[Frontiers in Bioscience, Landmark, 25, 874-892, Jan 1, 2020]

Applications of RNA characterisation in circulating tumour cells

Sara Hassan1-3, Tony Blick1-3, Elizabeth D. Williams1,2,4, Erik W. Thompson1-3

1School of Biomedical Sciences and Institute of Health and Biomedical Innovation (IHBI), Faculty of Health, Queensland University of Technology (QUT), Brisbane, Australia, 2Translational Research Institute, Brisbane, Australia, 3Invasion and Metastasis Unit, IHBI, QUT, Brisbane, Australia, 4Australian Prostate Cancer Research Centre – Queensland (APCRC-Q) and Queensland Bladder Cancer Initiative (QBCI), Brisbane, Australia


1. Abstract
2. Introduction
3. Beyond enumeration: RNA analysis of CTCs
4. Methodologies for RNA studies
    4.1. RT-qPCR
    4.2. RNA-ISH
    4.3. RNAseq
5. CTC-RNA signatures
6. Concluding remarks
7. References


Circulating tumour cells (CTCs) are shed into the bloodstream from both primary and secondary tumours and provide a non-invasive means to study tumor progression and response to treatment. Assessment of ribonucleic acid (RNA) and monitoring dynamic changes in gene expression profiles of CTCs extends their clinical and prognostic power and establish their role in guiding treatment. Among these methods, droplet digital (RT-ddPCR) technique provides a high sensitivity and detectibility of CTCs. RNA-sequencing (RNAseq) is the most comprehensive method, that would allow the simultaneous measurement of a large number of genes and theoretically the whole transcriptome. Since CTCs are heterogeneous in nature, single cell RNAseq methods are very valuable in assessing population dynamics and functional states of CTCs. While RNA in situ hybridization (RNA-ISH) is used relatively less frequently, it also allows for the assessment of expression of multiple genes within individual CTCs. Epithelial to Mesenchymal Transition (EMT) or Plasticity (EMP) is a major contributor to metastasis, providing a mechanism to allow cells to become migratory and invasive, and to survive in the bloodstream. Monitoring CTCs undergoing EMT may lead to improvement in their prognostic and predictive power. Here, we review various RNA analysis of CTCs and those that undergo EMT and their application in diagnosis, prognosis and treatment of cancers.


1. N. Kanwar and S. J. Done: Molecular Profiling and Significance of Circulating Tumor Cell Based Genetic Signatures. In: Isolation and Molecular Characterization of Circulating Tumor Cells. Springer, (2017)
DOI: 10.1007/978-3-319-55947-6_8

2. M.-x. Cao, Y.-p. Jiang, Y.-l. Tang and X.-h. Liang: The crosstalk between lncRNA and microRNA in cancer metastasis: orchestrating the epithelial-mesenchymal plasticity. Oncotarget, 8(7), 12472 (2017)
DOI: 10.18632/oncotarget.13957

3. M. Luo, M. Brooks and M. S Wicha: Epithelial-mesenchymal plasticity of breast cancer stem cells: implications for metastasis and therapeutic resistance. Current pharmaceutical design, 21(10), 1301-1310 (2015)
DOI: 10.2174/1381612821666141211120604
PMid:25506895 PMCid:PMC4498673

4. M. Yang, B. Ma, H. Shao, A. M. Clark and A. Wells: Macrophage phenotypic subtypes diametrically regulate epithelial-mesenchymal plasticity in breast cancer cells. BMC cancer, 16(1), 419 (2016)
DOI: 10.1186/s12885-016-2411-1
PMid:27387344 PMCid:PMC4936312

5. T. Ashworth: 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 (1869)

6. J.-M. Hou, M. G. Krebs, L. Lancashire, R. Sloane, A. Backen, R. K. Swain, L. Priest, A. Greystoke, C. Zhou and K. Morris: 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 (2012)
DOI: 10.1200/JCO.2010.33.3716

