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...