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Tumor Biology

, Volume 37, Issue 7, pp 9565–9578 | Cite as

Contribution of in vitro comparison of colorectal carcinoma cells from primary and metastatic lesions to elucidation of mechanisms of tumor progression and response to anticancer therapy

  • Lukáš Krbal
  • Veronika Hanušová
  • Jiří Soukup
  • Stanislav John
  • Petra Matoušková
  • Aleš Ryška
Original Article

Abstract

Colorectal cancer has been a leading cause of cancer-related morbidity and mortality. For the research and individualization of therapy, primary cell lines of the colorectal cancer appear to be still an invaluable tool. We evaluated the differences in metastatic potential between four isolated primary colon cancer cells and cells derived from their lymph node metastasis. These results were compared with correspond immortalized cells—SW480 and SW620, respectively. The ability to migrate was tested using real-time measurement in xCELLigence system. Expressions of molecules involved in adhesion and invasion processes were examined using RT-PCR and western blot analysis. Furthermore, impact of cytotoxic effect of selected chemotherapeutics (irinotecan, oxaliplatin) and biological therapy (bevacizumab, cetuximab, panitumumab) was assessed by the WST assay. As expected, cell lines derived from lymph node migrated more aggressively and higher expression of adhesion molecules ICAM-1, EpCAM, and N-cadherin was detected. The expression of MMP-2 and -9 was elevated, on the other hand, in cell lines derived from primary tumor cancer cells as well as the expression of miR-21, miR-29a, and miR-200a. The most pronounced cytotoxic effect has been recorded with oxaliplatin and irinotecan (IC50 = 48.23 resp. 0.11 μg/ml), especially in cells originating from lymph node metastases. In total, comparison of isolated cell lines and immortalized cell lines has shown many similarities, as well as several differences. Adhesion/invasion molecules and several miRNAs, which play an important role in tumor development and the invasive and migratory behavior, could be a useful therapeutic target in malignant colorectal cancer.

Keywords

Colorectal carcinoma Lymph node metastasis Tumor progression Anticancer therapy microRNA 

Abbreviations

VEGF

Vascular endothelial growth factor

EGFR

Epidermal growth factor receptor

miR

microRNA

MMP

Matrix metalloproteinases

EpCAM

Epithelial cell adhesion molecules

ICAM-1

Intercellular adhesion molecule 1

FBS

Fetal bovine serum

Notes

Acknowledgments

The authors would like to thank Svetlana Kopecka for her laboratory support in preparation of cell lines. This work was supported by Ministry of Health—Czech Republic, project IGA No. NT14150-3/2013.

