Tuberous sclerosis complex (TSC) is an inherited genetic disorder characterized by mutations in TSC1 or TSC2 class of tumor suppressers which impact several organs including the kidney. The renal manifestations are usually in the form of angiomyolipoma (AML, in 80% of the cases) and cystadenomas. mTOR inhibitors such as rapamycin and everolimus have shown efficacy in reducing the renal tumor burden. Early treatment prevents the progression of AML; however, the tumors regrow upon cessation of therapy implying a lifelong need for monitoring and management of this morbid disease. There is a critical need for development of imaging strategies to monitor response to therapy and progression of disease which will also facilitate development of newer targeted therapy. In this study we evaluated the potential of multiparametric ¹H magnetic resonance imaging (mpMRI) to monitor tumor response to therapy in a preclinical model of TSC, the transgenic mouse A/J Tsc2^+/- . We found 2-dimensional T₂-weighted sequence with 0.5 mm slice thickness to be optimal for detecting renal lesions as small as 0.016 mm³. Baseline characterization of lesions with MRI to assess physiological parameters such as cellularity and perfusion is critical for distinguishing between cystic and solid lesions. Everolimus treatment for three weeks maintained tumor growth at 36% from baseline, while control tumors displayed steady growth and were 70% larger than baseline at the end of therapy. Apparent diffusion coefficient, T₁ values and normalized T₂ intensity changes were also indictive of response to treatment. Our results indicate that standardization and implementation of improved MR imaging protocols will significantly enhance the utility of mpMRI in determining the severity and composition of renal lesions for better treatment planning.

In this work, we present a new ultrafast method for acquiring dynamic 2D EXchange SpectroscopY (EXSY) within a single acquisition. This technique reconstructs two-dimensional EXSY spectra from one-dimensional spectra based on the phase accrual during echo times. The Ultrafast-EXSY acquisition overcomes long acquisition times typically needed to acquire 2D NMR data by utilizing sparsity and phase dependence to dramatically undersample in the indirect time dimension. This allows for the acquisition of the 2D spectrum within a single shot. We have validated this method in simulations and hyperpolarized enzyme assay experiments separating the dehydration of pyruvate and lactate-to-pyruvate conversion. In a renal cell carcinoma cell (RCC) line, bidirectional exchange was observed. This new technique revealed decreased conversion of lactate-to-pyruvate with high expression of monocarboxylate transporter 4 (MCT4), known to correlate with aggressive cancer phenotypes. We also showed feasibility of this technique in vivo in a RCC model where bidirectional exchange was observed for pyruvate-lactate, pyruvate-alanine, and pyruvate-hydrate and were resolved in time. Broadly, the technique is well suited to investigate the dynamics of multiple exchange pathways and applicable to hyperpolarized substrates where chemical exchange has shown great promise across a range of disciplines.

Purpose

The balanced steady-state free precession sequence has been previously explored to improve the efficient use of nonrecoverable hyperpolarized ¹³C magnetization, but suffers from poor spectral selectivity and long acquisition time. The purpose of this study was to develop a novel metabolite-specific 3D bSSFP ("MS-3DSSFP") sequence with stack-of-spiral readouts for improved lactate imaging in hyperpolarized [1-¹³ C]pyruvate studies on a clinical 3T scanner.

Methods

Simulations were performed to evaluate the spectral response of the MS-3DSSFP sequence. Thermal ¹³C phantom experiments were performed to validate the MS-3DSSFP sequence. In vivo hyperpolarized [1-¹³ C], pyruvate studies were performed to compare the MS-3DSSFP sequence with metabolite-specific gradient echo ("MS-GRE") sequences for lactate imaging.

Results

Simulations, phantom, and in vivo studies demonstrate that the MS-3DSSFP sequence achieved spectrally selective excitation on lactate while minimally perturbing other metabolites. Compared with MS-GRE sequences, the MS-3DSSFP sequence showed approximately a 2.5-fold SNR improvement for lactate imaging in rat kidneys, prostate tumors in a mouse model, and human kidneys.