7. M. Cristofanilli, G. T. Budd, M. J. Ellis, A. Stopeck, J. Matera, M. C. Miller, J. M. Reuben, G. V. Doyle, W. J. Allard, L. W. M. M. Terstappen and D. F. Hayes: Circulating Tumor Cells, Disease Progression, and Survival in Metastatic Breast Cancer. New England Journal of Medicine, 351(8), 781-791 (2004)
DOI: 10.1056/NEJMoa040766

8. J. S. de Bono, H. I. Scher, R. B. Montgomery, C. Parker, M. C. Miller, H. Tissing, G. V. Doyle, L. W. W. M. Terstappen, K. J. Pienta and D. Raghavan: Circulating Tumor Cells Predict Survival Benefit from Treatment in Metastatic Castration-Resistant Prostate Cancer. Clinical Cancer Research, 14(19), 6302-6309 (2008)
DOI: 10.1158/1078-0432.CCR-08-0872

9. S. J. Cohen, C. J. Punt, N. Iannotti, B. H. Saidman, K. D. Sabbath, N. Y. Gabrail, J. Picus, M. Morse, E. Mitchell and M. C. Miller: Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer. Journal of clinical oncology, 26(19), 3213-3221 (2008)
DOI: 10.1200/JCO.2007.15.8923

10. R. Lampignano, H. Schneck, M. Neumann, T. Fehm and H. Neubauer: Enrichment, Isolation and Molecular Characterization of EpCAM-Negative Circulating Tumor Cells. In: Isolation and Molecular Characterization of Circulating Tumor Cells. Springer, (2017)
DOI: 10.1007/978-3-319-55947-6_10

11. N. Aceto, A. Bardia, D. T. Miyamoto, M. C. Donaldson, B. S. Wittner, J. A. Spencer, M. Yu, A. Pely, A. Engstrom, H. Zhu, B. W. Brannigan, R. Kapur, S. L. Stott, T. Shioda, S. L. Ramaswamy, Charles P., M. Toner, D. A. Haber and S. Maheswaran: Circulating Tumor Cell Clusters Are Oligoclonal Precursors of Breast Cancer Metastasis. Cell, 158(5), 1110-1122 (2014)
DOI: 10.1016/j.cell.2014.07.013
PMid:25171411 PMCid:PMC4149753

12. L. Allen Liotta, J. Kleinerman and G. M. Saldel: The significance of hematogenous tumor cell clumps in the metastatic process. Cancer research, 36(3), 889-894 (1976)

13. E. Hwang, J. H. Uh, H. S. Lee, C. H. Lee, S. J. Lee, S. H. Ahn, B. H. Son, J. W. Lee, J. H. Yu and N. J. Kwon: Cancer gene panel analysis of cultured circulating tumor cells and primary tumor tissue from patients with breast cancer. Oncology Letters, 13(6), 4627-4632 (2017)
DOI: 10.3892/ol.2017.6077
PMid:28599463 PMCid:PMC5452992

14. K. Pantel, R. H. Brakenhoff and B. Brandt: Detection, clinical relevance and specific biological properties of disseminating tumour cells. Nature reviews. Cancer, 8(5), 329 (2008)
DOI: 10.1038/nrc2375

15. K. Pantel and C. Alix-Panabières: Circulating tumour cells in cancer patients: challenges and perspectives. Trends in molecular medicine, 16(9), 398-406 (2010)
DOI: 10.1016/j.molmed.2010.07.001

16. M. Yu, S. Stott, M. Toner, S. Maheswaran and D. A. Haber: Circulating tumor cells: approaches to isolation and characterization. The Journal of cell biology, 192(3), 373-382 (2011)
DOI: 10.1083/jcb.201010021
PMid:21300848 PMCid:PMC3101098

17. L. Khoja, A. Backen, R. Sloane, L. Menasce, D. Ryder, M. Krebs, R. Board, G. Clack, A. Hughes and F. Blackhall: A pilot study to explore circulating tumour cells in pancreatic cancer as a novel biomarker. British journal of cancer, 106(3), 508 (2012)
DOI: 10.1038/bjc.2011.545
PMid:22187035 PMCid:PMC3273340

