Mechanisms of three-dimensional growth of thyroid cells during long-term simulated microgravity

Three-dimensional multicellular spheroids (MCS) of human cells are important in cancer research. We investigated possible mechanisms of MCS formation of thyroid cells. Both, normal Nthy-ori 3–1 thyroid cells and the poorly differentiated follicular thyroid cancer cells FTC-133 formed MCS within 7 and 14 days of culturing on a Random Positioning Machine (RPM), while a part of the cells continued to grow adherently in each culture. The FTC-133 cancer cells formed larger and numerous MCS than the normal cells. In order to explain the different behaviour, we analyzed the gene expression of IL6, IL7, IL8, IL17, OPN, NGAL, VEGFA and enzymes associated cytoskeletal or membrane proteins (ACTB, TUBB, PFN1, CPNE1, TGM2, CD44, FLT1, FLK1, PKB, PKC, ERK1/2, Casp9, Col1A1) as well as the amount of secreted proteins (IL-6, IL-7, IL-8, IL-17, OPN, NGAL, VEGFA). Several of these components changed during RPM-exposure in each cell line. Striking differences between normal and malignant cells were observed in regards to the expression of genes of NGAL, VEGFA, OPN, IL6 and IL17 and to the secretion of VEGFA, IL-17, and IL-6. These results suggest several gravi-sensitive growth or angiogenesis factors being involved in 3D formation of thyroid cells cultured under simulated microgravity.

[1]  Napoleone Ferrara,et al.  Blocking Vascular Endothelial Growth Factor-A Inhibits the Growth of Pituitary Adenomas and Lowers Serum Prolactin Level in a Mouse Model of Multiple Endocrine Neoplasia Type 1 , 2008, Clinical Cancer Research.

[2]  D. Grimm,et al.  The effects of weightlessness on the human organism and mammalian cells. , 2011, Current molecular medicine.

[3]  Sushovan Guha,et al.  The multifaceted roles of neutrophil gelatinase associated lipocalin (NGAL) in inflammation and cancer. , 2012, Biochimica et biophysica acta.

[4]  Marek Ancukiewicz,et al.  Efficacy, safety, and potential biomarkers of sunitinib monotherapy in advanced hepatocellular carcinoma: a phase II study. , 2009, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[5]  P. Pippia,et al.  5‐Lipoxygenase‐dependent apoptosis of human lymphocytes in the International Space Station: data from the ROALD experiment , 2012, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[6]  K. Mehta,et al.  Tissue transglutaminase as a central mediator in inflammation-induced progression of breast cancer , 2013, Breast Cancer Research.

[7]  A. Frilling,et al.  Growth regulation of normal thyroids and thyroid tumors in man. , 1990, Recent results in cancer research. Fortschritte der Krebsforschung. Progres dans les recherches sur le cancer.

[8]  M. Shakibaei,et al.  Simulated weightlessness changes the cytoskeleton and extracellular matrix proteins in papillary thyroid carcinoma cells , 2006, Cell and Tissue Research.

[9]  T. Kipari,et al.  Osteopontin--a molecule for all seasons. , 2002, QJM : monthly journal of the Association of Physicians.

[10]  Alamelu Sundaresan,et al.  The Impact of Simulated and Real Microgravity on Bone Cells and Mesenchymal Stem Cells , 2014, BioMed research international.

[11]  V. Velebný,et al.  Low molecular weight hyaluronan mediated CD44 dependent induction of IL-6 and chemokines in human dermal fibroblasts potentiates innate immune response. , 2014, Cytokine.

[12]  D. Grimm,et al.  Growing tissues in real and simulated microgravity: new methods for tissue engineering. , 2014, Tissue engineering. Part B, Reviews.

[13]  N. Hübner,et al.  Genomic Approach to Identify Factors That Drive the Formation of Three-Dimensional Structures by EA.hy926 Endothelial Cells , 2013, PloS one.

[14]  Felice Strollo,et al.  Chapter 4 Hormonal Changes in Humans During Spaceflight , 1999 .

[15]  Susan J. Crawford-Young,et al.  Effects of microgravity on cell cytoskeleton and embryogenesis. , 2006, The International journal of developmental biology.

[16]  Miho Suzuki,et al.  IL-6/IL-6 receptor system and its role in physiological and pathological conditions. , 2012, Clinical science.

[17]  M. Hughes-Fulford,et al.  Molecular mechanisms underlying the enhanced functions of three-dimensional hepatocyte aggregates. , 2014, Biomaterials.

[18]  D. Grimm,et al.  Human follicular and papillary thyroid carcinoma cells interact differently with human venous endothelial cells. , 1995, Thyroid : official journal of the American Thyroid Association.

[19]  W. Witke.  The role of profilin complexes in cell motility and other cellular processes. , 2004, Trends in cell biology.

[20]  M. Papotti,et al.  Poorly differentiated carcinoma of the thyroid: validation of the Turin proposal and analysis of IMP3 expression , 2010, Modern Pathology.

