoma. Ann Oncol 23(11):2907–2913. doi:10.1093/annonc/mds137

    Article  CAS  PubMed  Google Scholar 

  • Ilina O, Friedl P (2009) Mechanisms of collective cell migration at a glance. J Cell Sci 122(Pt 18):3203–3208. doi:10.1242/jcs.036525

    Article  CAS  PubMed  Google Scholar 

  • Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N (2002) Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci U S A 99(19):12293–12297. doi:10.1073/pnas.192461099

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jiang B, Mason J, Jewett A, Qian J, Ding Y, Cho WC, Zhang X, Man YG (2013) Cell budding from normal appearing epithelia: a predictor of colorectal cancer metastasis? Int J Biol Sci 9(1):119–133. doi:10.7150/ijbs.5441

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Katz AE, Olsson CA, Raffo AJ, Cama C, Perlman H, Seaman E, O’Toole KM, McMahon D, Benson MC, Buttyan R (1994) Molecular staging of prostate cancer with the use of an enhanced reverse transcriptase-PCR assay. Urology 43(6):765–775

    Article  CAS  PubMed  Google Scholar 

  • Ke Z, Lin M, Chen JF, Choi JS, Zhang Y, Fong A, Liang AJ, Chen SF, Li Q, Fang W, Zhang P, Garcia MA, Lee T, Song M, Lin HA, Zhao H, Luo SC, Hou S, Yu HH, Tseng HR (2015) Programming thermoresponsiveness of NanoVelcro substrates enables effective purification of circulating tumor cells in lung cancer patients. ACS Nano 9(1):62–70. doi:10.1021/nn5056282

    Article  CAS  PubMed  Google Scholar 

  • Kelley RK, Magbanua MJ, Butler TM, Collisson EA, Hwang J, Sidiropoulos N, Evason K, McWhirter RM, Hameed B, Wayne EM, Yao FY, Venook AP, Park JW (2015) Circulating tumor cells in hepatocellular carcinoma: a pilot study of detection, enumeration, and next-generation sequencing in cases and controls. BMC Cancer 15:206. doi:10.1186/s12885-015-1195-z

    Article  PubMed  PubMed Central  Google Scholar 

  • Kirby BJ, Jodari M, Loftus MS, Gakhar G, Pratt ED, Chanel-Vos C, Gleghorn JP, Santana SM, Liu H, Smith JP, Navarro VN, Tagawa ST, Bander NH, Nanus DM, Giannakakou P (2012) Functional characterization of circulating tumor cells with a prostate-cancer-specific microfluidic device. PLoS One 7(4):e35976. doi:10.1371/journal.pone.0035976

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Klein CA (2008) Cancer. The metastasis cascade. Science 321(5897):1785–1787. doi:10.1126/science.1164853

    Article  CAS  PubMed  Google Scholar 

  • Krebs MG, Sloane R, Priest L, Lancashire L, Hou JM, Greystoke A, Ward TH, Ferraldeschi R, Hughes A, Clack G, Ranson M, Dive C, Blackhall FH (2011) Evaluation and prognostic significance of circulating tumor cells in patients with non-small-cell lung cancer. J Clin Oncol 29(12):1556–1563. doi:10.1200/JCO.2010.28.7045

    Article  PubMed  Google Scholar 

  • Krebs MG, Renehan AG, Backen A, Gollins S, Chau I, Hasan J, Valle JW, Morris K, Beech J, Ashcroft L, Saunders MP, Dive C (2015) Circulating tumor cell enumeration in a phase II trial of a four-drug regimen in advanced colorectal cancer. Clin Colorectal Cancer 14(2):115–122 e111-112. doi:10.1016/j.clcc.2014.12.006

    Article  PubMed  Google Scholar 

  • Kulasingam V, Diamandis EP (2008) Strategies for discovering novel cancer biomarkers through utilization of emerging technologies. Nat Clin Pract Oncol 5(10):588–599. doi:10.1038/ncponc1187

    Article  CAS  PubMed  Google Scholar 

  • Lang JE, Scott JH, Wolf DM, Novak P, Punj V, Magbanua MJ, Zhu W, Mineyev N, Haqq CM, Crothers JR, Esserman LJ, Tripathy D, van’t Veer L, Park JW (2015) Expression profiling of circulating tumor cells in metastatic breast cancer. Breast Cancer Res Treat 149(1):121–131. doi:10.1007/s10549-014-3215-0