18. P. Paterlini-Brechot and N. L. Benali: Circulating tumor cells (CTC) detection: clinical impact and future directions. Cancer letters, 253(2), 180-204 (2007)
DOI: 10.1016/j.canlet.2006.12.014

19. T. M. Gorges and K. Pantel: Circulating tumor cells as therapy-related biomarkers in cancer patients. Cancer Immunology, Immunotherapy, 62(5), 931-939 (2013)
DOI: 10.1007/s00262-012-1387-1

20. N. J. Meropol, S. J. Cohen, G. V. Doyle, H. Tissing, L. W. M. M. Terstappen, M. C. Miller, C. J. A. Punt, N. Iannotti, B. H. Saidman, K. D. Sabbath, N. Y. Gabrail, J. Picus, M. A. Morse and E. Mitchell: Prognostic significance of circulating tumor cells in patients with metastatic colorectal cancer. Annals of Oncology, 20(7), 1223-1229 (2009)
DOI: 10.1093/annonc/mdn786

21. N. Bednarz-Knoll, C. Alix-Panabières and K. Pantel: Plasticity of disseminating cancer cells in patients with epithelial malignancies. Cancer and Metastasis Reviews, 31(3-4), 673-687 (2012)
DOI: 10.1007/s10555-012-9370-z

22. C. Alix-Panabières, S. Mader and K. Pantel: Epithelial-mesenchymal plasticity in circulating tumor cells. Journal of Molecular Medicine, 95(2), 133-142 (2017)
DOI: 10.1007/s00109-016-1500-6

23. M.-E. Francart, J. Lambert, A. M. Vanwynsberghe, E. W. Thompson, M. Bourcy, M. Polette and C. Gilles: Epithelial-mesenchymal plasticity and circulating tumor cells: Travel companions to metastases. Developmental Dynamics, 247(3), 432-450 (2018)
DOI: 10.1002/dvdy.24506

24. L. Pereira, J. M. Mariadason, R. D. Hannan and A. S. Dhillon: Implications of epithelial-mesenchymal plasticity for heterogeneity in colorectal cancer. Frontiers in oncology, 5 (2015)
DOI: 10.3389/fonc.2015.00013
PMid:25699236 PMCid:PMC4313606

25. D. J. Junk, R. Cipriano, B. L. Bryson, H. L. Gilmore and M. W. Jackson: Tumor microenvironmental signaling elicits epithelial-mesenchymal plasticity through cooperation with transforming genetic events. Neoplasia, 15(9), 1100-1109 (2013)
DOI: 10.1593/neo.131114
PMid:24027434 PMCid:PMC3769888

26. G. Hamilton and B. Rath: Mesenchymal-Epithelial Transition and Circulating Tumor Cells in Small Cell Lung Cancer. In: Isolation and Molecular Characterization of Circulating Tumor Cells. Springer, (2017)
DOI: 10.1007/978-3-319-55947-6_12

27. M. C. Karlsson, S. F. Gonzalez, J. Welin and J. Fuxe: Epithelial-Mesenchymal transition in cancer metastasis through the lymphatic system. Molecular Oncology (2017)
DOI: 10.1002/1878-0261.12092
PMid:28590032 PMCid:PMC5496496

28. Y. Peng, Y. Li, L. L. Gellert, X. Zou, J. Wang, B. Singh, R. Xu, L. Chiriboga, G. Daniels and R. Pan: Androgen receptor coactivator p44/Mep50 in breast cancer growth and invasion. Journal of cellular and molecular medicine, 14(12), 2780-2789 (2010)
DOI: 10.1111/j.1582-4934.2009.00936.x
PMid:19840198 PMCid:PMC3822728

29. A. Sistigu, F. Di Modugno, G. Manic and P. Nisticò: Deciphering the loop of epithelial-mesenchymal transition, inflammatory cytokines and cancer immunoediting. Cytokine & Growth Factor Reviews (2017)
DOI: 10.1016/j.cytogfr.2017.05.008