Conclusions

Improved lactate imaging using the MS-3DSSFP sequence in hyperpolarized [1-¹³ C]pyruvate studies was demonstrated in animals and humans. The MS-3DSSFP sequence could be applied for other clinical applications such as in the brain or adapted for imaging other metabolites such as pyruvate and bicarbonate.

The goal of the study was to establish early hyperpolarized (HP) ¹³ C MRI metabolic and perfusion changes that predict effective high-intensity focused ultrasound (HIFU) ablation and lead to improved adjuvant treatment of partially treated regions. To accomplish this a combined HP dual-agent (¹³ C pyruvate and ¹³ C urea) ¹³ C MRI/multiparametric ¹ H MRI approach was used to measure prostate cancer metabolism and perfusion 3-4 h, 1 d, and 5 d after exposure to ablative and sub-lethal doses of HIFU within adenocarcinoma of mouse prostate tumors using a focused ultrasound applicator designed for murine studies. Pathologic and immunohistochemical analysis of the ablated tumor demonstrated fragmented, non-viable cells and vasculature consistent with coagulative necrosis, and a mixture of destroyed tissue and highly proliferative, poorly differentiated tumor cells in tumor tissues exposed to sub-lethal heat doses in the ablative margin. In ablated regions, the intensity of HP ¹³ C lactate or HP ¹³ C urea and dynamic contrast-enhanced (DCE) MRI area under the curve images were reduced to the level of background noise by 3-4 h after treatment with no recovery by the 5 d time point in either case. In the tissues that received sub-lethal heat dose, there was a significant 60% ± 12.4% drop in HP ¹³ C lactate production and a significant 30 ± 13.7% drop in urea perfusion 3-4 h after treatment, followed by recovery to baseline by 5 d after treatment. DCE MRI K^trans showed a similar trend to HP ¹³ C urea, demonstrating a complete loss of perfusion with no recovery in the ablated region, while having a 40%-50% decrease 3-4 h after treatment followed by recovery to baseline values by 5 d in the margin region. The utility of the HP ¹³ C MR measures of perfusion and metabolism in optimizing focal HIFU, either alone or in combination with adjuvant therapy, deserves further testing in future studies.

Purpose

To compare the apparent diffusion coefficient (ADC) of hyperpolarized (HP) [¹³ C,¹⁵ N]urea to the ADC of endogenous water in healthy and fibrotic mouse liver.

Materials and methods

ADC measurements for water and [¹³ C]urea were made in agarose phantoms at 14.1T. Next, the ADC of water and injected HP [¹³ C,¹⁵ N]urea were measured in eight CD1 mouse livers before and after induction of liver fibrosis using CCl₄ . Liver fibrosis was quantified pathologically using the modified Brunt fibrosis score and compared to the measured ADC of water and urea.

Results

In cell-free phantoms with 12.5% agarose, water ADC was nearly twice the ADC of urea (1.93 × 10^-3 mm² /s vs. 1.00 × 10^-3 mm² /s). The mean ADC values of water and [¹³ C,¹⁵ N]urea in healthy mouse liver (±SD) were nearly identical [(0.75 ± 0.11) × 10^-3 mm² /s and (0.75 ± 0.22) × 10^-3 mm² /s, respectively]. Mean water and [¹³ C,¹⁵ N]urea ADC values in fibrotic liver (±SD) were (0.84 ± 0.22) × 10^-3 mm² /s and (0.75 ± 0.15) × 10^-3 mm² /s, respectively. Neither water nor urea ADCs were statistically different in the fibrotic livers compared to baseline (P = 0.14 and P = 0.99, respectively). Water and urea ADCs were positively correlated at baseline (R²  = 0.52 and P = 0.045) but not in fibrotic livers (R²  = 0.23 and P = 0.23).