    Article  CAS  PubMed  Google Scholar 

  • Lapin M, Tjensvoll K, Oltedal S, Buhl T, Gilje B, Smaaland R, Nordgard O (2016) MINDEC-an enhanced negative depletion strategy for circulating tumour cell enrichment. Sci Rep 6:28929. doi:10.1038/srep28929

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lawson DA, Bhakta NR, Kessenbrock K, Prummel KD, Yu Y, Takai K, Zhou A, Eyob H, Balakrishnan S, Wang CY, Yaswen P, Goga A, Werb Z (2015) Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells. Nature 526(7571):131–135. doi:10.1038/nature15260

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lee A, Park J, Lim M, Sunkara V, Kim SY, Kim GH, Kim MH, Cho YK (2014) All-in-one centrifugal microfluidic device for size-selective circulating tumor cell isolation with high purity. Anal Chem 86(22):11349–11356. doi:10.1021/ac5035049

    Article  CAS  PubMed  Google Scholar 

  • Lohr JG, Adalsteinsson VA, Cibulskis K, Choudhury AD, Rosenberg M, Cruz-Gordillo P, Francis JM, Zhang CZ, Shalek AK, Satija R, Trombetta JJ, Lu D, Tallapragada N, Tahirova N, Kim S, Blumenstiel B, Sougnez C, Lowe A, Wong B, Auclair D, Van Allen EM, Nakabayashi M, Lis RT, Lee GS, Li T, Chabot MS, Ly A, Taplin ME, Clancy TE, Loda M, Regev A, Meyerson M, Hahn WC, Kantoff PW, Golub TR, Getz G, Boehm JS, Love JC (2014) Whole-exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer. Nat Biotechnol 32(5):479–484. doi:10.1038/nbt.2892

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lorente D, Olmos D, Mateo J, Bianchini D, Seed G, Fleisher M, Danila DC, Flohr P, Crespo M, Figueiredo I, Miranda S, Baeten K, Molina A, Kheoh T, McCormack R, Terstappen LW, Scher HI, de Bono JS (2016) Decline in circulating tumor cell count and treatment outcome in advanced prostate cancer. Eur Urol. doi:10.1016/j.eururo.2016.05.023

    PubMed Central  Google Scholar 

  • Lu Y, Liang H, Yu T, Xie J, Chen S, Dong H, Sinko PJ, Lian S, Xu J, Wang J, Yu S, Shao J, Yuan B, Wang L, Jia L (2015) Isolation and characterization of living circulating tumor cells in patients by immunomagnetic negative enrichment coupled with flow cytometry. Cancer 121(17):3036–3045. doi:10.1002/cncr.29444

    Article  CAS  PubMed  Google Scholar 

  • Lu SH, Tsai WS, Chang YH, Chou TY, Pang ST, Lin PH, Tsai CM, Chang YC (2016) Identifying cancer origin using circulating tumor cells. Cancer Biol Ther 17(4):430–438. doi:10.1080/15384047.2016.1141839

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ludwig JA, Weinstein JN (2005) Biomarkers in cancer staging, prognosis and treatment selection. Nat Rev Cancer 5(11):845–856. doi:10.1038/nrc1739

    Article  CAS  PubMed  Google Scholar 

  • Magbanua MJ, Park JW (2013) Isolation of circulating tumor cells by immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS) for molecular profiling. Methods 64(2):114–118. doi:10.1016/j.ymeth.2013.07.029

    Article  CAS  PubMed  Google Scholar 

  • 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  Google Scholar 

  • Magbanua MJ, Carey LA, DeLuca A, Hwang J, Scott JH, Rimawi MF, Mayer EL, Marcom PK, Liu MC, Esteva FJ, Park JW, Rugo HS, Translational Breast Cancer Research C (2015) Circulating tumor cell analysis in metastatic triple-negative breast cancers. Clin Cancer Res 21(5):1098–1105. doi:10.1158/1078-0432.CCR-14-1948

    Article  CAS  PubMed  Google Scholar 

  • Man YG (2010) Aberrant leukocyte infiltration: a direct trigger for breast tumor invasion and metastasis. Int J Biol Sci 6(2):129–132