30. Luo, L. Wang, P. Wu, W. Gong, W. Chen, H. Zhao and Z. Zheng: Downregulated vimentin and upregulated E-cadherin in T1 stage non-small-cell lung cancer: does it suggest a mesenchymal-epithelial transition? Neoplasma (2017)
DOI: 10.4149/neo_2017_506

31. J. Cheng, B. Qin, B. Liu, T. Huang, Y. Li and L. Ma: Maternal embryonic leucine zipper kinase inhibits epithelial-mesenchymal transition by regulating transforming growth factor-β signaling. Oncology Letters, 13(6), 4794-4798 (2017)
DOI: 10.3892/ol.2017.6081
PMid:28588728 PMCid:PMC5452933

32. K. R. Fischer, A. Durrans, S. Lee, J. Sheng, F. Li, S. T. C. Wong, H. Choi, T. El Rayes, S. Ryu, J. Troeger, R. F. Schwabe, L. T. Vahdat, N. K. Altorki, V. Mittal and D. Gao: Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance. Nature, 527, 472 (2015)
DOI: 10.1038/nature15748
PMid:26560033 PMCid:PMC4662610

33. X. Ye, T. Brabletz, Y. Kang, G. D. Longmore, M. A. Nieto, B. Z. Stanger, J. Yang and R. A. Weinberg: Upholding a role for EMT in breast cancer metastasis. Nature, 547, E1 (2017)
DOI: 10.1038/nature22816
PMid:28682326 PMCid:PMC6283276

34. R. Kalluri and R. A. Weinberg: The basics of epithelial-mesenchymal transition. The Journal of clinical investigation, 119(6), 1420-1428 (2009)
DOI: 10.1172/JCI39104
PMid:19487818 PMCid:PMC2689101

35. J. Cursons, K.-J. Leuchowius, M. Waltham, E. Tomaskovic-Crook, M. Foroutan, C. P. Bracken, A. Redfern, E. J. Crampin, I. Street and M. J. Davis: Stimulus-dependent differences in signalling regulate epithelial-mesenchymal plasticity and change the effects of drugs in breast cancer cell lines. Cell Communication and Signaling, 13(1), 26 (2015)
DOI: 10.1186/s12964-015-0106-x
PMid:25975820 PMCid:PMC4432969

36. A. Sulaiman, Z. Yao and L. Wang: Re-evaluating the role of epithelial-mesenchymal-transition in cancer progression. Journal of biomedical research (2017)

37. E. W. Thompson and S. H. Nagaraj: Transition states that allow cancer to spread. In: Nature Publishing Group, (2018)
DOI: 10.1038/d41586-018-04403-x

38. M. Foroutan, D. D. Bhuva, R. Lyu, K. Horan, J. Cursons and M. J. Davis: Single sample scoring of molecular phenotypes. BMC Bioinformatics, 19(1), 404 (2018)
DOI: 10.1186/s12859-018-2435-4
PMid:30400809 PMCid:PMC6219008

39. D. Hanahan and R. A. Weinberg: Hallmarks of cancer: the next generation. cell, 144(5), 646-674 (2011)
DOI: 10.1016/j.cell.2011.02.013

40. M. Yu, A. Bardia, B. S. Wittner, S. L. Stott, M. E. Smas, D. T. Ting, S. J. Isakoff, J. C. Ciciliano, M. N. Wells and A. M. Shah: Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition. science, 339(6119), 580-584 (2013)
DOI: 10.1126/science.1228522
PMid:23372014 PMCid:PMC3760262

41. I. Pastushenko, A. Brisebarre, A. Sifrim, M. Fioramonti, T. Revenco, S. Boumahdi, A. Van Keymeulen, D. Brown, V. Moers, S. Lemaire, S. De Clercq, E. Minguijón, C. Balsat, Y. Sokolow, C. Dubois, F. De Cock, S. Scozzaro, F. Sopena, A. Lanas, N. D'Haene, I. Salmon, J.-C. Marine, T. Voet, P. A. Sotiropoulou and C. Blanpain: Identification of the tumour transition states occurring during EMT. Nature, 556(7702), 463-468 (2018)
DOI: 10.1038/s41586-018-0040-3

