Abstract A05: Evaluation of in vitro three dimensional breast cancer surrogates using histologic morphology and non-invasive imaging to monitor growth and viability throughout culture
Authors: Goliwas Kayla F. , Richter Jillian R. , Marshall Lauren E. , Berry Joel L. , Frost Andra R. 
Published: March 2017

Abstract
Introduction: Tumor dimensionality creates a dynamic three dimensional (3D) architecture that is influenced by the associated microenvironment, including stromal cells and the extracellular matrix. These paracrine interactions impact therapeutic efficacy and can alter drug response in vivo , yet most current in vitro models do not accurately recapitulate the dimensionality or the stromal microenvironment of human tumors. In vitro models that are more recapitulative of the human tumor microenvironment have broad applicability in evaluation of signaling pathways driving cancer progression, therapeutic efficacy, and mechanisms involved in therapeutic resistance and tumor recurrence. There is a great need to adapt traditional analytical methods, developed for two dimensional cell culture, for use in 3D tissue models. Herein, a novel perfusion bioreactor system is used to support the multi-week growth and development of 3D breast carcinoma tissue surrogates (measuring 1.0 cm in maximum dimension) consisting of breast carcinoma epithelial cell lines and cancer associated fibroblasts (CAF) in a supportive extracellular matrix. Further, non-invasive imaging techniques, commonly employed to evaluate in vivo animal model systems, were used to measure growth of the surrogates overtime.
Methods: 3D breast carcinoma surrogates were generated by incorporating MDA-MB-231 cells (tagged with GFP and luciferase) or MCF-7 cells (tagged with GFP and luciferase), with or without CAF, into an extracellular matrix. Surrogates were cultured in a perfusion bioreactor system for up to 3 weeks. Cell growth was measured on histologic sections of surrogates by counting the number of nucleated cells per surrogate cross-sectional area (cell density). Growth and viability were also determined in the same surrogates over time by using non-invasive fluorescence and luminescence imaging (IVIS 100 system).
Results: The use of a flow perfusion bioreactor system resulted in a marked increase in the cell density of surrogates compared to non-perfused surrogates (perfused: 93.3 nucleated cells/area vs. non-perfused: 32.1 nucleated cells/area) at 21 days culture. Fluorescence and luminescence imaging of surrogates, containing increasing concentrations of breast cancer epithelial cells, were imaged at day 0 to confirm a correlation between signal intensity and cell number using each imaging modality (GFP: R2=0.97, p&lt;0.01, Luciferase: R2=0.99, p=0.053 ). Next, fluorescence imaging of the same 3D breast carcinoma surrogates (containing breast carcinoma cells and CAF) overtime was completed at days 0, 7, and 14 of culture and showed an increase in signal (7.9 fold higher signal at day 14 compared to day 0) indicating growth throughout culture. Similar results were seen with imaging of the luciferase signal where the signal was 28.2 fold higher at day 14 compared to day 0.
Conclusions: The presence of perfusion allows the growth and development of a recapitulative breast carcinoma surrogate with a size similar to human breast carcinomas at the time of detection and with an appropriate tumor microenvironment. Non-invasive imaging methods have successfully been adapted to evaluate growth of the breast carcinoma surrogates throughout multi-week culture.
Future Directions: The use of these perfused breast carcinoma surrogates and imaging modalities in the evaluation of established and candidate cancer therapeutics will be assessed.
Citation Format: Kayla F. Goliwas, Jillian R. Richter, Lauren E. Marshall, Joel L. Berry, Andra R. Frost. Evaluation of in vitro three dimensional breast cancer surrogates using histologic morphology and non-invasive imaging to monitor growth and viability throughout culture. [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 A05.