Stromal alterations in ovarian cancers via wavelength dependent Second Harmonic Generation microscopy and optical scattering

Abstract

Background: Ovarian cancer remains the most deadly gynecological cancer with a poor aggregate survival rate; however, the specific rates are highly dependent on the stage of the disease upon diagnosis. Current screening and imaging tools are insufficient to detect early lesions and are not capable of differentiating the subtypes of ovarian cancer that may benefit from specific treatments.

Method: As an alternative to current screening and imaging tools, we utilized wavelength dependent collagen-specific Second Harmonic Generation (SHG) imaging microscopy and optical scattering measurements to probe the structural differences in the extracellular matrix (ECM) of normal stroma, benign tumors, endometrioid tumors, and low and high-grade serous tumors.

Results: The SHG signatures of the emission directionality and conversion efficiency as well as the optical scattering are related to the organization of collagen on the sub-micron size scale and encode structural information. The wavelength dependence of these readouts adds additional characterization of the size and distribution of collagen fibrils/fibers relative to the interrogating wavelengths. We found a strong wavelength dependence of these metrics that are related to significant structural differences in the collagen organization and are consistent with the dualistic classification of type I and II serous tumors. Moreover, type I endometrioid tumors have strongly differing ECM architecture than the serous malignancies. The SHG metrics and optical scattering measurements were used to form a linear discriminant model to classify the tissues, and we obtained high accuracy (>90%) between high-grade serous tumors from the other tissue types. High-grade serous tumors account for ~70% of ovarian cancers, and this delineation has potential clinical applications in terms of supplementing histological analysis, understanding the etiology, as well as development of an in vivo screening tool.

Conclusions: SHG and optical scattering measurements provide sub-resolution information and when combined provide superior diagnostic power over clinical imaging modalities. Additionally the measurements are able to delineate the different subtypes of ovarian cancer and may potentially assist in treatment protocols. Understanding the altered collagen assembly can supplement histological analysis and provide new insight into the etiology. These methods could become an in vivo screening tool for earlier detection which is important since ovarian malignancies can metastasize while undetectable by current clinical imaging resolution.

Keywords: Extracellular matrix (ECM); Optical scattering; Ovarian cancer; Second Harmonic Generation (SHG) imaging microscopy.

Fig. 1

Left column shows 3D renderings of forward directed SHG images of representative normal stroma, benign, LGS, endometrioid, and HGS ovarian tumors obtained at 890 nm excitation. The tissue sections were ~100 μm in thickness. Right column is representative H&E staining of the same tissue. Scale bar = 50 μm

Fig. 2

Wavelength dependence of the reduced scattering coefficient μ _s′ over the wavelength range used for SHG imaging (390–535 nm) for the normal stroma and ovarian tumors. The best fit to the scattering power law from the corresponding m factor is shown with the experimental data. All curves are the average response from normal n = 4; benign n = 4; endometrioid n = 3; LGS n = 4; and HGS n = 3. Error bars are standard error

Fig. 3

Extracted F_SHG/B_SHG emission directionality and SHG conversion efficiencies of the ovarian tissues obtained via Monte Carlo simulations. a Wavelength dependent F_SHG/B_SHG emission directionality response from 780 to 1160 nm (excitation wavelengths) and b Average F_SHG/B_SHG emission directionality at 988 nm, where best delineation between the tissues was obtained; p values showing significant differences are indicated. c Wavelength dependent SHG conversion efficiency response from 780 to 1070 nm excitation wavelengths. d Relative conversion efficiencies at 988 nm, where best delineation between tissues was obtained; p values showing significant differences are indicated. All curves are the average response from normal n = 4; benign n = 4, endometrioid n = 3; LGS n = 4; and HGS n = 3. Error bars are standard error

Fig. 4

Collagen fibril assembly based on the wavelength dependent phasematching response. TEM images of normal and HGS ovarian tissues. Cartoons of the LGS, endometrioid, and benign ovarian tissues