42. A. Markou, M. Lazaridou, P. Paraskevopoulos, S. Chen, M. Świerczewska, J. Budna, A. Kuske, T. M. Gorges, S. A. Joosse and T. Kroneis: Multiplex gene expression profiling of in vivo isolated circulating tumor cells in high-risk prostate cancer patients. Clinical chemistry, 64(2), 297-306 (2018)
DOI: 10.1373/clinchem.2017.275503

43. Yang, X. Du, G. Wang, Y. Sun, K. Chen, X. Zhu, A. J. Lazar, K. K. Hunt, R. E. Pollock and W. Zhang: Mesenchymal to epithelial transition in sarcomas. European journal of cancer, 50(3), 593-601 (2014)
DOI: 10.1016/j.ejca.2013.11.006

44. C. Li, M. Zhu, X. Lou, C. Liu, H. Chen, X. Lin, W. Ji, Z. Li and C. Su: Transcriptional factor OCT4 promotes esophageal cancer metastasis by inducing epithelial-mesenchymal transition through VEGF-C/VEGFR-3 signaling pathway. Oncotarget (2017)

45. S.-j. Zhou, F.-y. Liu, A.-h. Zhang, H.-f. Liang, Y. Wang, R. Ma, Y.-h. Jiang and N.-f. Sun: MicroRNA-199b-5p attenuates TGF-β1-induced epithelial-mesenchymal transition in hepatocellular carcinoma. British Journal of Cancer (2017)

46. A. Giordano, H. Gao, S. Anfossi, E. Cohen, M. Mego, B.-N. Lee, S. Tin, M. De Laurentiis, C. A. Parker and R. H. Alvarez: Epithelial-mesenchymal transition and stem cell markers in patients with HER2-positive metastatic breast cancer. Molecular cancer therapeutics, 11(11), 2526-2534 (2012)
DOI: 10.1158/1535-7163.MCT-12-0460
PMid:22973057 PMCid:PMC3500676

47. M. Mego, Z. Cierna, P. Janega, M. Karaba, G. Minarik, J. Benca, T. Sedlácková, G. Sieberova, P. Gronesova and D. Manasova: Relationship between circulating tumor cells and epithelial to mesenchymal transition in early breast cancer. BMC cancer, 15(1), 533 (2015)
DOI: 10.1186/s12885-015-1548-7
PMid:26194471 PMCid:PMC4509773

48. S. de Wit, M. Manicone, E. Rossi, R. Lampignano, L. Yang, B. Zill, A. Rengel-Puertas, M. Ouhlen, M. Crespo and A. M. S. Berghuis: EpCAMhigh and EpCAMlow circulating tumor cells in metastatic prostate and breast cancer patients. Oncotarget, 9(86), 35705 (2018)
DOI: 10.18632/oncotarget.26298
PMid:30479699 PMCid:PMC6235023

49. K. Chang, Y.-Y. Kong, B. Dai, D.-W. Ye, Y.-Y. Qu, Y. Wang, Z.-W. Jia and G.-X. Li: Combination of circulating tumor cell enumeration and tumor marker detection in predicting prognosis and treatment effect in metastatic castration-resistant prostate cancer. Oncotarget, 6(39), 41825 (2015)
DOI: 10.18632/oncotarget.6167
PMid:26497689 PMCid:PMC4747191

50. L. León-Mateos, H. Casas, A. Abalo, M. Vieito, M. Abreu, U. Anido, A. Gómez-Tato, R. López, M. Abal and L. Muinelo-Romay: Improving circulating tumor cells enumeration and characterization to predict outcome in first line chemotherapy mCRPC patients. Oncotarget (2017)
DOI: 10.18632/oncotarget.18025
PMid:28903376 PMCid:PMC5589615

51. M. Gogenur, T. Hillig and I. Gogenur: CytoTrack Analysis Reveals Low Presence of Circulating Tumor Cells in the Perioperative Period in Patients with Non-metastatic Colorectal Cancer. Anticancer Research, 37(6), 3099-3103 (2017)
DOI: 10.21873/anticanres.11666