[21]  R. Einspanier,et al.  Differential Gene Regulation under Altered Gravity Conditions in Follicular Thyroid Cancer Cells: Relationship between the Extracellular Matrix and the Cytoskeleton , 2011, Cellular Physiology and Biochemistry.

[22]  A. Cogoli,et al.  Neocartilage formation in 1 g, simulated, and microgravity environments: implications for tissue engineering. , 2010, Tissue engineering. Part A.

[23]  Jens Hauslage,et al.  Short‐term weightlessness produced by parabolic flight maneuvers altered gene expression patterns in human endothelial cells , 2012, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[24]  D. Grimm,et al.  Impact of Sunitinib on Human Thyroid Cancer Cells , 2013, Cellular Physiology and Biochemistry.

[25]  G. Melillo,et al.  Role of the VEGF/VEGFR axis in cancer biology and therapy. , 2012, Advances in cancer research.

[26]  Mario Passalacqua,et al.  Clinorotation-induced weightlessness influences the cytoskeleton of glial cells in culture , 2002, Brain Research.

[27]  G. Vunjak‐Novakovic,et al.  Tissue engineering of cartilage in space. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[28]  F. Strollo.  Hormonal changes in humans during spaceflight. , 1999, Advances in space biology and medicine.

[29]  G Gasset,et al.  The effect of weightlessness on cytoskeleton architecture and proliferation of human breast cancer cell line MCF-7. , 2001, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[30]  Napoleone Ferrara,et al.  Vascular Endothelial Growth Factor Signaling Pathways: Therapeutic Perspective , 2006, Clinical Cancer Research.

[31]  L. Claesson‐Welsh,et al.  VEGF receptor signalling ? in control of vascular function , 2006, Nature Reviews Molecular Cell Biology.

[32]  G. Schulze-Tanzil,et al.  Intraluminal Application of Vascular Endothelial Growth Factor Enhances Healing of Microvascular Anastomosis in a Rat Model , 2005, Journal of Vascular Research.

[33]  R. Wildgruber,et al.  A proteomic approach to analysing spheroid formation of two human thyroid cell lines cultured on a random positioning machine , 2011, Proteomics.

[34]  D. Grimm,et al.  Moderate alterations of the cytoskeleton in human chondrocytes after short‐term microgravity produced by parabolic flight maneuvers could be prevented by up‐regulation of BMP‐2 and SOX‐9 , 2015, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[35]  M. Mayo,et al.  Copine-I represses NF-κB transcription by endoproteolysis of p65 , 2008, Oncogene.

[36]  D. Grimm,et al.  Changes in Morphology, Gene Expression and Protein Content in Chondrocytes Cultured on a Random Positioning Machine , 2013, PloS one.

[37]  Borst,et al.  UvA-DARE ( Digital Academic Repository ) Technology and developments for the Random Positioning Machine , 2010 .

[38]  D. Grimm,et al.  Vascular endothelial growth factor induces extracellular matrix proteins and osteopontin in the umbilical artery. , 2008, Annals of vascular surgery.

[39]  V. Barresi,et al.  Neutrophil gelatinase-associated lipocalin (NGAL) immunohistochemical expression in follicular cell-derived thyroid tumors: a novel diagnostic tool? , 2012, Histology and histopathology.

[40]  G. Schulze-Tanzil,et al.  Weightlessness induced apoptosis in normal thyroid cells and papillary thyroid carcinoma cells via extrinsic and intrinsic pathways. , 2003, Endocrinology.

[41]  B. Snel,et al.  STRING: a web-server to retrieve and display the repeatedly occurring neighbourhood of a gene. , 2000, Nucleic acids research.

[42]  A. Cogoli,et al.  Modeled gravitational unloading induced downregulation of endothelin‐1 in human endothelial cells , 2007, Journal of cellular biochemistry.

[43]  P. Perestenko,et al.  Copines‐1, ‐2, ‐3, ‐6 and ‐7 show different calcium‐dependent intracellular membrane translocation and targeting , 2010, The FEBS journal.

[44]  Albert Sickmann,et al.  Identification of proteins involved in inhibition of spheroid formation under microgravity , 2015, Proteomics.

[45]  L. Kunz-Schughart,et al.  Multicellular tumor spheroids: intermediates between monolayer culture and in vivo tumor , 1999, Cell biology international.

[46]  M. Glogauer,et al.  Intracellular osteopontin is an integral component of the CD44‐ERM complex involved in cell migration , 2000, Journal of cellular physiology.

[47]  G. Schulze-Tanzil,et al.  Effects of simulated microgravity on thyroid carcinoma cells. , 2002, Journal of gravitational physiology : a journal of the International Society for Gravitational Physiology.

[48]  Tim Pohlemann,et al.  The liquid overlay technique is the key to formation of co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells. , 2011, Cytotherapy.

[49]  Jens Hauslage,et al.  Ground-based facilities for simulation of microgravity: organism-specific recommendations for their use, and recommended terminology. , 2013, Astrobiology.

