Three-Dimensional Lung Tumor Microenvironment Modulates Therapeutic Compound Responsiveness In Vitro – Implication for Drug Development

Three-dimensional (3D) cell culture is gaining acceptance in response to the need for cellular models that better mimic physiologic tissues. Spheroids are one such 3D model where clusters of cells will undergo self-assembly to form viable, 3D tumor-like structures. However, to date little is known about how spheroid biology compares to that of the more traditional and widely utilized 2D monolayer cultures. Therefore, the goal of this study was to characterize the phenotypic and functional differences between lung tumor cells grown as 2D monolayer cultures, versus cells grown as 3D spheroids. Eight lung tumor cell lines, displaying varying levels of epidermal growth factor receptor (EGFR) and cMET protein expression, were used to develop a 3D spheroid cell culture model using low attachment U-bottom plates. The 3D spheroids were compared with cells grown in monolayer for 1) EGFR and cMET receptor expression, as determined by flow cytometry, 2) EGFR and cMET phosphorylation by MSD assay, and 3) cell proliferation in response to epidermal growth factor (EGF) and hepatocyte growth factor (HGF). In addition, drug responsiveness to EGFR and cMET inhibitors (Erlotinib, Crizotinib, Cetuximab [Erbitux] and Onartuzumab [MetMab]) was evaluated by measuring the extent of cell proliferation and migration. Data showed that EGFR and cMET expression is reduced at day four of untreated spheroid culture compared to monolayer. Basal phosphorylation of EGFR and cMET was higher in spheroids compared to monolayer cultures. Spheroids showed reduced EGFR and cMET phosphorylation when stimulated with ligand compared to 2D cultures. Spheroids showed an altered cell proliferation response to HGF, as well as to EGFR and cMET inhibitors, compared to monolayer cultures. Finally, spheroid cultures showed exceptional utility in a cell migration assay. Overall, the 3D spheroid culture changed the cellular response to drugs and growth factors and may more accurately mimic the natural tumor microenvironment.

[1]  Soo-Jeong Choi,et al.  Anti-tumor activity of noble indirubin derivatives in human solid tumor models In Vitro , 2009, Archives of pharmacal research.

[2]  Andreas Krieg,et al.  Impact of the 3D Microenvironment on Phenotype, Gene Expression, and EGFR Inhibition of Colorectal Cancer Cell Lines , 2013, PloS one.

[3]  C. Verbeke,et al.  3D pancreatic carcinoma spheroids induce a matrix-rich, chemoresistant phenotype offering a better model for drug testing , 2013, BMC Cancer.

[4]  Kristiina Vuori,et al.  The phosphatidylinositol 3-kinase inhibitor, PX-866, is a potent inhibitor of cancer cell motility and growth in three-dimensional cultures , 2007, Molecular Cancer Therapeutics.

[5]  H. Poulsen,et al.  Targeting the Epidermal Growth Factor Receptor in Solid Tumor Malignancies , 2012, BioDrugs.

[6]  P. Friedl Prespecification and plasticity: shifting mechanisms of cell migration. , 2004, Current opinion in cell biology.

[7]  M. Willingham,et al.  A diphtheria toxin-epidermal growth factor fusion protein is cytotoxic to human glioblastoma multiforme cells. , 2003, Cancer research.

[8]  C. V. van Blitterswijk,et al.  Layer-by-layer tissue microfabrication supports cell proliferation in vitro and in vivo. , 2012, Tissue engineering. Part C, Methods.

[9]  G. Watanabe,et al.  Crosstalk to Stromal Fibroblasts Induces Resistance of Lung Cancer to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors , 2009, Clinical Cancer Research.

[10]  C. Croce,et al.  Cross-talk between MET and EGFR in non-small cell lung cancer involves miR-27a and Sprouty2 , 2013, Proceedings of the National Academy of Sciences.

[11]  F. Siannis,et al.  Assessment of somatic k-RAS mutations as a mechanism associated with resistance to EGFR-targeted agents: a systematic review and meta-analysis of studies in advanced non-small-cell lung cancer and metastatic colorectal cancer. , 2008, The Lancet. Oncology.

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

[13]  Walter Klepetko,et al.  Cell migration or cytokinesis and proliferation?--revisiting the "go or grow" hypothesis in cancer cells in vitro. , 2013, Experimental cell research.

[14]  W. Hait,et al.  Anticancer drug development: the grand challenges , 2010, Nature Reviews Drug Discovery.

[15]  Juergen Friedrich,et al.  Spheroid-based drug screen: considerations and practical approach , 2009, Nature Protocols.

[16]  Shuichi Takayama,et al.  High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array. , 2011, The Analyst.

[17]  Mitchell Ho,et al.  Rapid Generation of In Vitro Multicellular Spheroids for the Study of Monoclonal Antibody Therapy , 2011, Journal of Cancer.