52. T. Yokobori, H. Iinuma, T. Shimamura, S. Imoto, K. Sugimachi, H. Ishii, M. Iwatsuki, D. Ota, M. Ohkuma, T. Iwaya, N. Nishida, R. Kogo, T. Sudo, F. Tanaka, K. Shibata, H. Toh, T. Sato, G. F. Barnard, T. Fukagawa, S. Yamamoto, H. Nakanishi, S. Sasaki, S. Miyano, T. Watanabe, H. Kuwano, K. Mimori, K. Pantel and M. Mori: Plastin3 Is a Novel Marker for Circulating Tumor Cells Undergoing the Epithelial-Mesenchymal Transition and Is Associated with Colorectal Cancer Prognosis. Cancer Research, 73(7), 2059-2069 (2013)
DOI: 10.1158/0008-5472.CAN-12-0326

53. M. Ø. Agerbæk, S. R. Bang-Christensen, M.-H. Yang, T. M. Clausen, M. A. Pereira, S. Sharma, S. B. Ditlev, M. A. Nielsen, S. Choudhary, T. Gustavsson, P. H. Sorensen, T. Meyer, D. Propper, J. Shamash, T. G. Theander, A. Aicher, M. Daugaard, C. Heeschen and A. Salanti: The VAR2CSA malaria protein efficiently retrieves circulating tumor cells in an EpCAM-independent manner. Nature Communications, 9(1), 3279 (2018)
DOI: 10.1038/s41467-018-05793-2
PMid:30115931 PMCid:PMC6095877

54. T. T. Kwan, A. Bardia, L. M. Spring, A. Giobbie-Hurder, M. Kalinich, T. Dubash, T. Sundaresan, X. Hong, J. A. LiCausi and U. Ho: A digital RNA signature of circulating Tumor cells predicting early therapeutic response in localized and metastatic breast cancer. Cancer discovery, 8(10), 1286-1299 (2018)
DOI: 10.1158/2159-8290.CD-18-0432

55. X. Hong, R. J. Sullivan, M. Kalinich, T. T. Kwan, A. Giobbie-Hurder, S. Pan, J. A. LiCausi, J. D. Milner, L. T. Nieman and B. S. Wittner: Molecular signatures of circulating melanoma cells for monitoring early response to immune checkpoint therapy. Proceedings of the National Academy of Sciences, 115(10), 2467-2472 (2018)
DOI: 10.1073/pnas.1719264115
PMid:29453278 PMCid:PMC5877960

56. D. T. Miyamoto, R. J. Lee, M. Kalinich, J. A. LiCausi, Y. Zheng, T. Chen, J. D. Milner, E. Emmons, U. Ho and K. Broderick: An RNA-based digital circulating tumor cell signature is predictive of drug response and early dissemination in prostate cancer. Cancer discovery, 8(3), 288-303 (2018)
DOI: 10.1158/2159-8290.CD-16-1406
PMid:29301747 PMCid:PMC6342192

57. M. Kalinich, I. Bhan, T. T. Kwan, D. T. Miyamoto, S. Javaid, J. A. LiCausi, J. D. Milner, X. Hong, L. Goyal and S. Sil: An RNA-based signature enables high specificity detection of circulating tumor cells in hepatocellular carcinoma. Proceedings of the National Academy of Sciences, 114(5), 1123-1128 (2017)
DOI: 10.1073/pnas.1617032114
PMid:28096363 PMCid:PMC5293050

58. A. Markiewicz, J. Topa, A. Nagel, J. Skokowski, B. Seroczynska, T. Stokowy, M. Welnicka-Jaskiewicz and A. J. Zaczek: Spectrum of Epithelial-Mesenchymal Transition Phenotypes in Circulating Tumour Cells from Early Breast Cancer Patients. Cancers, 11(1), 59 (2019)
DOI: 10.3390/cancers11010059
PMid:30634453 PMCid:PMC6356662

