title: CD44 alternative splicing in gastric cancer cells is regulated by culture dimensionality and matrix stiffness.
abstract: Two-dimensional (2D) cultures often fail to mimic key architectural and physical features of the tumor microenvironment. Advances in biomaterial engineering allow the design of three-dimensional (3D) cultures within hydrogels that mimic important tumor-like features, unraveling cancer cell behaviors that would not have been observed in traditional 2D plastic surfaces. This study determined how 3D cultures impact CD44 alternative splicing in gastric cancer (GC) cells. In 3D cultures, GC cells lost expression of the standard CD44 isoform (CD44s), while gaining CD44 variant 6 (CD44v6) expression. This splicing switch was reversible, accelerated by nutrient shortage and delayed at lower initial cell densities, suggesting an environmental stress-induced response. It was further shown to be dependent on the hydrogel matrix mechanical properties and accompanied by the upregulation of genes involved in epithelial-mesenchymal transition (EMT), metabolism and angiogenesis. The 3D cultures reported here revealed the same CD44 alternative splicing pattern previously observed in human premalignant and malignant gastric lesions. These findings indicate that fundamental features of 3D cultures - such as soluble factors diffusion and mechanical cues - influence CD44 expression in GC cells. Moreover, this study provides a new model system to study CD44 dysfunction, whose role in cancer has been in the spotlight for decades.
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, 02115, USA; Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 4200-465, Porto, Portugal; Instituto de Engenharia Biomédica da Universidade do Porto (INEB), 4150-180, Porto, Portugal; Instituto de Investigação e Inovação em Saúde da Universidade do Porto (i3S), 4200-135, Porto, Portugal; Faculdade de Medicina da Universidade do Porto (FMUP)/Hospital S. João, 4200-319, Porto, Portugal.
authors.forename: Cristiana
authors.initials: C
authors.lastname: Branco da Cunha
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, 02115, USA; Dept. Orthopedic Surgery, Research Institute MOVE, VU University Medical Center, 1081, HV, Amsterdam, The Netherlands.
authors.forename: Darinka D
authors.initials: DD
authors.lastname: Klumpers
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, 02115, USA; Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, 02139, USA.
authors.forename: Sandeep T
authors.initials: ST
authors.lastname: Koshy
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, 02115, USA.
authors.forename: James C
authors.initials: JC
authors.lastname: Weaver
authors.affiliation: Department of Mechanical Engineering, Stanford University, Stanford, CA, 94305, USA.
authors.forename: Ovijit
authors.initials: O
authors.lastname: Chaudhuri
authors.affiliation: Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 4200-465, Porto, Portugal; Instituto de Investigação e Inovação em Saúde da Universidade do Porto (i3S), 4200-135, Porto, Portugal.
authors.forename: Raquel
authors.initials: R
authors.lastname: Seruca
authors.affiliation: Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 4200-465, Porto, Portugal; Instituto de Investigação e Inovação em Saúde da Universidade do Porto (i3S), 4200-135, Porto, Portugal; Faculdade de Medicina da Universidade do Porto (FMUP)/Hospital S. João, 4200-319, Porto, Portugal.
authors.forename: Fátima
authors.initials: F
authors.lastname: Carneiro
authors.affiliation: Instituto de Engenharia Biomédica da Universidade do Porto (INEB), 4150-180, Porto, Portugal; Instituto de Investigação e Inovação em Saúde da Universidade do Porto (i3S), 4200-135, Porto, Portugal; Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto (ICBAS), 4050-313, Porto, Portugal; Departamento de Engenharia Metalúrgica e Materiais, Faculdade de Engenharia da Universidade do Porto (FEUP), 4200-465, Porto, Portugal.
authors.forename: Pedro L
authors.initials: PL
authors.lastname: Granja
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, 02115, USA. Electronic address: mooneyd@seas.harvard.edu.
authors.forename: David J
authors.initials: DJ
authors.lastname: Mooney
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D065150:Cellular Reprogramming; D055119:Elastic Modulus; D005109:Extracellular Matrix / Q000378:metabolism*; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics*; D018384:Oxidative Stress; D051381:Rats; D013274:Stomach Neoplasms / Q000235:genetics* / Q000473:pathology
chemical_list: C497483:CD44 protein, human; D018960:Hyaluronan Receptors
keywords: Alternative splicing; CD44; Culture dimensionality; Extracellular matrix (ECM); Matrix stiffness