Increased progression of tumor-associated collagen signatures and increased local invasion with high collagen density. (a) TACS-1 in 8-week-old normal (wild-type; (i), (ii)) and collagen-dense (col1a1; (iii), (iv)) tumors showing more developed TACS-1 associated with density (early transition between TACS-1 and -2) while showing very early TACS-1 formation in wild-type tumors (shown with yellow arrowheads; white arrowhead indicates a TACS-1 region that is not shown since it is out of the focal plane). The displayed tumor regions ((ii) and (iv)) are at a Δz = 40 μm from collagen signatures ((i) and (iii)). Note the increased endogenous cellular autofluorescence associated with tumor cells in collagen-dense tissues when PyVT/wt (ii) and PyVT/Col1a1 (iv) tumors were imaged sequentially at the same power settings ((ii) versus (iv)). Representative of n = 4 pairs of tumors. (b) Tumors were imaged and MPE (pseudo-colored red) and SHG (pseudo-colored green) signals were separated. At 8 weeks tumors showed early TACS-3 regions and some local invasion in collagen-dense tumors (ii) while PyVT/wt tumors (i) were still primarily bound by collagen (TACS-2) and non-invasive. At 10 weeks, tumors from dense tissues (iv) displayed further regions of TACS-3 progression and an invasive phenotype, compared to control tissues (iii) that were largely non-invasive and had little collagen reorganization. Representative of n ≥ 6 tumors from each background. (c) Quantitative analysis of collagen fiber angles relative to the tumor boundary for 8-week (top) and 10-week (bottom) old animals. PyVT/wt animals displayed little TACS-3 and are primarily non-invasive with only 23% (8 weeks) and 24% (10 weeks) of their fibrils having angles outside of the TACS-2 distribution around 0° (that is less than -15° or more than 15°). In contrast PyVT/Col1a1 tumors were more invasive, possessing a broader fiber distribution and some regions of TACS-3 (distribution around 90°), with 46% and 51% of the fibrils distributed outside of the TACS-2 distribution (0°) at 8 weeks and 10 weeks, respectively (*indicates that the number of events associated with TACS-3/invasion (75° to 105°) was significantly greater). Calculated from at least 185 of tumor regions from at least 6 separate tumors. All scale bars are 25 μm.