An interplay between reaction-diffusion and cell-matrix adhesion regulates multiscale invasion in early breast carcinomatosis

Abstract

Equal contribution.The progression of cancer in the breast involves multiple reciprocal interactions between malignantly transformed epithelia, surrounding untransformed but affected stromal cells, and the extracellular matrix (ECM) that is remodelled during the process. A quantitative understanding of the relative contribution of such interactions to phenotypes associated with cancer cells can be arrived at through the construction of increasingly complex experimental and computational models. Herein, we introduce a multiscale 3D organo-and patho-typic model that approximates, to an unprecedented extent, the histopathological complexity of a tumor disseminating into its surrounding stromal milieu via both bulk and solitary motility dynamics. End-point and time-lapse microscopic observations of this model allow us to study the earliest steps of cancer invasion as well as the dynamical interactions between the epithelial and stromal compartments. We then construct an agent-based Cellular Potts model that incorporates constituents of the experimental model, as well as places them in similar spatial arrangements. The computational model, which comprises adhesion between cancer cells and the matrices, cell proliferation and apoptosis, and matrix remodeling through reaction-diffusion-based morphogen dynamics, is first trained to phenocopy controls run with the experimental model, wherein one or the other matrices have been removed. The trained computational model successfully predicts phenotypes of the experimental counterparts that are subjected to pharmacological treatments (inhibition of N-linked glycosylation and matrix metalloproteinase activity) and scaffold modulation (alteration of collagen density). Our results suggest that specific permissive regimes of cell-cell and cell-matrix adhesions operating in the context of a reaction-diffusionregulated ECM dynamics, promote multiscale invasion of breast cancer cells and determine the extent to which they migrate through their surrounding stroma. Preparation of cancer cell clusters:Normal/cancer cells were trypsinized using 1:5 diluted 0.25% Trypsin & 0.02% EDTA (HiMedia, TCL007). 30,000 cells per 200 μ L of defined medium (Blaschke et al., 1994) supplemented with 4% rBM (Corning, 354230) were cultured on 3% polyHEMA (Sigma, P3932) coated 96 well plate for 48 hours in a 37 o C humidified incubator with 5% carbon dioxide. 3D invasion assay:rBM-coated clusters were collected into 1.5 mL tubes, centrifuged briefly and supernatant is removed. Acid-extracted rat tail collagen (Gibco, A1048301) was neutralized on ice in the presence of 10X DMEM with 0.1N NaOH such that the final concentration of the collagen is 1 mg/mL. Pellet of clusters was resuspended in 50 μ L of neutralized collagen and seeded in 8-well chambered cover glass (Eppendorf 0030742036) and supplemented with defined medium. 3D cultures were grown in a 37 o C humidified incubator with 5% carbon dioxide. Processing of 3D invasive clusters:3D invasion cultures were washed with phosphate buffered saline pH 7...

ST6GAL1-mediated heterogeneity in sialic acid levels potentiates invasion of breast cancer epithelia

“…The ability of medium 2,6-Sial cells to adhere better to and invade more through ECM than high 2,6-Sial cells led us to hypothesize if the former invades in collective manner or through solitary mesenchymal movement where adhesion to ECM is crucial. To answer the question, we used a customized 3D assay, wherein clusters of cancer cells are coated with lrBM matrix and then embedded within fibrillar Type 1 collagen to mimic the collagen-rich stromal environment (31). In concurrence with our transwell experiments, single medium 2,6-Sial cells ( Fig 3A) were found to radially invade into the Type 1 collagen to a greater extent than high 2,6-Sial cancer cells ( Fig 3B).…”

“…Is greater adhesion of medium 2,6-Sial cancer cells to ECM causal to its enhanced ability for invasion when cultured in 3D separately, or in coculture with, high 2,6-Sial cells? To answer this, we resorted to a recently constructed computational model of cancer cell invasion (31) using the Compucell 3D simulation framework (32). Our model was demonstrated to simulate single cell-and collective-cell migration (individually and in combination, known as multiscale invasion) first through lrBM-like barriers and then collagen-like fibrillar environments, similar to the 3D invasion assay used above.…”

“…3D invasion assay was performed as described previously by our group (31) . Briefly, cancer clusters were made using 30,000 cells in a polyHEMA (Sigma, P3932) coated 96 well plated, defined medium (Blaschke etal, 1994) supplemented with 4% rBM.…”

“…The simulations performed were based from an earlier-established computational model in Compucell3D (31). Compucell3D (CC3D) is an environment that comprises the lattice-based GGH model(GGH: Glazier-Graner-Hogeweg also known as Cellular Potts model (CPM)) with solvers for partial differential equations in order to simulate biological processes, wherein molecules, cells and cellular ensembles may behave concurrently across distinct spatial scales (32,44).…”

“…The initial conditions and the behavior of cells in the model have been calibrated based on the invasive behavior of cancer cells in different types of ECM and upon treatment with pharmacological agents. The chief constituents of the model include cancer cells, nonfibrillar BM matrix, fibrillar Type 1 collagen matrix, degraded and newly synthesized cancer ECM, diffusible activator of ECM (such as MMPs) and its diffusible inhibitor (TIMP) (31). Contact energies related to adhesion between each of these constituents and diffusion and cooperative interaction between the molecules determines the behavior of the simulation and the end stage phenotype.…”