In Vivo Optical Metabolic Imaging of Long-Chain Fatty Acid Uptake in Orthotopic Models of Triple-Negative Breast Cancer

Simple Summary: A dysregulated metabolism is a hallmark of cancer. Once understood, tumor metabolic reprogramming can lead to targetable vulnerabilities, spurring the development of novel treatment strategies. Beyond the common observation that tumors rely heavily on glucose, building evidence indicates that a subset of tumors use lipids to maintain their proliferative or metastatic phenotype. This study developed an intra-vital microscopy method to quantify lipid uptake in breast cancer murine models using a fluorescently labeled palmitate molecule, Bodipy FL c16. This work highlights optical imaging's ability to both measure metabolic endpoints non-destructively and repeatedly, as well as inform small animal metabolic phenotyping beyond in vivo optical imaging of breast cancer alone. Targeting a tumor's metabolic dependencies is a clinically actionable therapeutic approach; however, identifying subtypes of tumors likely to respond remains difficult. The use of lipids as a nutrient source is of particular importance, especially in breast cancer. Imaging techniques offer the opportunity to quantify nutrient use in preclinical tumor models to guide development of new drugs that restrict uptake or utilization of these nutrients. We describe a fast and dynamic approach to image fatty acid uptake in vivo and demonstrate its relevance to study both tumor metabolic reprogramming directly, as well as the effectiveness of drugs targeting lipid metabolism. Specifically, we developed a quantitative optical approach to spatially and longitudinally map the kinetics of long-chain fatty acid uptake in in vivo murine models of breast cancer using a fluorescently labeled palmitate molecule, Bodipy FL c16. We chose intra-vital microscopy of mammary tumor windows to validate our approach in two orthotopic breast cancer models: a MYC-overexpressing, transgenic, triple-negative breast cancer (TNBC) model and a murine model of the 4T1 family. Following injection, Bodipy FL c16 fluorescence increased and reached its maximum after approximately 30 min, with the signal remaining stable during the 30-80 min post-injection period. We used the fluorescence at 60 min (Bodipy(60)), the mid-point in the plateau region, as a summary parameter to quantify Bodipy FL c16 fluorescence in subsequent experiments. Using our imaging platform, we observed a two- to four-fold decrease in fatty acid uptake in response to the downregulation of the MYC oncogene, consistent with findings from in vitro metabolic assays. In contrast, our imaging studies report an increase in fatty acid uptake with tumor aggressiveness (6NR, 4T07, and 4T1), and uptake was significantly decreased after treatment with a fatty acid transport inhibitor, perphenazine, in both normal mammary pads and in the most aggressive 4T1 tumor model. Our approach fills an important gap between in vitro assays providing rich metabolic information at static time points and imaging approaches visualizing metabolism in whole organs at a reduced resolution.

Automated summary

Targeting tumor metabolism is an effective therapeutic approach, with lipids playing a crucial role in certain cancer subtypes, particularly breast cancer. Imaging techniques, such as intra-vital microscopy with fluorescently labeled molecules, provide a dynamic and quantitative way to study metabolic pathways in vivo. Understanding and manipulating lipid uptake in tumors could lead to the development of new drugs that disrupt these metabolic dependencies and hinder tumor growth.