Introduction

Altered cellular metabolism has rapidly become a hallmark of cancer research as cancer cells are known to reprogram their metabolic pathways in order to support growth and proliferation [1]. In this regard, fluorescence lifetime imaging microscopy (FLIM) of the intrinsically fluorescent coenzyme NADH [2-3] has evolved as a method to detect cellular levels of ATP produced through oxidative phosphorylation. Additionally, hyperpolarized ¹³C-Magnetic resonance spectroscopy (¹³C-MRS) has proven to be an accurate method to detect the increase in aerobic glycolysis, or “Warburg effect [4]” which is associated with cancer metastasis [5]. Therefore, this study set out to investigate the metabolic shifts associated with cancer dormancy and metastasis in a series of murine non-metastatic, metastatic-dormant, and highly metastatic breast cancer cell lines.

Methods

Murine breast cancer cell lines (N=3/line) derived from a spontaneous mouse mammary tumor were separated based on metastatic potential: 67NR (nonmetastatic), 4T07 (metastatic-dormant), 4T1 (highly metastatic) , and individual 3D matrix gels with 8-million cells and collagen density of 2 mg/ml were prepared [6] in a dual (FLIM/MRS) bioreactor with temperature control [2]. NADH FLIM and hyperpolarized ¹³C-MRS were performed on each 3D cell culture in a single day. The 2D FLIM was conducted with a 20×air objective (NA = 0.75) (Nikon) on a custom multiphoton laser scanning system built around an inverted Nikon TE300 microscope (3). NADH was ex-cited with a wavelength of 740 nm and collected with a 450/70 bandpass filter (Semrock, Rochester, NY, USA). Collection times for each field of view (N=3) were 150 seconds, and data was analyzed using SPC Image (Becker and Hickl, Berlin, Germany). Hyperpolarized ¹³C-MRS measurements were conducted on a 4.7T preclinical magnetic resonance imaging system (Agilent Technologies, Santa Clara, CA) with a dual tuned ¹H/¹³C quadrature radio-frequency volume coil (Doty Scientific, Columbia, SC). Temperature of the cell culture inside the bioreactor was maintained at 36±1°C, and proton line widths were shimmed to ≤30Hz prior to pyruvate injection. [1-13C] Pyruvate (30 μl) (Cambridge Isotope Laboratories Inc., Tewksbury, MA) doped with 15 mM trityl radical (Ox063, GE Healthcare, Waukesha, WI) was hyperpolarized at 3.35T (HyperSense, Oxford Instruments, UK). Following solid-state polarization, the sample was rapidly heated with a 4 mL solvent containing 1.2 mL of 426 mM NaOH, 1.4 mL of 400 mM Tris base, and 1.4 mL of 250 mg/L EDTA. Liquid-state polarization values of 18-25% were achieved, and 150 μl was rapidly injected into the bioreactor via a cannula. Global ¹³C-MRS pulse-acquire spectra (FA/10°, TR/3000 ms, number of acquisitions = 90). Ratios of lactate to pyruvate (Lac/Pyr) were calculated from the Gaussian fit of the respective peak areas via prior knowledge and the AMARES algorithm within the jMRUI v5.2 software package [7-8], after zero-filling (4096) and the summation of the entire time course of ¹³C-MRS spectra. Results were represented as mean±SD, and differences between groups were assessed using Wilcoxon-Mann-Whitney-tests, with p≤0.05 considered to be significant.

Results

Cellular level FLIM measurements of NADH lifetime revealed no differences between mean, free, or bound NADH, which was indicative of constant oxidative phosphorylation across the non-metastatic, metastatic-dormant, and highly metastatic cell lines (Figure 1). Hyperpolarized ¹³C-MRS revealed that slow growing non-metastatic 67NR cell lines did not produce any quantifiable levels of lactate via aerobic glycolysis (Figure 2). However, differences in lactate peaks were clearly observed and quantified using hyperpolarized ¹³C-MRS (Figure 2) in all metastatic-dormant 4T07 cell cultures (Lac/Pyr: 1.8*10^-3±1.3*10^-3), and levels of lactate production were further increased (P<0.5) within highly metastatic 4T1 cells (Lac/Pyr: 4.3*10^-3±2.0*10^-3).

Conclusion

The combination of FLIM imaging of NADH and hyperpolarized ¹³C-MRS detected the real-time rates of oxidative phosphorylation and aerobic glycolysis across murine cell lines of variable metastatic potential. Initial FLIM analysis revealed no differences in free and bound NADH between cell lines, indicative of uniform ATP production through oxidative phosphorylation. However, hyperpolarized ¹³C-MRS measurements clearly detected a metabolic shift to aerobic glycolysis, through the detection of lactate production, which increased with greater breast cancer metastatic potential.

Acknowledgements

No acknowledgement found.