Mechanical characterizations by Atomic Force Microscopy (AFM)

AFM was used to quantify the local stiffness within the histology sections as well as engineering matrices for modulating local tension. Briefly, AFM indentation experiments were carried out with a MFP-3D AFM (Asylum Research, Santa Barbara, CA, USA). A special AFM tip, fitted with a borosilicate glass sphere (5 μm in diameter, 0.05 N × m⁻¹ of spring constant), was acquired from Novascan Tech. The AFM tip was brought down, with maximum strain set at 20% to indent the samples. Next, the Young’s Modulus was calculated. Tissue slides (3 frozen and 1 HE stained) from 3 patients for each category of mammographic density were used for the AFM profiling. H&E stained slide was used to select three regions per tissue compartment (e.g., epithelium, fat, collagen), which adds up to 9 regions per tissue section and 54 regions for the entire sample size. For each region, specified on the H&E stained slide, the corresponding region on the frozen samples was marked. Frozen samples were then thawed, and the size of each region was set at 80 μm × 80 μm. Each region was divided into 9 sub-regions for individual spot test. 3 spot measurements (e.g., triplet indentation), in each sub-region, were performed. These data were then plotted as the mean ± standard deviation (SD) in Fig. 1. The population profiling, for 81 point, is included in the supplementary Fig. 4 as a box plot. The same protocol was applied to measure spatial stiffness of histology sections as well as for calibrating stiffness of the engineered matrices for the embedded 3D cell culture model.