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Mammary fibroblasts remodel fibrillar collagen microstructure in a biomimetic nanocomposite hydrogel.

ABSTRACT: Architecture and microstructure of type I collagen fibers constitute central regulators of tumor invasion with aligned fibers providing a route for migration of stromal and cancer cells. Several different aspects of fibrillar collagen, such as stiffness, density, thickness, and pore size, may regulate migration of cancer cells, but determining effects of any one parameter requires clear decoupling of physical properties of collagen networks. The objective of this work is to develop and apply an in vitro three-dimensional (3D) tumor-extra cellular matrix (ECM) model with tunable physical parameters to define how stromal fibroblasts modulate collagen microstructure to control migration of breast cancer cells. We incorporated two different types of polyhedral oligomeric silsesquioxane (POSS) nano-molecules into a collagen/alginate matrix to induce different mechanisms of gelling. The resultant biomimetic, nanocomposite hydrogels show different collagen fibrillar microstructures while maintaining constant overall matrix stiffness, density, and porosimetry. Spheroids of human mammary fibroblasts embedded in these 3D matrices remodel the collagen network to varying extents based on differences in underlying matrix microstructures. The remodeled collagen matrix shows oriented, thicker fibrillar tracks, facilitating invasion of tumor cells. By decoupling effects of matrix stiffness and architecture, our nanocomposite hydrogels serve as robust platforms to investigate how biophysical properties of tumor environments control key processes regulating tumor progression in breast cancer and other malignancies. STATEMENT OF SIGNIFICANCE: Our manuscript demonstrates a new type of nanocomposite hydrogel with two different gelling mechanisms, produced by incorporating two types of polyhedral oligomeric silsesquioxane (POSS) nano-molecules into a collagen/alginate matrix. The resultant biomimetic hydrogels show different fibrillar collagen microstructures while maintaining constant overall matrix stiffness, density, and porosimetry. These gels allow us to uncouple effects of matrix stiffness versus architecture on migration and invasion of breast cancer cells and stromal fibroblasts. Upon embedding spheroids of human mammary fibroblasts (HMFs) and dissociated 231 breast cancer cells, we showed that HMFs remodeled the collagen network to differing extents dependent on starting matrix microstructures in each hydrogel. The remodeled collagen matrix showed aligned collagen fibers perpendicular to the surface of a spheroid with migrating HMFs following these fibers as occurs in tumors in vivo. To our knowledge, this is the first study showing significant different fibrillar collagen microstructures with constant collagen density and gel stiffness. This study establishes a new type of nanocomposite 3D hydrogels for studies of biophysical and cellular interactions in engineered tumor environments.

SUBMITTER: Liu C

PROVIDER: S-EPMC6291359 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Mammary fibroblasts remodel fibrillar collagen microstructure in a biomimetic nanocomposite hydrogel.

Liu Chun C Chiang Benjamin B Lewin Mejia Daniela D Luker Kathryn E KE Luker Gary D GD Lee Andre A

Acta biomaterialia 20181107

Architecture and microstructure of type I collagen fibers constitute central regulators of tumor invasion with aligned fibers providing a route for migration of stromal and cancer cells. Several different aspects of fibrillar collagen, such as stiffness, density, thickness, and pore size, may regulate migration of cancer cells, but determining effects of any one parameter requires clear decoupling of physical properties of collagen networks. The objective of this work is to develop and apply an ...[more]

PMID: 30414485

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Project description:UnlabelledNeovascularization is a hallmark of physiological and pathological tissue remodeling that is regulated in part by the extracellular matrix (ECM). Collagen I hydrogels or Matrigel are frequently used to study vascular network formation; however, in isolation these materials do not typically mimic the integrated effects of ECM structure and composition that may influence endothelial cells in vivo. Here, we have utilized microfabricated 3D culture models to control collagen I microstructure in the presence and absence of Matrigel and tested the effect of these variations on vascular network formation by human cerebral microvascular endothelial cells (hCMECs). Varied collagen microarchitecture was achieved by adjusting the gelation temperature and subsequently confirmed by structural analysis. Casting at colder temperature increased collagen fiber thickness and length, and inclusion of Matrigel further pronounced these differences. Interestingly, the presence of Matrigel affected vascular network formation by modulating hCMEC growth, whereas altered collagen fiber structure impacted the morphology and maturity of the developed vascular network. These differences were related to substrate-dependent changes in interleukin-8 (IL-8) secretion and were functionally relevant as vascular networks preformed in more fibrillar, Matrigel-containing hydrogels promoted angiogenic sprouting. Our studies indicate that collagen hydrogel microstructure and composition conjointly regulate vascular network formation with implications for translational and basic science approaches.Statement of significanceNeovascularization is a hallmark of both tissue homeostasis and disease and is in part regulated by cell remodeling that occurs in the extracellular matrix (ECM). The use of bio-mimetic hydrogel cell culture systems has been used to study the effects of the ECM on cell behavior. Here, we employ a hydrogel system that enables control over both the structure and composition of the ECM and subsequently investigated the effects that these have on blood vessel dynamics. Finally, we linked these differences to changes in protein secretion and the implications that this may play in scientific translation.

