Fluorescence lifetime imaging microscopy analysis of invading tumor cells. (a) Intensity and fluorescence lifetime imaging microscopy (FLIM) images of cells away from and near invasive TACS-3 regions showing increased fluorescent intensity and lifetime near invasive regions (left side of images). (b) FLIM images of tumors from 10-week-old PyVT/wt and PyVT/Col1a1 animals confirming the increased TACS-3 for collagen-dense tumors shown in Figure 5. (c) Increased fluorescent lifetimes for invading cells. Like stromal cells the second (long) and mean components are increased in invading cells. However, the short component is also increased in invading cells when compared to cells in the primary tumor mass. Note, 45 measurements for cells within the primary tumor mass and 45 measurements for invading cells adjacent to the tumor primary tumor mass were used to calculate lifetime values. (d) The second (long) component from cells within the primary tumor mass, invading tumor cells, and stromal cells showing a progressive increase as cells move from a primary epithelial tumor phenotype to a more migratory phenotype. *Indicates a statistically significant (p < 0.05) difference following analysis with one-way analysis of variance (ANOVA) with a post-hoc Tukey-Kramer test.