[18]  Joseph A DiMasi,et al.  Economics of new oncology drug development. , 2007, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[19]  Benjamin M Wu,et al.  Incorporation of multicellular spheroids into 3‐D polymeric scaffolds provides an improved tumor model for screening anticancer drugs , 2010, Cancer science.

[20]  T. Mitsudomi,et al.  Combined Therapy with Mutant-Selective EGFR Inhibitor and Met Kinase Inhibitor for Overcoming Erlotinib Resistance in EGFR-Mutant Lung Cancer , 2012, Molecular Cancer Therapeutics.

[21]  B. Al-Lazikani,et al.  Personalized Cancer Medicine: Molecular Diagnostics, Predictive biomarkers, and Drug Resistance , 2012, Clinical pharmacology and therapeutics.

[22]  C. Klein,et al.  A novel bispecific EGFR/Met antibody blocks tumor-promoting phenotypic effects induced by resistance to EGFR inhibition and has potent antitumor activity , 2013, Oncogene.

[23]  J. Christensen,et al.  c-Met as a target for human cancer and characterization of inhibitors for therapeutic intervention. , 2005, Cancer letters.

[24]  S. Marlovits,et al.  Impact of 3D-culture on the expression of differentiation markers and hormone receptors in growth plate chondrocytes as compared to articular chondrocytes. , 2009, International journal of molecular medicine.

[25]  X. Paoletti,et al.  Development of anti-cancer drugs. , 2010, Discovery medicine.

[26]  E. Shimizu,et al.  Lack of AKT activation in lung cancer cells with EGFR mutation is a novel marker of cetuximab sensitivity , 2012, Cancer biology & therapy.

[27]  E. Brown,et al.  Epithelial to mesenchymal transition is a determinant of sensitivity of non-small-cell lung carcinoma cell lines and xenografts to epidermal growth factor receptor inhibition. , 2005, Cancer research.

[28]  S. Eccles,et al.  Tumor spheroid-based migration assays for evaluation of therapeutic agents. , 2013, Methods in molecular biology.

[29]  G. V. Vande Woude,et al.  MET: a critical player in tumorigenesis and therapeutic target. , 2013, Cold Spring Harbor perspectives in biology.

[30]  I. Kasman,et al.  MetMAb, the one-armed 5D5 anti-c-Met antibody, inhibits orthotopic pancreatic tumor growth and improves survival. , 2007, Cancer research.

[31]  P. Workman,et al.  Resisting targeted therapy: fifty ways to leave your EGFR. , 2011, Cancer Cell.

[32]  Y. Sekido,et al.  Hepatocyte Growth Factor Reduces Susceptibility to an Irreversible Epidermal Growth Factor Receptor Inhibitor in EGFR-T790M Mutant Lung Cancer , 2009, Clinical Cancer Research.

[33]  R. Mattingly,et al.  Three-Dimensional Overlay Culture Models of Human Breast Cancer Reveal a Critical Sensitivity to Mitogen-Activated Protein Kinase Kinase Inhibitors , 2010, Journal of Pharmacology and Experimental Therapeutics.

[34]  Olga Ilina,et al.  Mechanisms of collective cell migration at a glance , 2009, Journal of Cell Science.

[35]  J. Beck,et al.  KRAS-mutated non-small cell lung cancer cells are responsive to either co-treatment with erlotinib or gefitinib and histone deacetylase inhibitors or single treatment with lapatinib. , 2011, Oncology reports.

[36]  L. Cazin,et al.  Hyaluronan hydrogel: an appropriate three-dimensional model for evaluation of anticancer drug sensitivity. , 2008, Acta Biomaterialia.

[37]  L. O’Driscoll,et al.  Three-dimensional cell culture: the missing link in drug discovery. , 2013, Drug discovery today.

[38]  M. Westphal,et al.  Erlotinib resistance in EGFR-amplified glioblastoma cells is associated with upregulation of EGFRvIII and PI3Kp110δ. , 2013, Neuro-oncology.

[39]  E. Giovannetti,et al.  Role of cMET expression in non-small-cell lung cancer patients treated with EGFR tyrosine kinase inhibitors. , 2008, Annals of oncology : official journal of the European Society for Medical Oncology.

[40]  R. Salgia,et al.  Role of MetMAb (OA-5D5) in c-MET active lung malignancies , 2011, Expert opinion on biological therapy.

[41]  J. Hainsworth,et al.  Randomized, double-blind, placebo-controlled, phase II trial of sorafenib and erlotinib or erlotinib alone in previously treated advanced non-small-cell lung cancer. , 2011, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[42]  D. Stolz,et al.  Cross-talk between Epidermal Growth Factor Receptor and c-Met Signal Pathways in Transformed Cells* , 2000, The Journal of Biological Chemistry.

