Differential Effects of Tissue Culture Coating Substrates on Prostate Cancer Cell Adherence, Morphology and Behavior

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-l-lysine, poly-l-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-l-lysine and poly-l-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement.

[1]  E. Fuchs,et al.  Actin dynamics and cell-cell adhesion in epithelia. , 2001, Current opinion in cell biology.

[2]  S. Farmer,et al.  Cell adhesion induces expression of growth-associated genes in suspension-arrested fibroblasts. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[3]  B. Knudsen,et al.  The impact of cell adhesion changes on proliferation and survival during prostate cancer development and progression , 2006, Journal of cellular biochemistry.

[4]  F M Watt,et al.  Regulation of development and differentiation by the extracellular matrix. , 1993, Development.

[5]  T. Webster,et al.  Mimicking the nanofeatures of bone increases bone-forming cell adhesion and proliferation , 2005 .

[6]  N. Balaban,et al.  Adhesion-dependent cell mechanosensitivity. , 2003, Annual review of cell and developmental biology.

[7]  Robert Langer,et al.  Principles of tissue engineering , 2014 .

[8]  A Benner,et al.  Active and inactive genes localize preferentially in the periphery of chromosome territories , 1996, The Journal of cell biology.

[9]  Bernd Hoffmann,et al.  One step ahead: role of filopodia in adhesion formation during cell migration of keratinocytes. , 2009, Experimental cell research.

[10]  W. Saltzman,et al.  Cell interactions with polymers , 2020, Principles of Tissue Engineering.

[11]  S. Svetina,et al.  The effect of substrate and adsorbed proteins on adhesion, growth and shape of CaCo‐2 cells , 2007, Cell biology international.

[12]  E. Ruoslahti,et al.  Superfibronectin is a functionally distinct form of fibronectin , 1994, Nature.

[13]  Y. Kariya,et al.  Polymerized Laminin-332 Matrix Supports Rapid and Tight Adhesion of Keratinocytes, Suppressing Cell Migration , 2012, PloS one.

[14]  P. Mattila,et al.  Filopodia: molecular architecture and cellular functions , 2008, Nature Reviews Molecular Cell Biology.

[15]  R. Hughes,et al.  Fibronectin synthesis and surface expression is correlated with cell morphology and adhesiveness in a cold-sensitive, G1-defective mutant of CHO cells. , 1981, Experimental cell research.

[16]  C. Damsky,et al.  Integrin signaling: it's where the action is. , 2002, Current opinion in cell biology.

[17]  E. Opara,et al.  Characteristics of Poly-L-Ornithine-coated alginate microcapsules. , 2005, Biomaterials.

[18]  H. Rodemann,et al.  Fibronectin and laminin increase resistance to ionizing radiation and the cytotoxic drug Ukrain® in human tumour and normal cells in vitro , 2003, International journal of radiation biology.

[19]  Arnoud Sonnenberg,et al.  Function and interactions of integrins , 2001, Cell and Tissue Research.

[20]  G. Bubley,et al.  Biology of prostate-specific antigen. , 2003, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[21]  Jennie B. Leach,et al.  Extracellular Matrix , 2015, Neuromethods.

[22]  Xiao Xu,et al.  The xCELLigence system for real-time and label-free monitoring of cell viability. , 2011, Methods in molecular biology.

[23]  R. Juliano,et al.  Integrin Signaling , 2005, Cancer and Metastasis Reviews.

[24]  J. Hoak,et al.  Diminished Platelet Adherence to Type V Collagen , 1983, Arteriosclerosis.

[25]  Jenny Zhu,et al.  Dynamic Monitoring of Cell Adhesion and Spreading on Microelectronic Sensor Arrays , 2005, Journal of biomolecular screening.

[26]  Yong Sik Chung,et al.  The effect of type II collagen coating of chitosan fibrous scaffolds on mesenchymal stem cell adhesion and chondrogenesis. , 2010, Acta biomaterialia.

[27]  Seong-Jin Kim,et al.  Raloxifene, a selective estrogen receptor modulator, induces apoptosis in androgen-responsive human prostate cancer cell line LNCaP through an androgen-independent pathway. , 2002, Cancer research.