[50]  W. Roos,et al.  The role of the cytoskeleton in sensing changes in gravity by nonspecialized cells , 2014, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[51]  M. Shibuya.  Involvement of Flt-1 (VEGF receptor-1) in cancer and preeclampsia , 2011, Proceedings of the Japan Academy. Series B, Physical and biological sciences.

[52]  M Hughes-Fulford,et al.  Function of the cytoskeleton in gravisensing during spaceflight. , 2003, Advances in space research : the official journal of the Committee on Space Research.

[53]  F. Strollo,et al.  Microgravity-induced apoptosis in cultured glial cells. , 2010, European journal of histochemistry : EJH.

[54]  D. Waugh,et al.  The Interleukin-8 Pathway in Cancer , 2008, Clinical Cancer Research.

[55]  C. Chi,et al.  Osteopontin promotes integrin activation through outside-in and inside-out mechanisms: OPN-CD44V interaction enhances survival in gastrointestinal cancer cells. , 2007, Cancer research.

[56]  Wei Liu,et al.  Osteopontin (OPN) expression in thyroid carcinoma. , 2010, Anticancer research.

[57]  A. Wells,et al.  Profilin-1 is a negative regulator of mammary carcinoma aggressiveness , 2007, British Journal of Cancer.

[58]  Jeffrey W. Clark,et al.  Efficacy, safety, and biomarkers of neoadjuvant bevacizumab, radiation therapy, and fluorouracil in rectal cancer: a multidisciplinary phase II study. , 2009, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[59]  Borst,et al.  Technology and Developments for the Random Positioning Machine, RPM , 2010 .

[60]  C. Dong,et al.  IL-17 family member cytokines: regulation and function in innate immunity. , 2010, Cytokine & growth factor reviews.

[61]  X. Lu,et al.  Overexpression of osteopontin induces angiogenesis of endothelial progenitor cells via the avβ3/PI3K/AKT/eNOS/NO signaling pathway in glioma cells. , 2011, European journal of cell biology.

[62]  A. Sickmann,et al.  Interaction of Proteins Identified in Human Thyroid Cells , 2013, International journal of molecular sciences.

[63]  G. Weber.  The cancer biomarker osteopontin: combination with other markers. , 2011, Cancer genomics & proteomics.

[64]  Astrid Horn,et al.  Spheroid formation of human thyroid cancer cells in an automated culturing system during the Shenzhou-8 Space mission. , 2013, Biomaterials.

[65]  Jeanne L. Becker,et al.  Using space-based investigations to inform cancer research on Earth , 2013, Nature Reviews Cancer.

[66]  Marian L. Lewis,et al.  Spaceflight alters microtubules and increases apoptosis in human lymphocytes (Jurkat) , 1998, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[67]  S. Löfgren,et al.  Expression of interleukin-17 in human colorectal cancer. , 2006, Anticancer research.

[68]  Ruth Hemmersbach,et al.  Differential gene expression profile and altered cytokine secretion of thyroid cancer cells in space , 2014, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[69]  S. Gaffen.  An overview of IL-17 function and signaling. , 2008, Cytokine.

[70]  M. Braun,et al.  Spheroid formation of human thyroid cancer cells under simulated microgravity: a possible role of CTGF and CAV1 , 2014, Cell Communication and Signaling.

[71]  D. Grimm,et al.  How and why does the proteome respond to microgravity? , 2011, Expert review of proteomics.

[72]  R. Grindeland,et al.  Cosmos 1887 mission overview: effects of microgravity on rat body and adrenal weights and plasma constituents , 1990, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[73]  N. Ferrara,et al.  The biology of VEGF and its receptors , 2003, Nature Medicine.

[74]  Marcel Egli,et al.  Simulated Microgravity: Critical Review on the Use of Random Positioning Machines for Mammalian Cell Culture , 2015, BioMed research international.

[75]  S. Baba,et al.  Profilin 1 overexpression in renal cell carcinoma , 2011, International journal of urology : official journal of the Japanese Urological Association.

[76]  G. Schulze-Tanzil,et al.  Simulated microgravity alters differentiation and increases apoptosis in human follicular thyroid carcinoma cells , 2002, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[77]  G. Kundu,et al.  Osteopontin promotes vascular endothelial growth factor-dependent breast tumor growth and angiogenesis via autocrine and paracrine mechanisms. , 2008, Cancer research.

[78]  A. Flyvbjerg,et al.  Plasma neutrophil gelatinase associated lipocalin (NGAL) is associated with kidney function in uraemic patients before and after kidney transplantation , 2012, BMC Nephrology.

[79]  D. Sheer,et al.  Characterisation of human thyroid epithelial cells immortalised in vitro by simian virus 40 DNA transfection. , 1989, British Journal of Cancer.

[80]  P. Herrlich,et al.  CD44: From adhesion molecules to signalling regulators , 2003, Nature Reviews Molecular Cell Biology.

[81]  Ruth Hemmersbach,et al.  Gravity‐sensitive signaling drives 3‐dimensional formation of multicellular thyroid cancer spheroids , 2012, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[82]  Ronald J. White,et al.  Humans in space , 2001, Nature.