Introduction

Glioblastoma multiforme (GBM) brain tumors are among the most lethal of all human cancers [1]. GBM diagnosis and staging rely on contrast-enhanced (CE) MRI to identify tumor-associated blood brain barrier (BBB) disruption; however, infiltrating tumor cells extend beyond BBB boundaries. Consequently, the tumor periphery is invisible on CE-MRI, leading to undertreatment [2],[3]. Here, we seek to combine recent advances in metabolic MRI methods, specifically deuterium metabolic imaging, with the known upregulation of amino acid (AA) transporters in GBM to detect glioma cells independent of BBB disruption, based on their greatly enhanced uptake of deuterium-labelled amino acids [4],[5]. We investigate the hypothesis that enhanced uptake of deuterated AAs in GBM can be measured with ²H MR; further, we provide proof-of-concept data in support of this method using a rodent a model of GBM.

Methods

Quantification of relaxation time constants and lower detection limit: Phantom preparation: A series of d₁₀-leucine phantoms spanning a concentration range of ~200 mM to 20 mM in low melting point agarose were prepared in 1.5 mL centrifuge tubes. T1 quantification: An inversion recovery experiment was used to quantify the T1 of the (-CD₃)₂, -CD₂, gamma-D, alpha-D, and HOD resonances; TR = 2500 ms, 40 exponentially-spaced inversion times ranging from 0.02 to 2 s. T2 quantification: A CPMG experiment was used to quantify T2 of the resonances identified above: TR = 2500 ms, 40 exponentially-spaced echo times ranging from 0.005 to 0.75 s. Detectability studies: ²H SPin-ECho full-Intensity Acquired Localized (SPECIAL) spectroscopy experiments were performed using 4x4x4 mm³ voxels (TR/TE = 250/4.27 ms).

In vivo detection of enhanced leucine uptake in glioma: Animal preparation: Tumor-bearing mice (n=3) were prepared by stereotactically injecting 50k GL261 cells into the dorsal striatum. Tumor-bearing and control (n=3) mice were injected with 500 µL of 100 mM d₁₀-leucine solution subcutaneously (Total dose = 35.3 mg/100 g). Time course ²H MRS: ²H SPECIAL spectra were collected in 5-minute increments up to 70 mins (TR/TE = 450/4.27 ms).

Data analysis: MRS resonance frequencies and amplitudes were estimated using Bayesian probability theory. Deuterated leucine concentrations were calculated based on an assumed endogenous HOD natural abundance of 16.35 mM. 

Results

The leucine-associated (-CD₃)₂ resonance and its T1 and T2 relaxation time constants were determined and used to optimize subsequent in vitro and in vivo detectability experiments ((-CD₃)₂: T1/T2 = 166/148 ms). Detectability at various concentrations was tested; as little as ~200 mM of d₁₀-leucine could be detected (SNR = 4.1) with a voxel size of 4x4x4 mm³ and a total scan time of 45 min. Enhanced uptake of d10-leucine in tumor tissue was shown over 70 minutes, with 5-minute resolution, following a subcutaneous injection (6-fold enhancement at 70 min. time point, Fig.1). Tumoral concentrations as large as 2.06 ± 0.28 mM (at 70 min.) were achieved using doses of 35.5 mg/100 g, while essentially no uptake was seen in normal-appearing brain.

Conclusions/Discussion

These experiments provide the first proof-of-principle data confirming that ²H MR can be used to measure enhanced AA uptake by GBM. The data support the affinity of GBM to circulating leucine, allowing for the possibility to detect glioma cells which infiltrate beyond BBB disruption. Thus, AA ²H MR has the potential to be a significant advance in the accurate and accessible detection of these aggressive brain tumors. Moreover, the success of this study lays a foundation for the study of differential AA metabolism in GBM.

Image/Figure:

Click to view full size

Image/Figure Caption:

Figure 1.  (a) SPECIAL ²H MR spectra of voxels representing tumor and control tissue (see voxel placement in (b)) over 70 minutes post-subcutaneous injection of d10-leucine (averaged into 10-minute intervals, lb = 10 Hz) show an increase in the leucine-associated peak at 0.9 ppm over time. (b) Concentrations of ²H leucine were calculated from (CD₃)₂ peak amplitudes, referenced to the natural abundance HOD peak. Grey dotted lines represent individual animals, while bolded lines represent averages. Increased concentrations of ²H leucine are seen in tumor compared to control tissue.

Author