59. A. A. Powell, A. H. Talasaz, H. Zhang, M. A. Coram, A. Reddy, G. Deng, M. L. Telli, R. H. Advani, R. W. Carlson and J. A. Mollick: Single cell profiling of circulating tumor cells: transcriptional heterogeneity and diversity from breast cancer cell lines. PloS one, 7(5), e33788 (2012)
DOI: 10.1371/journal.pone.0033788
PMid:22586443 PMCid:PMC3346739

60. A. Strati, A. Markou, C. Parisi, E. Politaki, D. Mavroudis, V. Georgoulias and E. Lianidou: Gene expression profile of circulating tumor cells in breast cancer by RT-qPCR. BMC cancer, 11, 422-422 (2011)
DOI: 10.1186/1471-2407-11-422
PMid:21967632 PMCid:PMC3224356

61. W. Onstenk, A. M. Sieuwerts, B. Mostert, Z. Lalmahomed, J. B. Bolt-de Vries, A. van Galen, M. Smid, J. Kraan, M. Van, V. de Weerd, R. Ramírez-Moreno, K. Biermann, C. Verhoef, D. J. Grünhagen, J. N. M. Ijzermans, J. W. Gratama, J. W. M. Martens, J. A. Foekens and S. Sleijfer: Molecular characteristics of circulating tumor cells resemble the liver metastasis more closely than the primary tumor in metastatic colorectal cancer. Oncotarget, 7(37), 59058-59069 (2016)
DOI: 10.18632/oncotarget.10175
PMid:27340863 PMCid:PMC5312295

62. M. Yu, D. T. Ting, S. L. Stott, B. S. Wittner, F. Ozsolak, S. Paul, J. C. Ciciliano, M. E. Smas, D. Winokur and A. J. Gilman: RNA sequencing of pancreatic circulating tumour cells implicates WNT signalling in metastasis. Nature, 487(7408), 510 (2012)
DOI: 10.1038/nature11217
PMid:22763454 PMCid:PMC3408856

63. D. T. Miyamoto, Y. Zheng, B. S. Wittner, R. J. Lee, H. Zhu, K. T. Broderick, R. Desai, D. B. Fox, B. W. Brannigan and J. Trautwein: RNA-Seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance. Science, 349(6254), 1351-1356 (2015)
DOI: 10.1126/science.aab0917
PMid:26383955 PMCid:PMC4872391

64. C. L. Chen, D. Mahalingam, P. Osmulski, R. R. Jadhav, C. M. Wang, R. J. Leach, T. C. Chang, S. D. Weitman, A. P. Kumar and L. Sun: Single-cell analysis of circulating tumor cells identifies cumulative expression patterns of EMT-related genes in metastatic prostate cancer. The Prostate, 73(8), 813-826 (2013)
DOI: 10.1002/pros.22625
PMid:23280481 PMCid:PMC4882087

65. S. C. Taylor, G. Laperriere and H. Germain: Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Scientific reports, 7(1), 2409 (2017)
DOI: 10.1038/s41598-017-02217-x
PMid:28546538 PMCid:PMC5445070

66. U. Andergassen, A. Kölbl, S. Hutter, K. Friese and U. Jeschke: Detection of circulating tumour cells from blood of breast cancer patients via RT-qPCR. Cancers, 5(4), 1212-1220 (2013)
DOI: 10.3390/cancers5041212
PMid:24202442 PMCid:PMC3875936

67. A. M. Sieuwerts, J. Kraan, J. Bolt-de Vries, P. van der Spoel, B. Mostert, J. W. Martens, J.-W. Gratama, S. Sleijfer and J. A. Foekens: Molecular characterization of circulating tumor cells in large quantities of contaminating leukocytes by a multiplex real-time PCR. Breast cancer research and treatment, 118(3), 455 (2009)
DOI: 10.1007/s10549-008-0290-0

68. R. V. L. Long Jin: In situ Hybridization: Methods and Applications. Journal of Clinical Laboratory Analysis 11, 2-9 (1997)
DOI: 10.1002/(SICI)1098-2825(1997)11:1<2::AID-JCLA2>3.0.CO;2-F

