Elucidation of mechanisms underlying collagen fibril assembly and matrix-induced guidance of cell fate will contribute to the design and expanded use of this biopolymer for research and clinical applications. Here, we define how Type I collagen oligomers affect in-vitro polymerization kinetics as well as fibril microstructure and mechanical properties of formed matrices. Monomers and oligomers were fractionated from acid-solubilized pig skin collagen and used to generate formulations varying in monomer/oligomer content or average polymer molecular weight (AMW). Polymerization half-times decreased with increasing collagen AMW and closely paralleled lag times, indicating that oligomers effectively served as nucleation sites. Furthermore, increasing AMW yielded matrices with increased interfibril branching and had no correlative effect on fibril density or diameter. These microstructure changes increased the stiffness of matrices as evidenced by increases in both shear storage and compressive moduli. Finally, the biological relevance of modulating collagen AMW was evidenced by the ability of cultured endothelial colony forming cells to sense associated changes in matrix physical properties and alter vacuole and capillary-like network formation. This work documents the importance of oligomers as another physiologically-relevant design parameter for development and standardization of polymerizable collagen formulations to be used for cell culture, regenerative medicine, and engineered tissue applications.
The dynamic process of vessel formation and remodeling is essential to embryonic development, post-natal tissue homeostasis and function, wound healing, and next-generation therapeutic vascularization and tissue engineering strategies. Here, uncommon collagen polymer building blocks, specified by their intermolecular cross-link composition, were used to tune the fibril microstructure and physical properties of collagen matrices for purposes of guiding three-dimensional (3D) lumenized vessel network formation by endothelial colony forming cells (ECFC) in vitro. A new and comprehensive 3D vessel morphometric analysis approach was also used to quantify vessel network morphology and architecture parameters. Results show that independent variation of collagen concentration (fibril density) and oligomer:monomer ratio (interfibril branching), two independent determinants of matrix stiffness, yield vessel networks with different architectures and persistence. Increases in fibril density decreased the overall number of vessel networks formed, while an increase in interfibril branching led to formation of highly branched vessel networks with increased vessel volume density. In general, increasing matrix stiffness, whether by modulation of fibril density or interfibril branching, contributed to increased lumen expansion and vessel segment elongation. Finally, oligomer, but not monomer, matrices induced maturation and stabilization (>14 days) of ECFC vessel networks, marked by changes in the temporal and spatial deposition of collagen type IV. Collectively, this work highlights that ECFC vessel morphogenesis is not dependent upon matrix stiffness alone, but rather the interplay of collagen fibril microstructure and matrix physical properties. Furthermore, it identifies oligomers and their associated intermolecular cross-links as new and important design parameters for vascular-inductive matrices for use in cell culture, regenerative medicine, and engineered tissue applications.
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.
While much progress has been made in the war on cancer, highly invasive cancers such as pancreatic cancer remain difficult to treat and anti-cancer clinical trial success rates remain low. One shortcoming of the drug development process that underlies these problems is the lack of predictive, pathophysiologically relevant preclinical models of invasive tumor phenotypes. While present-day 3D spheroid invasion models more accurately recreate tumor invasion than traditional 2D models, their shortcomings include poor reproducibility and inability to interface with automated, high-throughput systems. To address this gap, a novel 3D tumor-tissue invasion model which supports rapid, reproducible setup and user-definition of tumor and surrounding tissue compartments was developed. High-cell density tumor compartments were created using a custom-designed fabrication system and standardized oligomeric type I collagen to define and modulate ECM physical properties. Pancreatic cancer cell lines used within this model showed expected differential invasive phenotypes. Low-passage, patient-derived pancreatic cancer cells and cancer-associated fibroblasts were used to increase model pathophysiologic relevance, yielding fibroblast-mediated tumor invasion and matrix alignment. Additionally, a proof-of-concept multiplex drug screening assay was applied to highlight this model’s ability to interface with automated imaging systems and showcase its potential as a predictive tool for high-throughput, high-content drug screening.