Compliance with ethical standards

Conflict of interest

None.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

References

  1. 1.
    Gastrointestinal Tumor Study Group. Adjuvant therapy of colon cancer—results of a prospectively randomized trial. N Engl J Med. 1984;310(12):737–43. doi: 10.1056/NEJM198403223101201.CrossRefGoogle Scholar
  2. 2.
    Jass JR, Love SB, Northover JMA. A new prognostic classification of rectal cancer. Lancet. 1987;329(8545):1303–6. doi: 10.1016/S0140-6736(87)90552-6.CrossRefGoogle Scholar
  3. 3.
    Tang R, Wang JY, Chen JS, Chang-Chien CR, Tang S, Lin SE, et al. Survival impact of lymph node metastasis in TNM stage III carcinoma of the colon and rectum. J Am Coll Surg. 1995;180(6):705–12.PubMedGoogle Scholar
  4. 4.
    Achen MG, Stacker SA. Molecular control of lymphatic metastasis. Ann N Y Acad Sci. 2008;1131:225–34. doi: 10.1196/annals.1413.020.CrossRefPubMedGoogle Scholar
  5. 5.
    Kaneko I, Tanaka S, Oka S, Kawamura T, Hiyama T, Ito M, et al. Lymphatic vessel density at the site of deepest penetration as a predictor of lymph node metastasis in submucosal colorectal cancer. Dis Colon Rectum. 2007;50(1):13–21. doi: 10.1007/s10350-006-0745-5.CrossRefPubMedGoogle Scholar
  6. 6.
    Royston D, Jackson DG. Mechanisms of lymphatic metastasis in human colorectal adenocarcinoma. J Pathol. 2009;217(5):608–19. doi: 10.1002/path.2517.CrossRefPubMedGoogle Scholar
  7. 7.
    Fujii T, Tabe Y, Yajima R, Yamaguchi S, Tsutsumi S, Asao T, et al. Process of distant lymph node metastasis in colorectal carcinoma: implication of extracapsular invasion of lymph node metastasis. BMC Cancer. 2011;11:216. doi: 10.1186/1471-2407-11-216.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Aoyagi T, Terracina KP, Raza A, Takabe K. Current treatment options for colon cancer peritoneal carcinomatosis. World journal of gastroenterology : WJG. 2014;20(35):12493–500. doi: 10.3748/wjg.v20.i35.12493.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Palomares T, Alonso-Varona A, García-Alonso I, Portugal V. New strategies to enhance the efficacy of surgical treatment for colorectal liver metastasis. Colorectal Cancer - Surgery, Diagnostics and Treatment. 2014.Google Scholar
  10. 10.
    Aamodt R, Bondi J, Andersen SN, Bakka A, Bukholm G, Bukholm IR. The prognostic impact of protein expression of E-cadherin-catenin complexes differs between rectal and colon carcinoma. Gastroenterol Res Pract. 2010. doi: 10.1155/2010/616023.PubMedPubMedCentralGoogle Scholar
  11. 11.
    Ikeguchi M, Taniguchi T, Makino M, Kaibara N. Reduced E-cadherin expression and enlargement of cancer nuclei strongly correlate with hematogenic metastasis in colorectal adenocarcinoma. Scand J Gastroenterol. 2000;35(8):839–46.CrossRefPubMedGoogle Scholar
  12. 12.
    Lugli A, Zlobec I, Minoo P, Baker K, Tornillo L, Terracciano L, et al. Prognostic significance of the wnt signalling pathway molecules APC, beta-catenin and E-cadherin in colorectal cancer: a tissue microarray-based analysis. Histopathology. 2007;50(4):453–64. doi: 10.1111/j.1365-2559.2007.02620.x.CrossRefPubMedGoogle Scholar
  13. 13.
    Roca F, Mauro LV, Morandi A, Bonadeo F, Vaccaro C, Quintana GO, et al. Prognostic value of E-cadherin, beta-catenin, MMPs (7 and 9), and TIMPs (1 and 2) in patients with colorectal carcinoma. J Surg Oncol. 2006;93(2):151–60. doi: 10.1002/jso.20413.CrossRefPubMedGoogle Scholar
  14. 14.
    Ye Z, Zhou M, Tian B, Wu B, Li J. Expression of lncRNA-CCAT1, E-cadherin and N-cadherin in colorectal cancer and its clinical significance. Int J Clin Exp Med. 2015;8(3):3707–15.PubMedPubMedCentralGoogle Scholar
  15. 15.
    Zhuo HQ, Jiang KW, Dong LY, Zhu Y, Lu L, Lu Y, et al. Overexpression of N-cadherin is correlated with metastasis and worse survival in colorectal cancer patients. Chin Sci Bull. 2013;58(28-29):3529–34. doi: 10.1007/s11434-013-5813-3.CrossRefGoogle Scholar
  16. 16.
    Cristofanilli M, Charnsangavej C, Hortobagyi GN. Angiogenesis modulation in cancer research: novel clinical approaches. Nat Rev Drug Discov. 2002;1(6):415–26. doi: 10.1038/nrd819.CrossRefPubMedGoogle Scholar
  17. 17.
