Abstract B17: Quantification of three-dimensional collagen fiber organization as a sensitive indicator of cancerous changes
Authors: Liu Zhiyi , Arendt Lisa , Pouli Dimitra , Kuperwasser Charlotte , Georgakoudi Irene 
Published: March 2017

Abstract
Objectives: There is a lack of metrics that are representative of three-dimensional (3D) collagen fiber organization, despite the availability of high resolution 3D image stacks. This study aims to develop 3D orientation based biomarkers to quantify differences in the organization of collagen fibers between cancerous breast tissues and normal ones.
Methods: We have developed a new method to characterize the 3D orientation of collagen fibers in high-resolution image stacks using a weighted vector sum approach that is computationally efficient and entirely automated in its implementation. In addition, it enables assessment of collagen fiber orientation both locally, i.e. within a specific region of the tissue volume imaged, and globally, i.e. within the entire 3D image stack. Using this approach, we compare the collagen fiber organization between normal mammary glands and mammary tumors. Normal mammary tissues are collected from the fourth mammary gland of non-parous 12 week old FVB/N female mice, while mammary tumors are obtained from mice with breast cancer. These tumors are composed of cancer cells of human origin that have stromalized with mouse ECM and cancer associated fibroblasts as well as immune cells, which are transplanted into the glands of NOD/SCID mice. We use 800 nm as the excitation wavelength, and acquire second harmonic generation (SHG) images at 400 ± 10 nm. A total of 6 SHG 3D image stacks per group (3 stacks per tissue sample) are collected for analysis.
Results: The angular orientation distributions of the fibers indicate a “cross-hatched” organization in the normal tissues, while the formation of helical structures in the tumor specimens. This type of information is not possible to assess quantitatively if only 2D images are analyzed. In addition, we find that the collagen fibers of the tumor specimens exhibit significantly higher levels of alignment than the corresponding healthy mammary tissues. The presence of more highly aligned fibers within tumors is consistent with previous studies which assessed collagen organization using 2D images, even though 3D stacks were available. A possible reason for these changes in cancer may be associated with enhanced presence of newly synthesized collagen fibers.
Significance: These findings are promising in terms of identifying collagen organization as a biomarker for the detection of cancer. We anticipate that quantitative assessment of three-dimensional organization will be a useful quantitative biomarker for a broad range of cancers as well as more fundamental investigations that aim to unravel the organization of essential fiber-like structures in tissues and ultimately its impact on tissue function.
Citation Format: Zhiyi Liu, Lisa Arendt, Dimitra Pouli, Charlotte Kuperwasser, Irene Georgakoudi. Quantification of three-dimensional collagen fiber organization as a sensitive indicator of cancerous changes. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B17.