Synopsis

Keywords: Hyperpolarized MR (Non-Gas), Translational Studies

Metabolic imaging of hyperpolarized pyruvate can provide new insight into tumor progression and response to therapy. Pharmacokinetic modeling can be used to determine k_PL, the apparent rate constant for conversion of hyperpolarized pyruvate into lactate. In this work, we sought to characterize the effect of hyperpolarized pyruvate concentration on intracellular k_PL in ATC cell suspensions using a two-compartment pharmacokinetic model for k_PL quantification. Improved understanding of the effects of concentration on intracellular chemical conversion rates could lead to more accurate quantification of in vivo and clinical hyperpolarized MRI imaging biomarkers of metabolism.

Introduction

Hyperpolarized (HP) MRI is an emerging metabolic imaging method that permits the quantification of the conversion of HP [1-¹³C]-pyruvate into lactate^1-3, which can provide useful insight into the metabolic state of tumor tissue in real time. To date, HP ¹³C MRI has demonstrated strong clinical potential for characterization of tumor metabolism and response to therapy in a variety of cancer types^4-8. Pharmacokinetic (PK) modeling and analysis can be used to determine k_PL, the apparent rate constant for conversion of HP [1-¹³C]-pyruvate into lactate². However, further validation is needed in order to prove the potential of this metabolic imaging biomarker to guide cancer diagnosis and treatment.

In our previous work⁹, we evaluated intracellular k_PL in suspensions of Hth-83 anaplastic thyroid cancer (ATC) cells using ion chromatography-mass spectrometry (IC-MS) and HP nuclear magnetic resonance (NMR) imaging modalities; a closed PK model with two physical compartments (intracellular, extracellular) and two chemical pools (pyruvate, lactate) was used for quantification of intracellular k_PL values. Interestingly, IC-MS results revealed that the intracellular pyruvate and lactate pool sizes shifted dramatically, even when using 3 mM HP pyruvate in solution with cells. Improved understanding of the effects of pyruvate concentration on k_PL could lead to more robust analyses of in vivo and clinical HP MRI data. Therefore, the goal of this work was to capture effects of pyruvate concentration on intracellular k_PL using ATC cell suspensions and a closed two-compartment PK model.

Methods

Hyperpolarized NMR: HP [1-¹³C]-pyruvate was prepared using a HyperSense dissolution dynamic nuclear polarization system (Oxford Instruments, Abingdon, UK) as previously described¹⁰. All NMR experiments were conducted using a Spectrospin DPX-300 NMR spectrometer equipped with a broadband 10 mm NMR probe (Bruker, Billerica, MA, USA). Approximately 1×10⁷ Hth-83 ATC cells were suspended in 900 μL of cell media and added to a 10 mm Shigemi tube, along with 100 μL of D₂O¹¹. The Shigemi tube was lowered to isocenter of the NMR for ~15 minutes at 310 K in order to allow the ATC cells to equilibrate to body temperature. Aliquots of 40 mM HP [1-¹³C]-pyruvate were measured and diluted using standard PBS in order to achieve target concentrations of approximately 0.5, 1, 2, 3, 5 and 10 mM. A pulse-acquire sequence with a 10° excitation angle and a 2 s repetition time was used to acquire dynamic ¹³C spectra.

Pharmacokinetic Modeling: A closed PK model with two physical compartments (intracellular, extracellular) and two chemical pools (pyruvate, lactate) was used to analyze HP NMR data (Figure 1)². The following differential equations relate exchange between pyruvate and lactate in extracellular media (m) and within cells (c):

$$\frac{\partial P_m^*(t)}{\partial t}=\frac{k_{ecp}}{v_{m}}P_c^*(t)-(\frac{k_{ecp}}{v_{m}}+R_{Pyr})P_m^*(t)$$
$$\frac{\partial L_m^*(t)}{\partial t}=\frac{k_{ecl}}{v_{m}}L_c^*(t)-(\frac{k_{ecl}}{v_{m}}+R_{Lac})L_m^*(t)$$
$$\frac{\partial P_c^*(t)}{\partial t}=\frac{k_{ecp}}{v_{c}}P_m^*(t)+k_{LP}L_c^*(t)-(\frac{k_{ecp}}{v_{c}}+k_{PL}+R_{Pyr})P_c^*(t)$$
$$\frac{\partial L_c^*(t)}{\partial t}=\frac{k_{ecl}}{v_{c}}L_m^*(t)+k_{PL}P_c^*(t)-(\frac{k_{ecl}}{v_{c}}+k_{LP}+R_{Lac})L_c^*(t)$$
Here, k_ecp and k_ecl represent the transport of pyruvate and lactate across the cell membrane. v_m and v_c denote the extracellular and intracellular volume fraction. The apparent rate constant for chemical conversion of HP pyruvate to lactate is given by k_PL, and the reverse reaction rate is given by k_LP. R_Pyr and R_Lac denote losses due to T1 relaxation. Excitation losses were modeled as instantaneous following each excitation, and the total observed pyruvate (P*) and lactate (L*) signals were calculated as the sum of compartmental signals weighted by their respective volume fractions. To assess the uniqueness of the solutions, nuisance variables were fit to the data while k_PL was varied across a supraphysiological range of values. For these studies, k_PL values were selected at the point on the residual curve where k_PL begins to decrease by <1%. Analysis was conducted using MATLAB R2021a (The MathWorks, Natick, MA, USA).

Results

PK analysis of HP Hth-83 ATC in vivo experiments reveals that the highest k_PL was observed when testing the lowest concentration (0.5 mM). Figure 2 captures the distinct trend that exists between intracellular k_PL and concentration; as the final extracellular concentration continues to increase up to 10 mM, intracellular k_PL values decrease.

These effects can also be explored by analyzing the observed and fit HP pyruvate and lactate analysis curves for the contrasting 0.5 mM and 10 mM final concentration cases (Figure 3). There is a clear increase in the observed lactate AUC for the 0.5 mM final concentration case (Figure 3a, top, blue line), leading to an intracellular k_PL=1.185 s^-1 (Figure 3a, bottom). Contrastingly, there is a clear reduction in the observed lactate AUC for the 10 mM final concentration case (Figure 3b, top, blue line), leading to an intracellular k_PL=0.167 s^-1 (Figure 3b, bottom). The percent difference in the 0.5 and 10 mM k_PL values was found to be approximately 150%.

Discussion and Conclusion

In our previous work⁹, IC-MS data showed a dramatic shift in pyruvate and lactate pool sizes when using 3 mM concentrations of HP pyruvate in media. This lead us to hypothesize that the concentration of pyruvate may have an effect on intracellular k_PL. In this work, we show the distinct relationship between intracellular k_PL and concentration. Additional studies to assess the mechanism and duration of this effect are currently underway. Improved understanding of the effect of concentration on k_PL will allow for more reproducible quantification of k_PL and improved PK modeling of in vivo and clinical HP MRI data.

Acknowledgements

This work was supported by funding from the National Cancer Institute (R01CA211150, R01CA280980) and the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK105346) of the National Institutes of Health, and the Cancer Prevention and Research Institute of Texas (RP170366). The content is solely the responsibility of the authors and does not necessarily represent the official views of the sponsors.