    Moutasim K, Nystrom M, Thomas G (2011) Cell migration and invasion assays. In: Cree IA (ed) Cancer cell culture, vol 731. Methods in molecular biology. Humana Press, pp 333-343. doi: 10.1007/978-1-61779-080-5_27
  18. 18.
    Cree I (2011) Principles of cancer cell culture. In: Cree IA (ed) Cancer cell culture, vol 731. Methods in molecular biology. Humana Press, pp 13-26. doi: 10.1007/978-1-61779-080-5_2
  19. 19.
    Rusu D, Loret S, Peulen O, Mainil J, Dandrifosse G. Immunochemical, biomolecular and biochemical characterization of bovine epithelial intestinal primocultures. BMC Cell Biol. 2005;6:42. doi: 10.1186/1471-2121-6-42.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Ljung BM, Mayall B, Lottich C, Boyer C, Sylvester SS, Leight GS, et al. Cell dissociation techniques in human breast cancer—variations in tumor cell viability and DNA ploidy. Breast Cancer Res Treat. 1989;13(2):153–9.CrossRefPubMedGoogle Scholar
  21. 21.
    Mitra A, Mishra L, Li S. Technologies for deriving primary tumor cells for use in personalized cancer therapy. Trends Biotechnol. 2013;31(6):347–54.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Kanaan Z, Rai SN, Eichenberger MR, Roberts H, Keskey B, Pan J, et al. Plasma miR-21: a potential diagnostic marker of colorectal cancer. Ann Surg. 2012;256(3):544–51. doi: 10.1097/SLA.0b013e318265bd6f.CrossRefPubMedGoogle Scholar
  23. 23.
    Slaby O, Svoboda M, Michalek J, Vyzula R. MicroRNAs in colorectal cancer: translation of molecular biology into clinical application. Mol Cancer. 2009;8(1):102.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Schetter AJ, Okayama H, Harris CC. The role of microRNAs in colorectal cancer. Cancer J. 2012;18(3):244–52. doi: 10.1097/PPO.0b013e318258b78f.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Brunet Vega A, Pericay C, Moya I, Ferrer A, Dotor E, Pisa A, et al. microRNA expression profile in stage III colorectal cancer: circulating miR-18a and miR-29a as promising biomarkers. Oncol Rep. 2013;30(1):320–6. doi: 10.3892/or.2013.2475.PubMedGoogle Scholar
  26. 26.
    Omrane I, Benammar-Elgaaied A. The immune microenvironment of the colorectal tumor: involvement of immunity genes and microRNAs belonging to the TH17 pathway. Biochim Biophys Acta. 2015;1856(1):28–38. doi: 10.1016/j.bbcan.2015.04.001.PubMedGoogle Scholar
  27. 27.
    Ren A, Dong Y, Tsoi H, Yu J. Detection of miRNA as non-invasive biomarkers of colorectal cancer. Int J Mol Sci. 2015;16(2):2810–23.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Failli A, Consolini R, Legitimo A, Spisni R, Castagna M, Romanini A, et al. The challenge of culturing human colorectal tumor cells: establishment of a cell culture model by the comparison of different methodological approaches. Tumori. 2009;95(3):343–7.PubMedGoogle Scholar
  29. 29.
    Chougule P, Herlenius G, Hernandez NM, Patil PB, Xu B, Sumitran-Holgersson S. Isolation and characterization of human primary enterocytes from small intestine using a novel method. Scand J Gastroenterol. 2012;47(11):1334–43. doi: 10.3109/00365521.2012.708940.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Nakajima S, Doi R, Toyoda E, Tsuji S, Wada M, Koizumi M, et al. N-cadherin expression and epithelial-mesenchymal transition in pancreatic carcinoma. Clin Cancer Res Off J Ame Assoc Cancer Res. 2004;10(12 Pt 1):4125–33. doi: 10.1158/1078-0432.CCR-0578-03.CrossRefGoogle Scholar
  31. 31.
    Tsanou E, Peschos D, Batistatou A, Charalabopoulos A, Charalabopoulos K. The E-cadherin adhesion molecule and colorectal cancer. A global literature approach. Anticancer Res. 2008;28(6A):3815–26.PubMedGoogle Scholar
  32. 32.
    Paschos KA, Canovas D, Bird NC. The role of cell adhesion molecules in the progression of colorectal cancer and the development of liver metastasis. Cell Signal. 2009;21(5):665–74. doi: 10.1016/j.cellsig.2009.01.006.CrossRefPubMedGoogle Scholar
  33. 33.
    Maeda K, Kang SM, Sawada T, Nishiguchi Y, Yashiro M, Ogawa Y, et al. Expression of intercellular adhesion molecule-1 and prognosis in colorectal cancer. Oncol Rep. 2002;9(3):511–4.PubMedGoogle Scholar
  34. 34.
    Kelly CP, O'Keane JC, Orellana J, Schroy 3rd PC, Yang S, LaMont JT, et al. Human colon cancer cells express ICAM-1 in vivo and support LFA-1-dependent lymphocyte adhesion in vitro. The American journal of physiology. 1992;263(6 Pt 1):G864–70.PubMedGoogle Scholar
