Stroma Regulates Increased Epithelial Lateral Cell Adhesion in 3D Culture: A Role for Actin/Cadherin Dynamics

Abstract

BackgroundCell shape and tissue architecture are controlled by changes to junctional proteins and the cytoskeleton. How tissues control the dynamics of adhesion and cytoskeletal tension is unclear. We have studied epithelial tissue architecture using 3D culture models and found that adult primary prostate epithelial cells grow into hollow acinus-like spheroids. Importantly, when co-cultured with stroma the epithelia show increased lateral cell adhesions. To investigate this mechanism further we aimed to: identify a cell line model to allow repeatable and robust experiments; determine whether or not epithelial adhesion molecules were affected by stromal culture; and determine which stromal signalling molecules may influence cell adhesion in 3D epithelial cell cultures.Methodology/Principal FindingsThe prostate cell line, BPH-1, showed increased lateral cell adhesion in response to stroma, when grown as 3D spheroids. Electron microscopy showed that 9.4% of lateral membranes were within 20 nm of each other and that this increased to 54% in the presence of stroma, after 7 days in culture. Stromal signalling did not influence E-cadherin or desmosome RNA or protein expression, but increased E-cadherin/actin co-localisation on the basolateral membranes, and decreased paracellular permeability. Microarray analysis identified several growth factors and pathways that were differentially expressed in stroma in response to 3D epithelial culture. The upregulated growth factors TGFβ2, CXCL12 and FGF10 were selected for further analysis because of previous associations with morphology. Small molecule inhibition of TGFβ2 signalling but not of CXCL12 and FGF10 signalling led to a decrease in actin and E-cadherin co-localisation and increased paracellular permeability.Conclusions/SignificanceIn 3D culture models, paracrine stromal signals increase epithelial cell adhesion via adhesion/cytoskeleton interactions and TGFβ2-dependent mechanisms may play a key role. These findings indicate a role for stroma in maintaining adult epithelial tissue morphology and integrity.

Tumour cell invasion: an emerging role for basal epithelial cell extrusion

“…This could account for why pancreatic, colon and lung tumours that are driven by KRAS are typically metastatic with poor prognosis. The idea that tumours could metastasize independently of a primary tumour is alarming but not unheard of 51,52 . Indeed, molecular profiling of different tumour types suggests that some tumours develop with higher likelihoods of metastasizing than others 53–56 .…”

Assembly of a Three-Dimensional Multitype Bronchiole Coculture Model Using Magnetic Levitation

“…13,14,17,18,20,21 Three-dimensional cell culture techniques generally depend on promoting direct cell-cell interactions, using cell aggregation techniques like spheroids and pellet cultures, 22,23 or cell-matrix interactions, either with protein gels [24][25][26] or synthetic polymer scaffolds. 27,28 When used for coculture, 3D approaches have varied, including the formation of spheroids with multiple cell types, 29 encapsulated protein gels seeded with multiple cell types, 30 or hybrid methods of 3D cultures and monolayers. 31 In general, these methods have been successful in creating improved in vivo-like conditions, yet these methods often lack the spatial control necessary to organize cells into complex organotypic models that recapitulate tissue environments.…”

DNA hypermethylation in prostate cancer is a consequence of aberrant epithelial differentiation and hyperproliferation

“…30,51 In vitro differentiation of BPH-1 cells. BPH-1 cells were grown in 3D cultures under differentiating conditions as previously described 40,52,53 with some modifications: BPH-1 cells were seeded on a 50%(v/v) Matrigel plug and cultured in KSFM with 2% FCS, 2 mM L-glutamine, 10 nM R1881 and 10 nM b-estradiol, 5 ng/ml EGF, 1 mg/ml FGF, 4% (v/v) Matrigel, in co-culture with prostate stroma derived from a patient with high Gleason grade cancer. RNA extraction and qRT-PCR analysis.…”