3D collagen fibrillar microstructure guides pancreatic cancer cell phenotype and serves as a critical design parameter for phenotypic models of EMT

Abstract

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.

Axl-mediated activation of TBK1 drives epithelial plasticity in pancreatic cancer

“…EMT is a hallmark of metastasis in pancreatic cancer and is critical to cancer cell dissemination [36][37][38]. Within this morphological cellular program, epithelial cancer cells lose contact with the basement membrane and neighboring cells while gaining a more mesenchymal and invasive phenotype [36,39]. Our results show that tumors and isogenic cell lines from the KIC pancreatic GEMM that lack functional TBK1 are more epithelial in gene expression and morphology than KIC tumors containing wild-type Tbk1.…”

“…It is estimated that nearly 90% of cancer mortalities are due to metastases [35], yet TBK1 studies to date have evaluated the effect of TBK1 inhibition only on primary tumor burden. EMT is a hallmark of metastasis in pancreatic cancer and is critical to cancer cell dissemination [36][37][38]. Within this morphological cellular program, epithelial cancer cells lose contact with the basement membrane and neighboring cells while gaining a more mesenchymal and invasive phenotype [36,39].…”

Cancer Associated Fibroblast (CAF) regulation of PDAC parenchymal (CPC) and CSC phenotypes is modulated by ECM composition.

“…Given the above described crosstalk between CAFs and cancer cells and the reported role of the ECM composition in regulating the phenotypes of both PDAC CPCs [5] [18] and CSCs [5], we evaluated the role of the ECM composition on the effect of two concentrations of Conditioned Medium (CM) derived from CAFs isolated from a PDAC patient on the growth, invasive capacity and vascular morphology of each cell type. The experiments were performed with the cells cultured on either 90% Matrigel:10% Collagen I or 20%Matrigel:80% Collagen I since these combinations very well mimic the ECM of early and late stages of PDAC progression, respectively (see Materials and Methods and protocol scheme in Supplemental Figure 1).…”

“…Here, we analyzed the role of different ECM compositions in modulating the effect of CAF-derived signals from conditioned medium of primary cultured CAFs derived from patients with PDAC on the parenchymal (CPC) and CSC populations. These experiments were performed in a previously described three-dimensional (3D) organotypic model of PDAC [5] [18] mimicking the ECM of early (low collagen I levels) and late (high collagen I levels) stage PDAC tumors.…”

Designer hydrogels: Shedding light on the physical chemistry of the pancreatic cancer microenvironment

“…Voytik-Harbin and colleagues examined EMT in PCCs using 3D collagen oligomer gels mixed with different ratios of Matrigel while maintaining constant matrix stiffness [51]. PCCs exhibited more spindle-shaped and single-celled morphology when cultured in soft (100 Pa) collagen oligomer gels, as opposed to round clusters when grew in Matrigel.…”