CLSM images show nuclear morphology of Saos-2 and hOB cells tested. (a) R and CV values were calculated from 11 artificially generated templates mimicking nuclear deformations of actual cancer cells (a1). 50 images of each template with various orientations, scale, and software-generated noise were tested. This artificially generated population was processed by our software and the resulting distribution of R and CV values were used to identify 5 main regions of deformation: R1 (R1, CV1 ≤ 0.1, 0.1): no deformation, R2 (0.1, 0.1 < R2, CV2 ≤ 0.2, 0.2): low deformation-more compact, R3 (0.2, 0 < R3, CV3 ≤ 0.5, 0.3) low deformation-less compact, R4 (0, 0.3 < R4, CV4 ≤ 0.2, 0.5) high deformation-more compact and R5 (0.2, 0.3 < R5, CV5 ≤ 0.5, 0.5) high deformation-less compact. (a2) Using the regions identified in (a1), the average nuclear deformability of a population was quantified through a deformation score (DS) calculated by assigning each cell a weight based on the region it fell in (R1 = 1, R2 = 2, R3 = 3, R4 = 4, R5 = 5), and then these weights were averaged for all cells in each population. A deformation score less than or equal to 3 indicates little or no deformation and that the population is non-cancerous. The R and CV values for Saos-2 and hOB cells cultured on Control and P4G4 surfaces are: for hOB, DS = 1.04 on Control and 1.22 on P4G4, and for Saos-2, DS = 1.58 on Control and 3.55 on P4 G4. On the P4G4 surface, 97% of individual hOB cells had DS ≤ 3, and 70% of individual Saos-2 cells had DS > 3. On the Control surface, 99% of hOB cells and 96% of Saos-2 cells had DS ≤ 3, meaning that they were minimally deformed or not deformed at all. (b) Confocal micrographs of Saos-2 nuclei on Control (b1) show no deformations while on P4G4 surface (b2) they show extensive deformations resulting in lobulated “t” and “+” like shapes. Micrographs of hOB nuclei reveal that they do not deform on Control (b3) and minimally deform on P4G4 (b4) surfaces.