Medical Physics Seminar – Monday, March 24, 2014
Second Harmonic Generation and Optical Scattering Probes of the ECM in Ovarian Cancer
Paul Campagnola, PhD
Professor, Depts of Biomedical Engineering & Medical Physics, LOCI, UW-School of Medicine & Public Health, Madison, WI - USA
A profound remodeling of the extracellular matrix occurs in human ovarian cancer. Here we show that characteristic changes in the architecture of collagen (concentration, fibril/fiber structure) and isoform distribution (Col I vs Col III) in ovarian cancer can be probed quantitatively by several metrics based on Second Harmonic Generation (SHG) imaging microscopy. We first compared the collagen organization in human ex vivo normal ovaries and high grade serous carcinomas using forward-backward (F/B) directional and polarization anisotropy measurements, which are reflective of the fibril and fiber assembly respectively. We found that the stromal collagen is extensively remodeled in the form of new collagen synthesis. We have also compared the collagen structure in borderline, lowgrade, and papillary serous tumors and found intermediate behavior between normal and high grade disease. Lastly, we have measured the spectral dependence of the reduced scattering coefficient in normal ovaries and several types of malignancies and found the respective spectral slopes are consistent with the changes seen by SHG imaging. Taken together, changes in collagen may be an important biomarker in ovarian cancer that can be measured by SHG directional measurements and optical scattering. We also interrogate the structure of in vitro fibrillar models of the stroma comprised of mixtures of Col I and III, where the latter is up-regulated in ovarian cancer. The SHG intensity and forward-backward ratios, decrease with increasing Col III content, consistent with decreased phasematching due to more randomized structures. We also probe the net collagen α-helix pitch angle within the gel mixtures using a new pixel based polarization resolved approach. The extracted pitch angles are consistent with those of peptide models and the method has sufficient sensitivity to differentiate Col I from the Col I/Col III mixtures. The pixel-based approach is also used to determine the SHG signal polarization anisotropy and we found that increased Col III results in decreased alignment of the dipole moments within the focal volume. Lastly, the mixed gels show decreased SHG circular dichroism (CD), indicating the overall chirality is decreased upon Col III incorporation. Collectively, the measurements and analysis all indicate that incorporation of Col III results in decreased organization across several levels of collagen assembly.