Multidrug resistance characterization in multicellular tumour spheroids from two human lung cancer cell lines

BackgroundMost of the knowledge about the mechanisms of multidrug resistance in lung cancer has been achieved through the use of cell lines isolated from tumours cultivated either in suspensions of isolated cells or in monolayers and following exposition to different cytostatic agents. However, tumour cell lines growing as multicellular tumour spheroids (MTS) frequently develop multicellular resistance in a drug-independent form. The aim of this study was to characterize the phenotypic and functional differences between two human NSCLC cell lines (INER-37 and INER-51) grown as traditional monolayer cultures versus as MTS.MethodsAfter 72 hours treatment with anticancer drugs, chemosensitivity in monolayers and tumour spheroids cultures was assessed using MTT assay. Reverse transcription-polymerase chain reaction was employed to detect the mRNAs of multidrug resistance-related genes. The expression of P-gp was analyzed by immunohistochemical staining and cell cycle profiles were analyzed using FACS.ResultsThe results indicate that when grown as MTS each lung cancer cell line had different morphologies as well as and abrogation of cell proliferation with decrease of the G2/M phase. Also, MTS acquired multicellular resistance to several chemotherapeutic agents in only a few days of culture which were accomplished by significant changes in the expression of MDR-related genes.ConclusionOverall, the MTS culture changed the cellular response to drugs nevertheless each of the cell lines studied seems to implement different mechanisms to acquire multicellular resistance.

[1]  J. Carlsson,et al.  Relations between ph, oxygen partial pressure and growth in cultured cell spheroids , 1988, International journal of cancer.

[2]  J. Jardillier,et al.  Multicellular resistance: a paradigm for clinical resistance? , 2000, Critical reviews in oncology/hematology.

[3]  J. Hescheler,et al.  Modulation of intrinsic P-glycoprotein expression in multicellular prostate tumor spheroids by cell cycle inhibitors. , 2002, Biochimica et biophysica acta.

[4]  L. Kunz-Schughart,et al.  Multicellular tumor spheroids: an underestimated tool is catching up again. , 2010, Journal of biotechnology.

[5]  I. Tannock,et al.  Drug penetration in solid tumours , 2006, Nature Reviews Cancer.

[6]  H. H. Evans,et al.  Etoposide sensitivity and topoisomerase II activity in Chinese hamster V79 monolayers and small spheroids. , 1991, International journal of radiation biology.

[7]  J. P. Freyer,et al.  Influence of glucose and oxygen supply conditions on the oxygenation of multicellular spheroids. , 1986, British Journal of Cancer.

[8]  I. Tannock,et al.  The penetration of anticancer drugs through tumor tissue as a function of cellular adhesion and packing density of tumor cells. , 2006, Cancer research.

[9]  P. Borst.  Genetic mechanisms of drug resistance. A review. , 1991, Acta oncologica.

[10]  Jacques Ferlay,et al.  GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC Cancer Base No. 11 [Internet] , 2013 .

[11]  B. Chauffert,et al.  Circumvention of confluence‐dependent resistance in a human multi‐drug‐resistant colon‐cancer cell line , 1995, International journal of cancer.

[12]  J. Freyer.  Role of necrosis in regulating the growth saturation of multicellular spheroids. , 1988, Cancer research.

[13]  S. Simon,et al.  Cell biological mechanisms of multidrug resistance in tumors. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[14]  S. Cole.  Targeting multidrug resistance protein 1 (MRP1, ABCC1): past, present, and future. , 2014, Annual review of pharmacology and toxicology.

[15]  C. Mathers,et al.  Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008 , 2010, International journal of cancer.

[16]  R. Sutherland.  Cell and environment interactions in tumor microregions: the multicell spheroid model. , 1988, Science.

[17]  S. Hahn,et al.  Epidermal growth factor receptor tyrosine kinase inhibitors vs conventional chemotherapy in non-small cell lung cancer harboring wild-type epidermal growth factor receptor: a meta-analysis. , 2014, JAMA.

[18]  B. Teicher,et al.  Gene Expression Analysis of Tumor Spheroids Reveals a Role for Suppressed DNA Mismatch Repair in Multicellular Resistance to Alkylating Agents , 2004, Molecular and Cellular Biology.

[19]  P. Olive,et al.  Drug and radiation resistance in spheroids: cell contact and kinetics , 1994, Cancer and Metastasis Reviews.

[20]  C. Mathers,et al.  GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 [Internet]. Lyon, France: International Agency for Research on Cancer , 2013 .