  35. 35.
    Zhang YY, Chen B, Ding YQ. Metastasis-associated factors facilitating the progression of colorectal cancer. Asian Pac J Cancer Prev: APJCP. 2012;13(6):2437–44.CrossRefPubMedGoogle Scholar
  36. 36.
    Zucker S, Vacirca J. Role of matrix metalloproteinases (MMPs) in colorectal cancer. Cancer Metastasis Rev. 2004;23(1-2):101–17.CrossRefPubMedGoogle Scholar
  37. 37.
    Kheirelseid EAH, Miller N, Chang KH, Nugent M, Kerin MJ. Clinical applications of gene expression in colorectal cancer. J Gastrointest Oncol. 2013;4(2):144–57.PubMedPubMedCentralGoogle Scholar
  38. 38.
    Triulzi T, Tagliabue E, Casalini P, Iorio MV. microRNA: New Players in Metastatic Process. Oncogene and Cancer - From Bench to Clinic. 2013.Google Scholar
  39. 39.
    Chan SH, Wang LH. Regulation of cancer metastasis by microRNAs. J Biomed Sci. 2015;22:9. doi: 10.1186/s12929-015-0113-7.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Berrino F, De Angelis R, Sant M, Rosso S, Lasota MB, Coebergh JW, et al. Survival for eight major cancers and all cancers combined for European adults diagnosed in 1995–99: results of the EUROCARE-4 study. Lancet Oncol. 2007;8(9):773–83. doi: 10.1016/S1470-2045(07)70245-0.CrossRefPubMedGoogle Scholar
  41. 41.
    Dahan L, Sadok A, Formento JL, Seitz J, Kovacic H. Modulation of cellular redox state underlies antagonism between oxaliplatin and cetuximab in human colorectal cancer cell lines. Br J Pharmacol. 2009;158(2):610–20.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Saltz LB, Niedzwiecki D, Hollis D, Goldberg RM, Hantel A, Thomas JP, et al. Irinotecan fluorouracil plus leucovorin is not superior to fluorouracil plus leucovorin alone as adjuvant treatment for stage III colon cancer: results of CALGB 89803. J Clin Oncol: Off J Ame Soc Clin Oncol. 2007;25(23):3456–61. doi: 10.1200/JCO.2007.11.2144.CrossRefGoogle Scholar
  43. 43.
    Van Cutsem E, Labianca R, Bodoky G, Barone C, Aranda E, Nordlinger B, et al. Randomized phase III trial comparing biweekly infusional fluorouracil/leucovorin alone or with irinotecan in the adjuvant treatment of stage III colon cancer: PETACC-3. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2009;27(19):3117–25.CrossRefGoogle Scholar
  44. 44.
    Arnould S, Hennebelle I, Canal P, Bugat R, Guichard S. Cellular determinants of oxaliplatin sensitivity in colon cancer cell lines. Eur J Cancer. 2003;39(1):112–9.CrossRefPubMedGoogle Scholar
  45. 45.
    Ikehata M, Ogawa M, Yamada Y, Tanaka S, Ueda K, Iwakawa S. Different effects of epigenetic modifiers on the cytotoxicity induced by 5-fluorouracil, irinotecan or oxaliplatin in colon cancer cells. Biol Pharm Bull. 2014;37(1):67–73. doi: 10.1248/bpb.b13-00574.CrossRefPubMedGoogle Scholar
  46. 46.
    O'Connell MJ. Oxaliplatin or irinotecan as adjuvant therapy for colon cancer: the results are in. J Clin Oncol : Off J Am Soc Clin Oncol. 2009;27(19):3082–4. doi: 10.1200/JCO.2009.22.2919.CrossRefGoogle Scholar
  47. 47.
    Luca T, Barresi V, Privitera G, Musso N, Caruso M, Condorelli DF, et al. In vitro combined treatment with cetuximab and trastuzumab inhibits growth of colon cancer cells. Cell Prolif. 2014;47(5):435–47. doi: 10.1111/cpr.12125.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Lukáš Krbal
    • 1
  • Veronika Hanušová
    • 2
  • Jiří Soukup
    • 1
  • Stanislav John
    • 2
    • 3
  • Petra Matoušková
    • 4
  • Aleš Ryška
    • 1
  1. 1.The Fingerland Department of PathologyFaculty of Medicine and Faculty Hospital in Hradec Králové, Charles University in PragueHradec KraloveCzech Republic
  2. 2.Department of Medical Biology and GeneticsFaculty of Medicine in Hradec Králové, Charles University in PragueHradec KrálovéCzech Republic
  3. 3.Department of Oncology and RadiotherapyFaculty of Medicine and Faculty Hospital in Hradec Králové, Charles University in PragueHradec KraloveCzech Republic
  4. 4.Department of Biochemical SciencesFaculty of Pharmacy in Hradec Králové, Charles University in PragueHradec KrálovéCzech Republic

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