    Article  PubMed  PubMed Central  Google Scholar 

  • Marchetti A, Del Grammastro M, Felicioni L, Malatesta S, Filice G, Centi I, De Pas T, Santoro A, Chella A, Brandes AA, Venturino P, Cuccurullo F, Crino L, Buttitta F (2014) Assessment of EGFR mutations in circulating tumor cell preparations from NSCLC patients by next generation sequencing: toward a real-time liquid biopsy for treatment. PLoS One 9(8):e103883. doi:10.1371/journal.pone.0103883

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Massard C, Loriot Y, Fizazi K (2011) Carcinomas of an unknown primary origin–diagnosis and treatment. Nat Rev Clin Oncol 8(12):701–710. doi:10.1038/nrclinonc.2011.158

    Article  CAS  PubMed  Google Scholar 

  • Mazel M, Jacot W, Pantel K, Bartkowiak K, Topart D, Cayrefourcq L, Rossille D, Maudelonde T, Fest T, Alix-Panabieres C (2015) Frequent expression of PD-L1 on circulating breast cancer cells. Mol Oncol 9(9):1773–1782. doi:10.1016/j.molonc.2015.05.009

    Article  CAS  PubMed  Google Scholar 

  • Mehlen P, Puisieux A (2006) Metastasis: a question of life or death. Nat Rev Cancer 6(6):449–458. doi:10.1038/nrc1886

    Article  CAS  PubMed  Google Scholar 

  • 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. doi:10.1158/1078-0432.CCR-04-1110

    Article  PubMed  Google Scholar 

  • Mitchell MJ, King MR (2013) Fluid shear stress sensitizes cancer cells to receptor-mediated apoptosis via trimeric death receptors. New J Phys 15:015008. doi:10.1088/1367-2630/15/1/015008

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Miyamoto DT, Zheng Y, Wittner BS, Lee RJ, Zhu H, Broderick KT, Desai R, Fox DB, Brannigan BW, Trautwein J, Arora KS, Desai N, Dahl DM, Sequist LV, Smith MR, Kapur R, Wu CL, Shioda T, Ramaswamy S, Ting DT, Toner M, Maheswaran S, Haber DA (2015) RNA-Seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance. Science 349(6254):1351–1356. doi:10.1126/science.aab0917

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Molnar B, Ladanyi A, Tanko L, Sreter L, Tulassay Z (2001) Circulating tumor cell clusters in the peripheral blood of colorectal cancer patients. Clin Cancer Res 7(12):4080–4085

    CAS  PubMed  Google Scholar 

  • Nagrath S, Sequist LV, Maheswaran S, Bell DW, Irimia D, Ulkus L, Smith MR, Kwak EL, Digumarthy S, Muzikansky A, Ryan P, Balis UJ, Tompkins RG, Haber DA, Toner M (2007) Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 450(7173):1235–1239. doi:10.1038/nature06385

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nakazawa M, Lu C, Chen Y, Paller CJ, Carducci MA, Eisenberger MA, Luo J, Antonarakis ES (2015) Serial blood-based analysis of AR-V7 in men with advanced prostate cancer. Ann Oncol 26(9):1859–1865. doi:10.1093/annonc/mdv282

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nel I, Jehn U, Gauler T, Hoffmann AC (2014) Individual profiling of circulating tumor cell composition in patients with non-small cell lung cancer receiving platinum based treatment. Transl Lung Cancer Res 3(2):100–106. doi:10.3978/j.issn.2218-6751.2014.03.05

    CAS  PubMed  PubMed Central  Google Scholar 

  • Neves RP, Raba K, Schmidt O, Honisch E, Meier-Stiegen F, Behrens B, Mohlendick B, Fehm T, Neubauer H, Klein CA, Polzer B, Sproll C, Fischer JC, Niederacher D, Stoecklein NH (2014) Genomic high-resolution profiling of single CKpos/CD45neg flow-sorting purified circulating tumor cells from patients with metastatic breast cancer. Clin Chem 60(10):1290–1297. doi:10.1373/clinchem.2014.222331

