The Feasibly of Functionally Guided Intensity Modulated Radiation Therapy Treatment Planning Using Perfluoropropane-Enhanced MRI for Lung Cancer

Radiotherapy is commonly used to treat lung cancer. However, radiation induced damage to lung tissue is a major limiting factor to its use. To minimize normal tissue lung toxicity from conformal radiotherapy treatment planning, we investigated the use of Perfluoropropane(PFP)-enhanced MR imaging to assess and guide the sparing of functioning lung. Fluorine Enhanced MRI using Perfluoropropane(PFP) is a dynamic multi-breath steady state technique enabling quantitative and qualitative assessments of lung function(1).

Imaging data was obtained from studies previously acquired in the Duke Image Analysis Laboratory. All studies were approved by the Duke IRB. The data was de-identified for this project, which was also approved by the Duke IRB. Subjects performed several breath-holds at total lung capacity(TLC) interspersed with multiple tidal breaths(TB) of Perfluoropropane(PFP)/oxygen mixture. Additive wash-in intensity images were created through the summation of the wash-in phase breath-holds. Additionally, model based fitting was utilized to create parametric images of lung function(1).

Varian Eclipse treatment planning software was used for putative treatment planning. For each subject two plans were made, a standard plan, with no regional functional lung information considered other than current standard models. Another was created using functional information to spare functional lung while maintaining dose to the target lesion. Plans were optimized to a prescription dose of 60 Gy to the target over the course of 30 fractions.

A decrease in dose to functioning lung was observed when utilizing this functional information compared to the standard plan for all five subjects. PFP-enhanced MR imaging is a feasible method to assess ventilatory lung function and we have shown how this can be incorporated into treatment planning to potentially decrease the dose to normal tissue.

A Model of Lung Tumor Angiogenesis in a Biomimetic Poly(ethylene glycol)-based Hydrogel System

Tumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. A major outstanding challenge associated with studying tumor angiogenesis is that existing preclinical models are limited in their recapitulation of in vivo cellular organization in 3D. This disparity highlights the need for better approaches to study the dynamic interplay of relevant cells and signaling molecules as they are organized in the tumor microenvironment. In this thesis, we combined 3D culture of lung adenocarcinoma cells with adjacent 3D microvascular cell culture in 2-layer cell-adhesive, proteolytically-degradable poly(ethylene glycol) (PEG)-based hydrogels to study tumor angiogenesis and the impacts of neovascularization on tumor cell behavior.

In initial studies, 344SQ cells, a highly metastatic, murine lung adenocarcinoma cell line, were characterized alone in 3D in PEG hydrogels. 344SQ cells formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media that significantly increased with exposure to transforming growth factor beta 1 (TGF-β1), a potent tumor progression-promoting factor. Vascular cells alone in hydrogels formed tubule networks with localized activated TGF-β1. To study cancer cell-vascular cell interactions, the engineered 2-layer tumor angiogenesis model with 344SQ and vascular cell layers was employed. Large, invasive 344SQ clusters developed at the interface between the layers, and were not evident further from the interface or in control hydrogels without vascular cells. A modified model with spatially restricted 344SQ and vascular cell layers confirmed that observed 344SQ cluster morphological changes required close proximity to vascular cells. Additionally, TGF-β1 inhibition blocked endothelial cell-driven 344SQ migration.

Two other lung adenocarcinoma cell lines were also explored in the tumor angiogenesis model: primary tumor-derived metastasis-incompetent, murine 393P cells and primary tumor-derived metastasis-capable human A549 cells. These lung cancer cells also formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media. Epithelial morphogenesis varied for the primary tumor-derived cell lines compared to 344SQ cells, with far less epithelial organization present in A549 spheroids. Additionally, 344SQ cells secreted the highest concentration of two of the three angiogenic growth factors assessed. This finding correlated to 344SQ exhibiting the most pronounced morphological response in the tumor angiogenesis model compared to the 393P and A549 cell lines.

Overall, this dissertation demonstrates the development of a novel 3D tumor angiogenesis model that was used to study vascular cell-cancer cell interactions in lung adenocarcinoma cell lines with varying metastatic capacities. Findings in this thesis have helped to elucidate the role of vascular cells in tumor progression and have identified differences in cancer cell behavior in vitro that correlate to metastatic capacity, thus highlighting the usefulness of this model platform for future discovery of novel tumor angiogenesis and tumor progression-promoting targets.