Alteration of Cellular Behavior and Response to PI3K Pathway Inhibition by Culture in 3D Collagen Gels

Most investigations into cancer cell drug response are performed with cells cultured on flat (2D) tissue culture plastic. Emerging research has shown that the presence of a three-dimensional (3D) extracellular matrix (ECM) is critical for normal cell behavior including migration, adhesion, signaling, proliferation and apoptosis. In this study we investigate differences between cancer cell signaling in 2D culture and a 3D ECM, employing real-time, live cell tracking to directly observe U2OS human osteosarcoma and MCF7 human breast cancer cells embedded in type 1 collagen gels. The activation of the important PI3K signaling pathway under these different growth conditions is studied, and the response to inhibition of both PI3K and mTOR with PI103 investigated. Cells grown in 3D gels show reduced proliferation and migration as well as reduced PI3K pathway activation when compared to cells grown in 2D. Our results quantitatively demonstrate that a collagen ECM can protect U2OS cells from PI103. Overall, our data suggests that 3D gels may provide a better medium for investigation of anti-cancer drugs than 2D monolayers, therefore allowing better understanding of cellular response and behavior in native like environments.

[1]  R. Jain,et al.  Role of extracellular matrix assembly in interstitial transport in solid tumors. , 2000, Cancer research.

[2]  P. Lequerica-Fernández,et al.  Expression of MMP-7 and MT1-MMP in oral squamous cell carcinoma as predictive indicator for tumor invasion and prognosis. , 2007, Journal of Oral Pathology & Medicine.

[3]  W. Dalton,et al.  Cell adhesion mediated drug resistance (CAM-DR): role of integrins and resistance to apoptosis in human myeloma cell lines. , 1999, Blood.

[4]  mTORC2 Caught in a SINful Akt. , 2006, Developmental cell.

[5]  J. Engelman,et al.  The PI3K pathway as drug target in human cancer. , 2010, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[6]  A. Anderson,et al.  A hybrid mathematical model of solid tumour invasion: the importance of cell adhesion. , 2005, Mathematical medicine and biology : a journal of the IMA.

[7]  P. Pandolfi,et al.  Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer. , 2008, The Journal of clinical investigation.

[8]  J. Erler,et al.  Reversing Hypoxic Cell Chemoresistance in Vitro Using Genetic and Small Molecule Approaches Targeting Hypoxia Inducible Factor-1 , 2006, Molecular Pharmacology.

[9]  Muhammad H Zaman,et al.  Bioengineering approaches to study multidrug resistance in tumor cells. , 2011, Integrative biology : quantitative biosciences from nano to macro.

[10]  R. Bonnecaze,et al.  Extracellular matrix stiffness and architecture govern intracellular rheology in cancer. , 2009, Biophysical journal.

[11]  D. Fisher,et al.  A CD44 survival pathway triggers chemoresistance via lyn kinase and phosphoinositide 3-kinase/Akt in colon carcinoma cells. , 2001, Cancer research.

[12]  D. Sabatini.  mTOR and cancer: insights into a complex relationship , 2006, Nature Reviews Cancer.

[13]  Christian A. Rees,et al.  Molecular portraits of human breast tumours , 2000, Nature.

[14]  R. Shelton,et al.  Comparison of bone marrow cell growth on 2D and 3D alginate hydrogels , 2005, Journal of materials science. Materials in medicine.

[15]  J. McKay,et al.  Matrix metalloproteinase 13 activity is associated with poor prognosis in colorectal cancer , 2002, Journal of clinical pathology.

[16]  K. Griffith,et al.  Expression of Bcl-xL in ovarian carcinoma is associated with chemoresistance and recurrent disease. , 2005, Gynecologic oncology.

[17]  L. Griffith,et al.  Capturing complex 3D tissue physiology in vitro , 2006, Nature Reviews Molecular Cell Biology.

[18]  William Pao,et al.  Identifying genotype-dependent efficacy of single and combined PI3K- and MAPK-pathway inhibition in cancer , 2009, Proceedings of the National Academy of Sciences.

[19]  Neal Rosen,et al.  The BAD protein integrates survival signaling by EGFR/MAPK and PI3K/Akt kinase pathways in PTEN-deficient tumor cells. , 2005, Cancer cell.

[20]  Kenneth M. Yamada,et al.  Cell migration in 3D matrix. , 2005, Current opinion in cell biology.

[21]  L. Pajor,et al.  Frequent occurrence of low grade cases among metastatic gastrointestinal stromal tumours , 2003, Journal of clinical pathology.

[22]  R. Roth,et al.  PRAS40 Regulates mTORC1 Kinase Activity by Functioning as a Direct Inhibitor of Substrate Binding* , 2007, Journal of Biological Chemistry.

[23]  G. Neufeld,et al.  A novel asymmetric 3D in-vitro assay for the study of tumor cell invasion , 2009, BMC Cancer.

[24]  Frederick Grinnell,et al.  Fibroblast biology in three-dimensional collagen matrices. , 2003, Trends in cell biology.

[25]  Kenneth M. Yamada,et al.  Modeling Tissue Morphogenesis and Cancer in 3D , 2007, Cell.

