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

Abstract

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 (Bodipy60), 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.

A Spectroscopic Technique to Simultaneously Characterize Fatty Acid Uptake, Mitochondrial Activity, Vascularity, and Oxygen Saturation for Longitudinal Studies In Vivo

“…Multi-parametric metabolic measurements improve the ability to distinguish tissue types. Previously, we have not observed statistically significant differences in TMRE or Bodipy uptake between 67NR and 4T1 tumor lines [ 16 , 30 , 31 ] using our optical toolbox. The findings in this study match previous work.…”

“…Bodipy FL C16 (Ex: 488 nm, Em: 510 nm) and TMRE (Ex: 555 nm, Em: 585 nm) intensity were measured and reported as the average intensity of wavelengths within 2.5 nm of the emission peak. Fluorophore concentrations were chosen to be biologically relevant based on empirical measurements reported previously [ 16 , 22 ]. As expected, both Bodipy FL C16 and TMRE fluorescence increase linearly as the concentration of fluorophore increases (R 2 = 0.9721and R 2 = 0.9183, respectively).…”

“…Phantom pairs contained Bodipy FL C16 at 200 nM, 400 nM, and 600 nM. Concentrations are based on biologically relevant fluorophore concentrations in previous in vivo microscopy studies [ 16 ]. To appropriately compare non-turbid and turbid phantoms, a scaling factor was applied to all non-turbid spectra based on a ratio of the peak intensity of the phantom containing Bodipy FL C16 at 200 nM, low-scatter phantom spectrum, and the fluorophore in a non-turbid phantom containing Bodipy FL C16 at 200 nM, no-scatter phantom spectrum.…”

“…Dysregulated lipid metabolism is an important mechanism for tumor recurrence following therapy [ 12 , 13 ]. Triple Negative Breast Cancers (TNBC), a subtype with particularly poor clinical outcomes [ 14 , 15 ], show increased reliance on fats as an energy source compared to other subtypes of breast cancer [ 16 ]. Higher dependence on both oxidative phosphorylation and fatty acid β-oxidation has been demonstrated in Doxorubicin-resistant TNBC cells [ 17 ].…”

Empirical method for rapid quantification of intrinsic fluorescence signals of key metabolic probes from optical spectra measured on tissue-mimicking turbid medium

“…We envision that our method will be generally applicable to other metabolic probes such as bodipy to report tumor fatty acid uptake. 25 However, when a new probe such as bodipy is introduced, additional tests have to be done to ensure the new probe will be optically and biologically compatible with the existing probes. To implement general use of the empirical model with existing commercial optical filters for spectral data processing, we evaluated the effect of the CWL bands used for correction and their bandwidths on the accuracy of metabolic probe quantification.…”