    CAS  PubMed  Google Scholar 

  • Ozkumur E, Shah AM, Ciciliano JC, Emmink BL, Miyamoto DT, Brachtel E, Yu M, Chen PI, Morgan B, Trautwein J, Kimura A, Sengupta S, Stott SL, Karabacak NM, Barber TA, Walsh JR, Smith K, Spuhler PS, Sullivan JP, Lee RJ, Ting DT, Luo X, Shaw AT, Bardia A, Sequist LV, Louis DN, Maheswaran S, Kapur R, Haber DA, Toner M (2013) Inertial focusing for tumor antigen-dependent and -independent sorting of rare circulating tumor cells. Sci Transl Med 5(179):179ra147. doi:10.1126/scitranslmed.3005616

    Article  CAS  Google Scholar 

  • Paget S (1989) The distribution of secondary growths in cancer of the breast. 1889. Cancer Metastasis Rev 8(2):98–101

    CAS  PubMed  Google Scholar 

  • Pailler E, Adam J, Barthelemy A, Oulhen M, Auger N, Valent A, Borget I, Planchard D, Taylor M, Andre F, Soria JC, Vielh P, Besse B, Farace F (2013) Detection of circulating tumor cells harboring a unique ALK rearrangement in ALK-positive non-small-cell lung cancer. J Clin Oncol 31(18):2273–2281. doi:10.1200/JCO.2012.44.5932

    Article  PubMed  Google Scholar 

  • Pardoll DM (2012) The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 12(4):252–264. doi:10.1038/nrc3239

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Peeters DJ, Brouwer A, Van den Eynden GG, Rutten A, Onstenk W, Sieuwerts AM, Van Laere SJ, Huget P, Pauwels P, Peeters M, Vermeulen PB, Dirix LY (2015) Circulating tumour cells and lung microvascular tumour cell retention in patients with metastatic breast and cervical cancer. Cancer Lett 356(2 Pt B):872–879. doi:10.1016/j.canlet.2014.10.039

    Article  CAS  PubMed  Google Scholar 

  • Podsypanina K, Du YC, Jechlinger M, Beverly LJ, Hambardzumyan D, Varmus H (2008) Seeding and propagation of untransformed mouse mammary cells in the lung. Science 321(5897):1841–1844. doi:10.1126/science.1161621

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Postow MA, Callahan MK, Wolchok JD (2015) Immune checkpoint blockade in cancer therapy. J Clin Oncol 33(17):1974–1982. doi:10.1200/JCO.2014.59.4358

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Poveda A, Kaye SB, McCormack R, Wang S, Parekh T, Ricci D, Lebedinsky CA, Tercero JC, Zintl P, Monk BJ (2011) Circulating tumor cells predict progression free survival and overall survival in patients with relapsed/recurrent advanced ovarian cancer. Gynecol Oncol 122(3):567–572. doi:10.1016/j.ygyno.2011.05.028

    Article  PubMed  Google Scholar 

  • 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  PubMed Central  Google Scholar 

  • Qiao YY, Lin KX, Zhang Z, Zhang DJ, Shi CH, Xiong M, Qu XH, Zhao XH (2015) Monitoring disease progression and treatment efficacy with circulating tumor cells in esophageal squamous cell carcinoma: a case report. World J Gastroenterol 21(25):7921–7928. doi:10.3748/wjg.v21.i25.7921

    Article  PubMed  PubMed Central  Google Scholar 

  • Ran R, Li L, Wang M, Wang S, Zheng Z, Lin PP (2013) Determination of EGFR mutations in single cells microdissected from enriched lung tumor cells in peripheral blood. Anal Bioanal Chem 405(23):7377–7382. doi:10.1007/s00216-013-7156-y

    Article  CAS  PubMed  Google Scholar 

  • Reategui E, Aceto N, Lim EJ, Sullivan JP, Jensen AE, Zeinali M, Martel JM, Aranyosi AJ, Li W, Castleberry S, Bardia A, Sequist LV, Haber DA, Maheswaran S, Hammond PT, Toner M, Stott SL (2015) Tunable nanostructured coating for the capture and selective release of viable circulating tumor cells. Adv Mater 27(9):1593–1599. doi:10.1002/adma.201404677

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Reyes EE, VanderWeele DJ, Isikbay M, Duggan R, Campanile A, Stadler WM, Vander Griend DJ, Szmulewitz RZ (2014) Quantitative characterization of androgen receptor protein expression and cellular localization in circulating tumor cells from patients with metastatic castration-resistant prostate cancer. J Transl Med 12:313. doi:10.1186/s12967-014-0313-z