[28]  D E Ingber,et al.  Fibronectin controls capillary endothelial cell growth by modulating cell shape. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Hua Zhang,et al.  Adhesion, proliferation, and gene expression profile of human umbilical vein endothelial cells cultured on bilayered polyelectrolyte coatings composed of glycosaminoglycans , 2010, Biointerphases.

[30]  T. Wight,et al.  The extracellular matrix: an active or passive player in fibrosis? , 2011, American journal of physiology. Gastrointestinal and liver physiology.

[31]  J. Brugge,et al.  Sensing the environment: a historical perspective on integrin signal transduction , 2002, Nature Cell Biology.

[32]  P. Vogt,et al.  Adhesion, Vitality and Osteogenic Differentiation Capacity of Adipose Derived Stem Cells Seeded on Nitinol Nanoparticle Coatings , 2013, PloS one.

[33]  P. McKeown-Longo,et al.  Fibronectin's III-1 module contains a conformation-dependent binding site for the amino-terminal region of fibronectin. , 1994, The Journal of biological chemistry.

[34]  J. Planell,et al.  Effect of blasting treatment and Fn coating on MG63 adhesion and differentiation on titanium: a gene expression study using real-time RT-PCR , 2011, Journal of materials science. Materials in medicine.

[35]  L. Jennings,et al.  Integrin-mediated signal transduction , 1998, Cellular and Molecular Life Sciences CMLS.

[36]  C. Nicolini,et al.  Modifications of chromatin structure and gene expression following induced alterations of cellular shape. , 2004, The international journal of biochemistry & cell biology.

[37]  Luca Wurfel,et al.  Principles Of Tissue Engineering , 2016 .

[38]  J. Amédée,et al.  Effect of surface roughness of the titanium alloy Ti-6Al-4V on human bone marrow cell response and on protein adsorption. , 2001, Biomaterials.

[39]  T. Bretschneider,et al.  The Diaphanous-related formin dDia2 is required for the formation and maintenance of filopodia , 2005, Nature Cell Biology.

[40]  S. Cross,et al.  TMPRSS2 fusions in prostate cancer , 2008 .

[41]  Anna V. Taubenberger,et al.  Phenotypic Characterization of Prostate Cancer LNCaP Cells Cultured within a Bioengineered Microenvironment , 2012, PloS one.

[42]  E. Marani,et al.  Adhesion and proliferation of human Schwann cells on adhesive coatings. , 2004, Biomaterials.

[43]  J. Small,et al.  Building the actin cytoskeleton: filopodia contribute to the construction of contractile bundles in the lamella , 2008, The Journal of cell biology.

[44]  J. Alderman,et al.  The surface energy of various biomaterials coated with adhesion molecules used in cell culture. , 2007, Colloids and Surfaces B: Biointerfaces.

[45]  W. Mark Saltzman,et al.  CHAPTER 19 – CELL INTERACTIONS WITH POLYMERS , 2000 .

[46]  Mitsuru Nenoi,et al.  Regulation of , 2004 .

[47]  Etienne Hanon,et al.  The Use of Real-Time Cell Analyzer Technology in Drug Discovery: Defining Optimal Cell Culture Conditions and Assay Reproducibility with Different Adherent Cellular Models , 2011, Journal of biomolecular screening.

[48]  G. Murphy,et al.  LNCaP model of human prostatic carcinoma. , 1983, Cancer research.

[49]  Gower,et al.  Fibronectin influences cellular proliferation and apoptosis similarly in LNCaP and PC-3 prostate cancer cell lines. , 2000, Urologic oncology.

[50]  J. Workman,et al.  Alteration of nucleosome structure as a mechanism of transcriptional regulation. , 1998, Annual review of biochemistry.

[51]  R. Andersen,et al.  Strongylophorine-26, a Rho-dependent inhibitor of tumor cell invasion that reduces actin stress fibers and induces nonpolarized lamellipodial extensions , 2005, Molecular Cancer Therapeutics.

[52]  Y. Lange,et al.  Cell Adhesion to Fibronectin Regulates Membrane Lipid Biosynthesis through 5′-AMP-activated Protein Kinase* , 1997, The Journal of Biological Chemistry.

[53]  D. Hocking,et al.  Fibronectin matrix assembly enhances adhesion-dependent cell growth. , 1998, Journal of cell science.