[26]  Patricia J. Keely,et al.  Use of three-dimensional collagen gels to study mechanotransduction in T47D breast epithelial cells , 2005, Biological Procedures Online.

[27]  Kenneth M. Yamada,et al.  Taking Cell-Matrix Adhesions to the Third Dimension , 2001, Science.

[28]  Roger T. Bonnecaze,et al.  Cancer Cell Migration: Integrated Roles of Matrix Mechanics and Transforming Potential , 2011, PloS one.

[29]  Susan G Hilsenbeck,et al.  Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. , 2008, Journal of the National Cancer Institute.

[30]  D. Miklavčič,et al.  Cell–Cell Electrofusion: Optimization of Electric Field Amplitude and Hypotonic Treatment for Mouse Melanoma (B16-F1) and Chinese Hamster Ovary (CHO) Cells , 2010, The Journal of Membrane Biology.

[31]  J. Blenis,et al.  mTORC1-Mediated Control of Protein Translation , 2010 .

[32]  Konstantinos Vougas,et al.  The proteome profile of the human osteosarcoma U2OS cell line. , 2008, Cancer genomics & proteomics.

[33]  K. Shokat,et al.  PI-103, a dual inhibitor of Class IA phosphatidylinositide 3-kinase and mTOR, has antileukemic activity in AML , 2008, Leukemia.

[34]  Y. Hishikawa,et al.  Overexpression of metallothionein correlates with chemoresistance to cisplatin and prognosis in esophageal cancer. , 1997, Oncology.

[35]  Cynthia A. Reinhart-King,et al.  Tensional homeostasis and the malignant phenotype. , 2005, Cancer cell.

[36]  David M Sabatini,et al.  Defining the role of mTOR in cancer. , 2007, Cancer cell.

[37]  E. Armanasco,et al.  Plasma MMP‐9 (92 kDa‐MMP) activity is useful in the follow‐up and in the assessment of prognosis in breast cancer patients , 2003, International journal of cancer.

[38]  K. Black,et al.  Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma , 2006, Molecular Cancer.

[39]  Pier Paolo Pandolfi,et al.  The PTEN–PI3K pathway: of feedbacks and cross-talks , 2008, Oncogene.

[40]  C. Arteaga,et al.  Herceptin-induced inhibition of phosphatidylinositol-3 kinase and Akt Is required for antibody-mediated effects on p27, cyclin D1, and antitumor action. , 2002, Cancer research.

[41]  Kenneth M. Yamada,et al.  Cell–matrix adhesion , 2007, Journal of cellular physiology.

[42]  F. Zunino,et al.  Overview of tumor cell chemoresistance mechanisms. , 2005, Methods in molecular medicine.

[43]  G. Ligresti,et al.  Extracellular Matrix: A Matter of Life and Death , 2008, Connective tissue research.

[44]  D Fujimoto,et al.  Pyridinoline, a non-reducible crosslink of collagen. Quantitative determination, distribution, and isolation of a crosslinked peptide. , 1978, Journal of biochemistry.

[45]  F. Miller,et al.  Factors affecting growth and drug sensitivity of mouse mammary tumor lines in collagen gel cultures. , 1985, Cancer research.

[46]  W. Dalton,et al.  Cell Adhesion Mediated Drug Resistance (CAM-DR): Role of Integrins and Resistance to Apoptosis in Human Myeloma Cell Lines , 1999 .

[47]  F. Pampaloni,et al.  The third dimension bridges the gap between cell culture and live tissue , 2007, Nature Reviews Molecular Cell Biology.

[48]  R. Kiss,et al.  Can anti‐migratory drugs be screened in vitro? A review of 2D and 3D assays for the quantitative analysis of cell migration , 2007, Medicinal research reviews.

[49]  J. Blenis,et al.  mTORC1: A Signaling Integration Node Involved in Cell Growth , 2009 .

[50]  R K Craig,et al.  Methods in molecular medicine. , 1987, British medical journal.

[51]  Dewi Harjanto,et al.  Matrix Mechanics and Receptor—Ligand Interactions in Cell Adhesion , 2010 .

[52]  Alissa M. Weaver,et al.  Tumor Morphology and Phenotypic Evolution Driven by Selective Pressure from the Microenvironment , 2006, Cell.

[53]  William A Weiss,et al.  A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma. , 2006, Cancer cell.

[54]  M. Ernstoff.  mTOR Pathway and mTOR Inhibitors in Cancer Therapy , 2011 .

[55]  E. Golemis,et al.  Fibroblast-derived 3D matrix differentially regulates the growth and drug-responsiveness of human cancer cells. , 2008, Matrix biology : journal of the International Society for Matrix Biology.

[56]  D. Lauffenburger,et al.  Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[57]  D. Herbage,et al.  Collagen-based biomaterials as 3D scaffold for cell cultures: applications for tissue engineering and gene therapy , 2000, Medical and Biological Engineering and Computing.

[58]  C. Sotiriou,et al.  Gene expression profiles derived from fine needle aspiration correlate with response to systemic chemotherapy in breast cancer , 2002, Breast Cancer Research.