[43]  C. Ries,et al.  Comparison of 3D and 2D tumor models reveals enhanced HER2 activation in 3D associated with an increased response to trastuzumab , 2009, Oncogene.

[44]  H. Uramoto,et al.  Expression of selected gene for acquired drug resistance to EGFR-TKI in lung adenocarcinoma. , 2011, Lung cancer.

[45]  L. Windus,et al.  Chemokine receptor expression on integrin-mediated stellate projections of prostate cancer cells in 3D culture. , 2013, Cytokine.

[46]  A. Hopkins Network pharmacology: the next paradigm in drug discovery. , 2008, Nature chemical biology.

[47]  N. Hiraoka,et al.  Prognostic significance of overexpression of c-Met oncoprotein in cholangiocarcinoma , 2011, British Journal of Cancer.

[48]  Y. Yatabe,et al.  Hepatocyte growth factor induces gefitinib resistance of lung adenocarcinoma with epidermal growth factor receptor-activating mutations. , 2008, Cancer research.

[49]  Ravi Salgia,et al.  Synergism of EGFR and c-Met pathways, cross-talk and inhibition, in non-small cell lung cancer , 2008, Journal of carcinogenesis.

[50]  Aimin Zhou,et al.  Development, validation and pilot screening of an in vitro multi-cellular three-dimensional cancer spheroid assay for anti-cancer drug testing. , 2013, Bioorganic & medicinal chemistry.

[51]  Hai Hu,et al.  Loss of BRCA1 leads to an increase in epidermal growth factor receptor expression in mammary epithelial cells, and epidermal growth factor receptor inhibition prevents estrogen receptor-negative cancers in BRCA1-mutant mice , 2011, Breast Cancer Research.

[52]  I. Kola,et al.  The State of Innovation in Drug Development , 2008, Clinical pharmacology and therapeutics.

[53]  Hiroaki Sakurai,et al.  Transient Suppression of Ligand-mediated Activation of Epidermal Growth Factor Receptor by Tumor Necrosis Factor-α through the TAK1-p38 Signaling Pathway* , 2007, Journal of Biological Chemistry.

[54]  L. Windus,et al.  In vivo biomarker expression patterns are preserved in 3D cultures of Prostate Cancer. , 2012, Experimental cell research.

[55]  Deborah S. Barkauskas,et al.  Dual MET–EGFR combinatorial inhibition against T790M-EGFR-mediated erlotinib-resistant lung cancer , 2008, British Journal of Cancer.

[56]  Shinji Takeuchi,et al.  Paracrine Receptor Activation by Microenvironment Triggers Bypass Survival Signals and ALK Inhibitor Resistance in EML4-ALK Lung Cancer Cells , 2012, Clinical Cancer Research.

[57]  Tobias Schmelzle,et al.  Engineering tumors with 3D scaffolds , 2007, Nature Methods.

[58]  R. Sun,et al.  HGF stimulates proliferation through the HGF/c-Met pathway in nasopharyngeal carcinoma cells. , 2012, Oncology letters.

[59]  M. Westphal,et al.  A Novel One-Armed Anti-c-Met Antibody Inhibits Glioblastoma Growth In vivo , 2006, Clinical Cancer Research.

[60]  D. Barnes Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum-free cell culture , 1982, The Journal of cell biology.

[61]  F. Hirsch,et al.  Hepatocyte growth factor expression in EGFR-mutant lung cancer with intrinsic and acquired resistance to tyrosine kinase inhibitors in a Japanese cohort. , 2011, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[62]  I. Okamoto,et al.  Differential roles of trans-phosphorylated EGFR, HER2, HER3, and RET as heterodimerisation partners of MET in lung cancer with MET amplification , 2011, British Journal of Cancer.

[63]  S. Yano,et al.  Hepatocyte Growth Factor Induces Resistance to Anti-Epidermal Growth Factor Receptor Antibody in Lung Cancer , 2012, Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer.

[64]  Luc Stoppini,et al.  OrganDots – an organotypic 3D tissue culture platform for drug development , 2012, Expert opinion on drug discovery.

[65]  M. Meyerson,et al.  PTEN loss contributes to erlotinib resistance in EGFR-mutant lung cancer by activation of Akt and EGFR. , 2009, Cancer research.

[66]  M. Park,et al.  From Tpr-Met to Met, tumorigenesis and tubes , 2007, Oncogene.

[67]  J. Siegfried,et al.  Dual Blockade of EGFR and c-Met Abrogates Redundant Signaling and Proliferation in Head and Neck Carcinoma Cells , 2011, Clinical Cancer Research.

[68]  N. Steimberg,et al.  Modelling tissues in 3D: the next future of pharmaco-toxicology and food research? , 2009, Genes & Nutrition.

[69]  Maria Vinci,et al.  Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation , 2012, BMC Biology.