Conclusion

ADC of injected hyperpolarized urea in healthy liver reflects a smaller change as compared to free solution than ADC of water. This may reflect differences in cellular compartmentalization of the two compounds. No significant change in ADC of either water or urea were observed in relatively mild stages of liver fibrosis.

Level of evidence

1 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2018;47:141-151.

There is an unmet clinical need for new and robust imaging biomarkers to distinguish indolent from aggressive prostate cancer. Hallmarks of aggressive tumors such as a decrease in extracellular pH (pH_e) can potentially be used to identify aggressive phenotypes. In this study, we employ an optimized, high signal-to-noise ratio hyperpolarized (HP) ¹³C pH_e imaging method to discriminate between indolent and aggressive disease in a murine model of prostate cancer. Transgenic adenocarcinoma of the mouse prostate (TRAMP) mice underwent a multiparametric MR imaging exam, including HP [¹³C] bicarbonate MRI for pH_e, with ¹H apparent diffusion coefficient (ADC) mapping and HP [1-¹³C] pyruvate MRI to study lactate metabolism. Tumor tissue was excised for histological staining and qRT-PCR to quantify mRNA expression for relevant glycolytic enzymes and transporters. We observed good separation in pH_e between low- and high-grade tumor regions, with high-grade tumors demonstrating a lower pH_e. The pH_e also correlated strongly with monocarboxylate transporter Mct4 gene expression across all tumors, suggesting that lactate export via MCT4 is associated with acidification in this model. Our results implicate extracellular acidification as an indicator of indolent-to-aggressive transition in prostate cancer and suggest feasibility of HP pH_e imaging to detect high-grade, clinically significant disease in men as part of a multiparametric MRI examination.

Vorinostat is a histone deacetylase (HDAC) inhibitor that inhibits cell proliferation and induces apoptosis in solid tumors, and is in clinical trials for the treatment of glioblastoma (GBM). The goal of this study was to assess whether hyperpolarized ¹³ C MRS and magnetic resonance spectroscopic imaging (MRSI) can detect HDAC inhibition in GBM models. First, we confirmed HDAC inhibition in U87 GBM cells and evaluated real-time dynamic metabolic changes using a bioreactor system with live vorinostat-treated or control cells. We found a significant 40% decrease in the ¹³ C MRS-detectable ratio of hyperpolarized [1-¹³ C]lactate to hyperpolarized [1-¹³ C]pyruvate, [1-¹³ C]Lac/Pyr, and a 37% decrease in the pseudo-rate constant, k_PL , for hyperpolarized [1-¹³ C]lactate production, in vorinostat-treated cells compared with controls. To understand the underlying mechanism for this finding, we assessed the expression and activity of lactate dehydrogenase (LDH) (which catalyzes the pyruvate to lactate conversion), its associated cofactor nicotinamide adenine dinucleotide, the expression of monocarboxylate transporters (MCTs) MCT1 and MCT4 (which shuttle pyruvate and lactate in and out of the cell) and intracellular lactate levels. We found that the most likely explanation for our finding that hyperpolarized lactate is reduced in treated cells is a 30% reduction in intracellular lactate levels that occurs as a result of increased expression of both MCT1 and MCT4 in vorinostat-treated cells. In vivo ¹³ C MRSI studies of orthotopic tumors in mice also showed a significant 52% decrease in hyperpolarized [1-¹³ C]Lac/Pyr when comparing vorinostat-treated U87 GBM tumors with controls, and, as in the cell studies, this metabolic finding was associated with increased MCT1 and MCT4 expression in HDAC-inhibited tumors. Thus, the ¹³ C MRSI-detectable decrease in hyperpolarized [1-¹³ C]lactate production could serve as a biomarker of response to HDAC inhibitors.

