Figure 7.

Type III collagen (Col3) deficiency alters collagen matrix in fibroblast-derived matrices and the stromal matrix and increases myofibroblast density and alignment in murine (4T1) tumors. A: Second harmonic (SHG) two-photon imaging was used to analyze the fibrillar collagen intensity and organization in Col3^+/+ and Col3^−/− fibroblast-derived matrices. White signal represents collagen fibers. Fibroblasts were isolated from six embryos. B: Collagen signal intensity (percentage SHG-positive area; five images taken per individual fibroblast matrix) was analyzed using ImageJ software. C: Linearity was analyzed by generating fast Fourier transform (FFT) plots in ImageJ software for each image. Signal that produced a more elongate ellipse represents more aligned, organized fibers. D: Linearity was calculated using the FFT plots. E: Hematoxylin and eosin sections of 14-day-old tumors (0.5 × 10⁶ cells injected). F: SHG imaging was used to visualize the fibrillar collagen in central portions of paraffin-embedded 4T1 tumor sections from Col3^+/+ and Col3^+/− mice. G: Sections of 4T1 tumors from Col3^+/+ and Col3^+/− mice (seven per genotype) were stained for α-smooth muscle actin (α-SMA), to label myofibroblasts. Five random images that did not contain tumor edge were taken per tumor. Representative images are shown. Green, α-SMA; blue, DAPI. H and I: ImageJ software was used to quantify the intensity of α-SMA staining (α-SMA–positive area; H) and alignment (I). Data represent means ± SEM. n = 3 Col3^+/+ and Col3^−/− embryos (A). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. Original magnification, ×20 (E–G). ECM, extracellular matrix.