Mass transport within collagen-based matrices is critical to tissue development, repair, and pathogenesis as well as the design of next generation tissue engineering strategies. This work shows how collagen precursors, specified by intermolecular cross-link composition, provide independent control of collagen matrix mechanical and transport properties. Collagen matrices were prepared from tissue-extracted monomers or oligomers. Viscoelastic behavior was measured in oscillatory shear and unconfined compression. Matrix permeability and diffusivity were measured using gravity-driven permeametry and integrated optical imaging, respectively. Both collagen types showed an increase in stiffness and permeability hindrance with increasing collagen concentration (fibril density); however, different physical property-concentration relationships were noted. Diffusivity wasn’t affected by concentration for either collagen type over the range tested. In general, oligomer matrices exhibited a substantial increase in stiffness and only a modest decrease in transport properties when compared to monomer matrices prepared at the same concentration. The observed differences in viscoelastic and transport properties were largely attributed to increased levels of interfibril branching within oligomer matrices. The ability to relate physical properties to relevant microstructure parameters, including fibril density and interfibril branching, is expected to advance the understanding of cell-matrix signaling as well as facilitate model-based prediction and design of matrix-based therapeutic strategies.
The depletion of ARF6 in epithelial cysts causes a striking inversion of glandular orientation. This requires temporal Rac1 inactivation and is accompanied in basement membrane cultures by improperly assembled laminins. In collagen I, these inverted cysts promote integrin-linked fibril linearization reminiscent of matrix remodeling in disease.
Angiopoietin-like 2 (ANGPTL2) has been reported to induce sprouting angiogenesis; however, its role in vasculogenesis, the de novo lumenization of endothelial cells (EC), remains unexplored. We sought to investigate the potential role of ANGPTL2 in regulating human cord blood derived endothelial colony forming cell (ECFC) vasculogenesis through siRNA mediated inhibition of ANGPTL2 gene expression. We found that ECFCs in which ANGPTL2 was diminished displayed a 3-fold decrease in in vitro lumenal area whereas addition of exogenous ANGPTL2 protein domains to ECFCs lead to increased lumen formation within a 3 dimensional collagen assay of vasculogenesis. ECFC migration was attenuated by 36% via ANGPTL2 knockdown (KD) although proliferation and apoptosis were not affected. We subsequently found that JNK, but not ERK1/2, phosphorylation was decreased upon ANGPTL2 KD, and expression of MT1-MMP, known to be regulated by JNK and a critical regulator of EC migration and 3D lumen formation, was decreased in lumenized structures in vitro derived from ANGPTL2 silenced ECFCs. Treatment of ECFCs in 3D collagen matrices with either a JNK inhibitor or exogenous rhTIMP-3 (an inhibitor of MT1-MMP activity) resulted in a similar phenotype of decreased vascular lumen formation as observed with ANGPTL2 KD, whereas stimulation of JNK activity increased vasculogenesis. Based on gene silencing, pharmacologic, cellular, and biochemical approaches, we conclude that ANGPTL2 positively regulates ECFC vascular lumen formation likely through its effects on migration and in part by activating JNK and increasing MT1-MMP expression.
Metastasis is the primary cause of cancer mortality. the primary tumors of colorectal cancer (cRc) often metastasize to the liver. In this study, we have collected 122 samples from 45 CRC patients. Among them, 32 patients have primary tumors, adjacent normal tissues, and matched liver metastases. thirteen patients have primary tumors without distant metastasis and matched normal tissues. characterization of these samples was conducted by whole-exome and RnA sequencing and SNP6.0 analysis. Our results revealed no significant difference in genetic alterations including common oncogenic mutations, whole genome mutations and copy number variations between primary and metastatic tumors. We then assembled gene co-expression networks and identified metastasiscorrelated gene networks of immune-suppression, epithelial-mesenchymal transition (eMt) and angiogenesis as the key events and potentially synergistic drivers associated with cRc metastasis. Further independent cohort validation using published datasets has verified that these specific gene networks are up regulated throughout the tumor progression. the gene networks of eMt, angiogenesis, immune-suppression and t cell exhaustion are closely correlated with the poor patient outcome and intrinsic anti-PD-1 resistance. These results offer insights of combinational strategy for the treatment of metastatic cRc. CRC is the third most common cancer in world with second highest cancer-related mortality worldwide 1. In US alone, it is estimated that approximately 137,000 people are diagnosed, and more than 50,000 are dead from CRC each year. CRC primary tumors often metastasize to the liver, which accounts for most of CRC related death. The molecular mechanism of tumor metastasis remains poorly understood. It is believed to be a multiple step process that includes cells to detach from their original site and invade the neighboring submucosa, extravasate and survive in the vasculature and metastatic site, and eventually reestablish tumor in alien organ 2. Prevention
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