[21]  S. Cole,et al.  Transport of glutathione and glutathione conjugates by MRP1. , 2006, Trends in pharmacological sciences.

[22]  R. Durand.  Slow penetration of anthracyclines into spheroids and tumors: a therapeutic advantage? , 2008, Cancer Chemotherapy and Pharmacology.

[23]  P. Bunn,et al.  Recent clinical advances in lung cancer management. , 2014, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[24]  M. Gassmann,et al.  Regulation of the multidrug resistance transporter P‐glycoprotein in multicellular tumor spheroids by hypoxia‐inducible factor‐1 and reactive oxygen species , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[25]  Gerhard Christofori,et al.  Cell adhesion and signalling by cadherins and Ig-CAMs in cancer , 2004, Nature Reviews Cancer.

[26]  Masatoshi Watanabe,et al.  Three-dimensional cellular spheroid formation provides human prostate tumor cells with tissue-like features. , 2007, Anticancer research.

[27]  Ponce de León Valeria,et al.  Changes in P-glycoprotein activity are mediated by the growth of a tumour cell line as multicellular spheroids , 2005, Cancer Cell International.

[28]  L. Stewart,et al.  Adjuvant chemotherapy, with or without postoperative radiotherapy, in operable non-small-cell lung cancer: two meta-analyses of individual patient data , 2010, The Lancet.

[29]  R. Barrera-Rodríguez,et al.  Characterization of human NSCLC cell line with innate etoposide‐resistance mediated by cytoplasmic localization of topoisomerase II alpha , 2005, Cancer science.

[30]  G. Hofmann,et al.  Biochemical and pharmacological properties of p170 and p180 forms of topoisomerase II. , 1989, Biochemistry.

[31]  Kjell Johnson,et al.  Three-Dimensional Lung Tumor Microenvironment Modulates Therapeutic Compound Responsiveness In Vitro – Implication for Drug Development , 2014, PloS one.

[32]  A. Oloumi,et al.  Changes in subcellular distribution of topoisomerase IIalpha correlate with etoposide resistance in multicell spheroids and xenograft tumors. , 2000, Cancer research.

[33]  M. Plummer,et al.  International agency for research on cancer. , 2020, Archives of pathology.

[34]  Matthias Gutekunst,et al.  Three‐dimensional models of cancer for pharmacology and cancer cell biology: Capturing tumor complexity in vitro/ex vivo , 2014, Biotechnology journal.

[35]  J. Carlsson,et al.  Relations between the penetration, binding and average concentration of cytostatic drugs in human tumour spheroids , 2004, Cancer Chemotherapy and Pharmacology.

[36]  G Vallancien,et al.  Gene expression profiles of bladder cancers: evidence for a striking effect of in vitro cell models on gene patterns , 2002, British Journal of Cancer.

[37]  J. Hescheler,et al.  Development of an intrinsic P‐glycoprotein‐mediated doxorubicin resistance in quiescent cell layers of large, multicellular prostate tumor spheroids , 1998, International journal of cancer.

[38]  I. Roninson,et al.  Drug resistance conferred by MDR1 expression in spheroids formed by glioblastoma cell lines. , 1997, Anticancer research.

[39]  David Harrington,et al.  Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. , 2002, The New England journal of medicine.

[40]  T. Kwok,et al.  The relationship between tumour geometry and the response of tumour cells to cytotoxic drugs — An in vitro study using EMT6 multicellular spheroids , 1985, International journal of cancer.

[41]  M. Kool,et al.  A family of drug transporters: the multidrug resistance-associated proteins. , 2000, Journal of the National Cancer Institute.

[42]  R. Ganapathi,et al.  DNA topoisomerase II enzymes as molecular targets for cancer chemotherapy. , 2010, Current cancer drug targets.

[43]  A. Tomida,et al.  Glucose‐regulated stresses induce resistance to camptothecin in human cancer cells , 1996, International journal of cancer.

[44]  J. Sandoval,et al.  Three-dimensional neuroblastoma cell culture: proteomic analysis between monolayer and multicellular tumor spheroids , 2008, Pediatric Surgery International.

[45]  H. H. Evans,et al.  Cell killing and DNA damage by etoposide in Chinese hamster V79 monolayers and spheroids: influence of growth kinetics, growth environment and DNA packaging. , 1993, British journal of cancer.

[46]  M. Clynes,et al.  Generation of lung cancer cell line variants by drug selection or cloning. , 2011, Methods in molecular biology.