69. R. Payne, F. Wang, N. Su, J. Krell, A. Zebrowski, E. Yagüe, X. Ma, Y. Luo and R. Coombes: Viable circulating tumour cell detection using multiplex RNA in situ hybridisation predicts progression-free survival in metastatic breast cancer patients. British journal of cancer, 106(11), 1790 (2012)
DOI: 10.1038/bjc.2012.137
PMid:22538972 PMCid:PMC3364118

70. B. L. Khoo, S. C. Lee, P. Kumar, T. Z. Tan, M. E. Warkiani, S. G. Ow, S. Nandi, C. T. Lim and J. P. Thiery: Short-term expansion of breast circulating cancer cells predicts response to anti-cancer therapy. Oncotarget, 6(17), 15578 (2015)
DOI: 10.18632/oncotarget.3903
PMid:26008969 PMCid:PMC4558172

71. D. Ramsköld, S. Luo, Y.-C. Wang, R. Li, Q. Deng, O. R. Faridani, G. A. Daniels, I. Khrebtukova, J. F. Loring and L. C. Laurent: Full-Length mRNA-Seq from single cell levels of RNA and individual circulating tumor cells. Nature biotechnology, 30(8), 777 (2012)
DOI: 10.1038/nbt.2282
PMid:22820318 PMCid:PMC3467340

72. D. T. Ting, B. S. Wittner, M. Ligorio, N. V. Jordan, A. M. Shah, D. T. Miyamoto, N. Aceto, F. Bersani, B. W. Brannigan and K. Xega: Single-cell RNA sequencing identifies extracellular matrix gene expression by pancreatic circulating tumor cells. Cell reports, 8(6), 1905-1918 (2014)
DOI: 10.1016/j.celrep.2014.08.029
PMid:25242334 PMCid:PMC4230325

73. J. George, J. S. Lim, S. J. Jang, Y. Cun, L. Ozretić, G. Kong, F. Leenders, X. Lu, L. Fernández-Cuesta and G. Bosco: Comprehensive genomic profiles of small cell lung cancer. Nature, 524(7563), 47 (2015)
DOI: 10.1038/nature14664
PMid:26168399 PMCid:PMC4861069

74. F. Castro-Giner, M. C. Scheidmann and N. Aceto: Beyond Enumeration: Functional and Computational Analysis of Circulating Tumor Cells to Investigate Cancer Metastasis. Frontiers in Medicine, 5, 34 (2018)
DOI: 10.3389/fmed.2018.00034
PMid:29520361 PMCid:PMC5827555

76. Y. Li, S. Wu and F. Bai: Molecular characterization of circulating tumor cells—from bench to bedside. In: Seminars in cell & developmental biology. Elsevier, (2017)

Abbreviations AR: androgen receptor cDNA: complementary DNA CTC: circulating tumour cell ctDNA: circulating tumour DNA EGFR: epidermal growth factor receptor EMP: epithelial mesenchymal plasticity EMT: epithelial mesenchymal transition ER: oestrogen receptor HCC: hepatocellular carcinoma HER2: human epidermal growth factor receptor 2 ICC: immunocytochemistry MET: mesenchymal epithelial transition NHV: normal healthy volunteer OS: overall survival PFS: progression-free survival PSA: prostate specific antigen RNA: ribonucleic acid RNA-ISH: RNA- in situ hybridisation RNAseq: RNA sequencing RT-ddPCR: reverse transcriptase-droplet digital polymerase chain reaction RT-qPCR: reverse transcriptase-quantitative polymerase chain reaction SNP: single nucleotide polymorphism

Key Words: CTC, Metastasis, EMT, Tumour Heterogeneity, RNA analysis, CTC score, Review

Send correspondence to: Sara Hassan, School of Biomedical Sciences and Institute of Health & Biomedical Innovation (IHBI), Faculty of Health, Queensland University of Technology (QUT), Brisbane, Australia, Tel: 61 424500957, E-mail: sara.hassan@hdr.qut.edu.au