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Rossille D, Gressier M, Damotte D, Maucort-Boulch D, Pangault C, Semana G, Le Gouill S, Haioun C, Tarte K, Lamy T, Milpied N, Fest T, Groupe Ouest-Est des Leucemies et Autres Maladies du S, Groupe Ouest-Est des Leucemies et Autres Maladies du S (2014) High level of soluble programmed cell death ligand 1 in blood impacts overall survival in aggressive diffuse large B-cell lymphoma: results from a French multicenter clinical trial. Leukemia 28(12):2367–2375. doi:10.1038/leu.2014.137

    Article  CAS  PubMed  Google Scholar 

  • Sarioglu AF, Aceto N, Kojic N, Donaldson MC, Zeinali M, Hamza B, Engstrom A, Zhu H, Sundaresan TK, Miyamoto DT, Luo X, Bardia A, Wittner BS, Ramaswamy S, Shioda T, Ting DT, Stott SL, Kapur R, Maheswaran S, Haber DA, Toner M (2015) A microfluidic device for label-free, physical capture of circulating tumor cell clusters. Nat Methods 12(7):685–691. doi:10.1038/nmeth.3404

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Scher HI, Heller G, Molina A, Attard G, Danila DC, Jia X, Peng W, Sandhu SK, Olmos D, Riisnaes R, McCormack R, Burzykowski T, Kheoh T, Fleisher M, Buyse M, de Bono JS (2015) Circulating tumor cell biomarker panel as an individual-level surrogate for survival in metastatic castration-resistant prostate cancer. J Clin Oncol 33(12):1348–1355. doi:10.1200/JCO.2014.55.3487

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Seal SH (1964) A sieve for the isolation of cancer cells and other large cells from the blood. Cancer 17:637–642

    Article  CAS  PubMed  Google Scholar 

  • Siegel RL, Miller KD, Jemal A (2016) Cancer statistics, 2016. CA Cancer J Clin 66(1):7–30. doi:10.3322/caac.21332

    Article  PubMed  Google Scholar 

  • Smerage JB, Hayes DF (2008) The prognostic implications of circulating tumor cells in patients with breast cancer. Cancer Investig 26(2):109–114. doi:10.1080/07357900701784204

    Article  Google Scholar 

  • 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 32(31):3483–3489. doi:10.1200/JCO.2014.56.2561

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Smith B, Selby P, Southgate J, Pittman K, Bradley C, Blair GE (1991) Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction. Lancet 338(8777):1227–1229

    Article  CAS  PubMed  Google Scholar 

  • Stott SL, Hsu CH, Tsukrov DI, Yu M, Miyamoto DT, Waltman BA, Rothenberg SM, Shah AM, Smas ME, Korir GK, Floyd FP Jr, Gilman AJ, Lord JB, Winokur D, Springer S, Irimia D, Nagrath S, Sequist LV, Lee RJ, Isselbacher KJ, Maheswaran S, Haber DA, Toner M (2010a) Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. Proc Natl Acad Sci U S A 107(43):18392–18397. doi:10.1073/pnas.1012539107

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Stott SL, Lee RJ, Nagrath S, Yu M, Miyamoto DT, Ulkus L, Inserra EJ, Ulman M, Springer S, Nakamura Z, Moore AL, Tsukrov DI, Kempner ME, Dahl DM, Wu CL, Iafrate AJ, Smith MR, Tompkins RG, Sequist LV, Toner M, Haber DA, Maheswaran S (2010b) Isolation and characterization of circulating tumor cells from patients with localized and metastatic prostate cancer. Sci Transl Med 2(25):25ra23. doi:10.1126/scitranslmed.3000403

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Sun W, Yuan X, Tian Y, Wu H, Xu H, Hu G, Wu K (2015) Non-invasive approaches to monitor EGFR-TKI treatment in non-small-cell lung cancer. J Hematol Oncol 8:95. doi:10.1186/s13045-015-0193-6

    Article  PubMed  PubMed Central  Google Scholar 

  • Tanaka F, Yoneda K, Kondo N, Hashimoto M, Takuwa T, Matsumoto S, Okumura Y, Rahman S, Tsubota N, Tsujimura T, Kuribayashi K, Fukuoka K, Nakano T, Hasegawa S (2009) Circulating tumor cell as a diagnostic marker in primary lung cancer. Clin Cancer Res 15(22):6980–6986. doi:10.1158/1078-0432.CCR-09-1095