Project description:Pancreatic cancer, one of the deadliest cancers, is characterized by high rates of metastasis and intense desmoplasia, both of which are associated with changes in fibrillar type I collagen composition and microstructure. Epithelial to mesenchymal transition (EMT), a critical step of metastasis, also involves a change in extracellular matrix (ECM) context as cells detach from basement membrane (BM) and engage interstitial matrix (IM). The objective of this work was to develop and apply an in-vitro three-dimensional (3D) tumor-ECM model to define how ECM composition and biophysical properties modulate pancreatic cancer EMT. Three established pancreatic ductal adenocarcinoma (PDAC) lines were embedded within 3D matrices prepared with type I collagen Oligomer (IM) at various fibril densities to control matrix stiffness or Oligomer and Matrigel combined at various ratios while maintaining constant matrix stiffness. Evaluation of cell morphology and protein expression at both the cellular- and population-levels revealed a spectrum of matrix-driven EMT phenotypes that were dependent on ECM composition and architecture as well as initial PDAC phenotype. In general, exposure to fibrillar IM was sufficient to drive EMT, with cells displaying spindle-shaped morphology and mesenchymal markers, and non-fibrillar BM promoted more epithelial behavior. When cultured within low density Oligomer, only a subpopulation of epithelial BxPC-3 cells displayed EMT while mesenchymal MiaPaCa-2 cells displayed more uniform spindle-shaped morphologies and mesenchymal marker expression. Interestingly, as IM fibril density increased, associated changes in spatial constraints and matrix stiffness resulted in all PDAC lines growing as tight clusters; however mesenchymal marker expression was maintained. Collectively, the comparison of these results to other in-vitro tumor models highlights the role of IM fibril microstructure in guiding EMT heterogeneity and showcases the potential of standardized 3D matrices such as Oligomer to serve as robust platforms for mechanistic study of metastasis and creation of predictive drug screening models.

Project description:ImportanceSpontaneous coronary artery dissection (SCAD) is an increasingly recognized nonatherosclerotic cause of acute myocardial infarction enriched among individuals with early-onset myocardial infarction but is of unclear etiology.ObjectiveTo assess which genes contribute to the development of SCAD.Design, setting, and participantsTo prioritize genes influencing risk for SCAD, whole-exome sequencing was performed among individuals with SCAD in the discovery and replication cohorts from a tertiary care hospital outpatient specialty clinic, and gene set enrichment analyses were also performed for disruptive coding variants. All patients were sequentially enrolled beginning July 2013. Aggregate prevalence of rare disruptive variants for prioritized gene sets was compared between individuals with SCAD with population-based controls comprising 46 468 UK Biobank participants with whole-exome sequencing. Complementary mice models were used for in vivo validation. Analysis took place between June 2020 and January 2021.Main outcomes and measuresThe frequency and identity of rare genetic variants in individuals with SCAD.ResultsOf 130 patients, 109 (83.8%) were female (26 of 32 [81.2%] in the discovery cohort and 83 of 98 [84.7%] in the replication cohort) with mean (SD) age at first SCAD event of 48.41 (8.76) years in the discovery cohort and 47.74 (10.09) years in the replication cohort. Across all patients with SCAD, rare disruptive variants were found within 10 collagen genes (COL3A1, COL5A1, COL4A1, COL6A1, COL5A2, COL12A1, COL4A5, COL1A1, COL1A2, and COL27A1) were 17-fold (P = 1.5 × 10-9) enriched among individuals with SCAD compared with a background of 2506 constrained genes expressed in coronary artery. Furthermore, compared with individuals from the UK Biobank, individuals with SCAD were 1.75-fold (P = .04) more likely to carry disruptive rare variants within fibrillar collagen genes. Complementary mice models haploinsufficient for Col3a1 or Col5a1, the 2 most common collagen gene variants identified in SCAD cases, demonstrated increased risk of arterial dissection and increased size of arterial diameters especially in female mice, with resulting changes in collagen fibril organization and diameter.Conclusions and relevanceUnbiased gene discovery in patients with SCAD with independent human and murine validation highlights the role of the extracellular matrix dysfunction in SCAD.

Dataset Information

Mammary fibroblasts remodel fibrillar collagen microstructure in a biomimetic nanocomposite hydrogel.

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Mammary fibroblasts remodel fibrillar collagen microstructure in a biomimetic nanocomposite hydrogel.

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