Differentiation of Col I and Col III isoforms in stromal models of ovarian cancer by analysis of second harmonic generation polarization and emission directionality

Abstract

A profound remodeling of the extracellular matrix occurs in many epithelial cancers. In ovarian cancer, the minor collagen isoform of Col III becomes upregulated in invasive disease. Here we use second harmonic generation (SHG) imaging microscopy to probe structural differences in fibrillar models of the ovarian stroma comprised of mixtures of Col I and III. The SHG intensity and forward-backward ratios decrease with increasing Col III content, consistent with decreased phasematching due to more randomized structures. We further probe the net collagen α-helix pitch angle within the gel mixtures using what is believed to be a new pixel-based polarization-resolved approach that combines and extends previous analyses. 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. We further developed the pixel-based approach to extract the SHG signal polarization anisotropy from the same polarization-resolved image matrix. Using this approach, we found that increased Col III results in decreased alignment of the dipole moments within the focal volume. Collectively, the SHG measurements and analysis all indicate that incorporation of Col III results in decreased organization across several levels of collagen organization. Furthermore, the findings suggest that the collagen isoforms comingle within the same fibrils, in good agreement with ultrastructural data. The pixel-based polarization analyses (both excitation and emission) afford determination of structural properties without the previous requirement of having well-aligned fibers, and the approaches should be generally applicable in tissue.

Figure 1

Single SHG optical sections of Col I/Col III gels, increasing from 0, 5, 10, 15, 20, and 40% Col III with the balance of Col I (a–f). The raw intensities are shown, which decrease with increasing Col III content. (Inset, panel f) For viewing purposes, the contrast for 40% Col III was stretched. Scale bar = 50 μm.

Figure 2

Forward/backward ratios (a and b) as a function of depth for the series of Col I/III gels. After thresholding, the intensities were integrated over the field of view for three series of experiments. The overall trend is that the F/B ratio decreases with increasing Col III content. The statistical analysis is in Table 1. To see this figure in color, go online.

Figure 3

Flowchart of the processes used to extract the α-helix pitch angle and SHG polarization anisotropy, beginning with 324 images of 18 × 18 polarization excitation and collection angles. To see this figure in color, go online.

Figure 4

Pixel maps of the p and q parameters (obtained by Eq. 11) for 100% Col I (top panel) and 60% Col I/40% Col III (bottom panel) along with the resulting histograms of the distribution. To see this figure in color, go online.

Figure 5

Using the most probable P and Q values, SHG polarization responses are reconstructed by Eq. 12 for mixed different percentage type-III collagen gels. The result for 100% Col I is nearly identical to that previously obtained by the single-axis molecular model. To see this figure in color, go online.

Figure 6

Pixel-based SHG anisotropy responses are determined by using θ_o (Eq. 11) to create a transformed image matrix, M2, of equivalent fiber axes and then calculating β every 10°. The result for 100% Col I is nearly identical to that previously obtained by performing the anisotropy on a fiber-by-fiber basis. To see this figure in color, go online.