    Article  CAS  PubMed  Google Scholar 

  • Tien YW, Kuo HC, Ho BI, Chang MC, Chang YT, Cheng MF, Chen HL, Liang TY, Wang CF, Huang CY, Shew JY, Chang YC, Lee EY, Lee WH (2016) A high circulating tumor cell count in portal vein predicts liver metastasis from periampullary or pancreatic cancer: a high portal venous CTC count predicts liver metastases. Medicine 95(16):e3407. doi:10.1097/MD.0000000000003407

    Article  PubMed  PubMed Central  Google Scholar 

  • Tsao DA, Yang MJ, Chang HJ, Yen LC, Chiu HH, Hsueh EJ, Chen YF, Lin SR (2010) A fast and convenient new technique to detect the therapeutic target, K-ras mutant, from peripheral blood in non-small cell lung cancer patients. Lung Cancer 68(1):51–57. doi:10.1016/j.lungcan.2009.05.023

    Article  PubMed  Google Scholar 

  • van de Wouw AJ, Janssen-Heijnen ML, Coebergh JW, Hillen HF (2002) Epidemiology of unknown primary tumours; incidence and population-based survival of 1285 patients in Southeast Netherlands, 1984–1992. Eur J Cancer 38(3):409–413

    Article  PubMed  Google Scholar 

  • Vona G, Sabile A, Louha M, Sitruk V, Romana S, Schutze K, Capron F, Franco D, Pazzagli M, Vekemans M, Lacour B, Brechot C, Paterlini-Brechot P (2000) Isolation by size of epithelial tumor cells : a new method for the immunomorphological and molecular characterization of circulatingtumor cells. Am J Pathol 156(1):57–63. doi:10.1016/S0002-9440(10)64706-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang ZP, Eisenberger MA, Carducci MA, Partin AW, Scher HI, Ts’o PO (2000) Identification and characterization of circulating prostate carcinoma cells. Cancer 88(12):2787–2795

    Article  CAS  PubMed  Google Scholar 

  • Welinder C, Jansson B, Lindell G, Wenner J (2015) Cytokeratin 20 improves the detection of circulating tumor cells in patients with colorectal cancer. Cancer Lett 358(1):43–46. doi:10.1016/j.canlet.2014.12.024

    Article  CAS  PubMed  Google Scholar 

  • Williams ES, Rodriguez-Bravo V, Chippada-Venkata U, De Ia I-VJ, Gong Y, Galsky M, Oh W, Cordon-Cardo C, Domingo-Domenech J (2015) Generation of prostate cancer patient derived xenograft models from circulating tumor cells. J Vis Exp 105:53182. doi:10.3791/53182

    Google Scholar 

  • 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  PubMed Central  Google Scholar 

  • Zheng X, Carstens JL, Kim J, Scheible M, Kaye J, Sugimoto H, Wu CC, LeBleu VS, Kalluri R (2015) Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature 527(7579):525–530. doi:10.1038/nature16064

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhou MD, Hao S, Williams AJ, Harouaka RA, Schrand B, Rawal S, Ao Z, Brenneman R, Gilboa E, Lu B, Wang S, Zhu J, Datar R, Cote R, Tai YC, Zheng SY (2014) Separable bilayer microfiltration device for viable label-free enrichment of circulating tumour cells. Sci Rep 4:7392. doi:10.1038/srep07392

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Genomics Research Center, Academia Sinica, 128, Sec. 2, Academic Rd., Nankang, Taipei, 115, Taiwan

    Jia-Yang Chen & Ying-Chih Chang

Authors
  1. Jia-Yang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ying-Chih Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ying-Chih Chang .

Editor information

Editors and Affiliations

  1. University of California San Francisco, San Francisco, California, USA

    Mark Jesus M. Magbanua

  2. University of California San Francisco, San Francisco, California, USA

    John W. Park

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer International Publishing AG

About this chapter

Cite this chapter

Chen, JY., Chang, YC. (2017). Strategies for Isolation and Molecular Profiling of Circulating Tumor Cells. In: Magbanua, M., Park, J. (eds) Isolation and Molecular Characterization of Circulating Tumor Cells. Advances in Experimental Medicine and Biology, vol 994. Springer, Cham. https://doi.org/10.1007/978-3-319-55947-6_2

Download citation

Publish with us

Policies and ethics

Search

Navigation