title: Three-dimensional lung tumor microenvironment modulates therapeutic compound responsiveness in vitro--implication for drug development.
abstract: 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.
authors.affiliation: Biologics Research, Biotechnology Center of Excellence, Janssen R&D, LLC, Pharmaceutical Companies of Johnson & Johnson, Spring House, Pennsylvania, United States of America.
authors.forename: Jason E
authors.initials: JE
authors.lastname: Ekert
authors.affiliation: Arbor Analytics, LLC, Ann Arbor, Michigan, United States of America.
authors.forename: Kjell
authors.initials: K
authors.lastname: Johnson
authors.affiliation: Biologics Research, Biotechnology Center of Excellence, Janssen R&D, LLC, Pharmaceutical Companies of Johnson & Johnson, Spring House, Pennsylvania, United States of America.
authors.forename: Brandy
authors.initials: B
authors.lastname: Strake
authors.affiliation: Biologics Research, Biotechnology Center of Excellence, Janssen R&D, LLC, Pharmaceutical Companies of Johnson & Johnson, Spring House, Pennsylvania, United States of America.
authors.forename: Jose
authors.initials: J
authors.lastname: Pardinas
authors.affiliation: Biologics Research, Biotechnology Center of Excellence, Janssen R&D, LLC, Pharmaceutical Companies of Johnson & Johnson, Spring House, Pennsylvania, United States of America.
authors.forename: Stephen
authors.initials: S
authors.lastname: Jarantow
authors.affiliation: Biologics Research, Biotechnology Center of Excellence, Janssen R&D, LLC, Pharmaceutical Companies of Johnson & Johnson, Spring House, Pennsylvania, United States of America.
authors.forename: Robert
authors.initials: R
authors.lastname: Perkinson
authors.affiliation: Biologics Research, Biotechnology Center of Excellence, Janssen R&D, LLC, Pharmaceutical Companies of Johnson & Johnson, Spring House, Pennsylvania, United States of America.
authors.forename: David C
authors.initials: DC
authors.lastname: Colter
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use*; D018929:Cell Culture Techniques / Q000379:methods*; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D055808:Drug Discovery; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000473:pathology*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D019859:Proto-Oncogene Proteins c-met / Q000037:antagonists & inhibitors / Q000378:metabolism; D015203:Reproducibility of Results; D018874:Spheroids, Cellular / Q000187:drug effects / Q000473:pathology; D016896:Treatment Outcome; D014407:Tumor Cells, Cultured; D059016:Tumor Microenvironment / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D008024:Ligands; D047428:Protein Kinase Inhibitors; D004815:Epidermal Growth Factor; D017228:Hepatocyte Growth Factor; D066246:ErbB Receptors; D019859:Proto-Oncogene Proteins c-met