Purpose

Image-guided prostate biopsies are routinely acquired in the diagnosis and treatment monitoring of prostate cancer, yielding useful tissue for identifying metabolic biomarkers and therapeutic targets. We developed an optimized biopsy tissue culture protocol in combination with [1,6-¹³ C₂ ]glucose labeling and quantitative high-resolution NMR to measure glycolysis and tricarboxcylic acid (TCA) cycle activity in freshly acquired living human prostate biopsies.

Methods

We acquired 34 MRI-ultrasound fusion-guided prostate biopsies in vials on ice from 22 previously untreated patients. Within 15 min, biopsies were transferred to rotary tissue culture in 37°C prostate medium containing [1,6-¹³ C₂ ]glucose. Following 24 h of culture, tissue lactate and glutamate pool sizes and fractional enrichments were quantified using quantitative ¹ H high resolution magic angle spinning Carr-Purcell-Meiboom-Gill (CPMG) spectroscopy at 1°C with and without ¹³ C decoupling. Lactate effluxed from the biopsy tissue was quantified in the culture medium using quantitative solution-state high-resolution NMR.

Results

Lactate concentration in low-grade cancer (1.15 ± 0.78 nmol/mg) and benign (0.74 ± 0.15 nmol/mg) biopsies agreed with prior published measurements of snap-frozen biopsies. There was substantial fractional enrichment of [3-¹³ C]lactate (≈70%) and [4-¹³ C]glutamate (≈24%) in both low-grade cancer and benign biopsies. Although a significant difference in tissue [3-¹³ C]lactate fractional enrichment was not observed, lactate efflux was significantly higher (P < 0.05) in low-grade cancer biopsies (0.55 ± 0.14 nmol/min/mg) versus benign biopsies (0.31 ± 0.04 nmol/min/mg).

Conclusion

A protocol was developed for quantification of lactate production-efflux and TCA cycle activity in single living human prostate biopsies, allowing metabolic labeling on a wide spectrum of human tissues (e.g., metastatic, post-non-surgical therapy) from patients not receiving surgery.

70-90% of low-grade gliomas and secondary glioblastomas are characterized by mutations in isocitrate dehydrogenase 1 (IDHmut). IDHmut produces the oncometabolite 2-hydroxyglutarate (2HG), which drives tumorigenesis in these tumors. The phosphoinositide-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway represents an attractive therapeutic target for IDHmut gliomas, but noninvasive indicators of drug target modulation are lacking. The goal of this study was therefore to identify magnetic resonance spectroscopy (MRS)-detectable metabolic biomarkers associated with IDHmut glioma response to the dual PI3K/(mTOR) inhibitor XL765. ¹H-MRS of two cell lines genetically modified to express IDHmut showed that XL765 induced a significant reduction in several intracellular metabolites including 2HG. Importantly, examination of an orthotopic IDHmut tumor model showed that enhanced animal survival following XL765 treatment was associated with a significant in vivo ¹H-MRS detectable reduction in 2HG but not with significant inhibition in tumor growth. Further validation is required, but our results indicate that 2HG could serve as a potential noninvasive MRS-detectable metabolic biomarker of IDHmut glioma response to PI3K/mTOR inhibition.

Although lower grade gliomas are driven by mutations in the isocitrate dehydrogenase 1 (IDH1) gene and are less aggressive than primary glioblastoma, they nonetheless generally recur. IDH1-mutant patients are increasingly being treated with temozolomide, but early detection of response remains a challenge and there is a need for complementary imaging methods to assess response to therapy prior to tumor shrinkage. The goal of this study was to determine the value of magnetic resonance spectroscopy (MRS)-based metabolic changes for detection of response to temozolomide in both genetically engineered and patient-derived mutant IDH1 models. Using ¹H MRS in combination with chemometrics identified several metabolic alterations in temozolomide-treated cells, including a significant increase in steady-state glutamate levels. This was confirmed in vivo, where the observed ¹H MRS increase in glutamate/glutamine occurred prior to tumor shrinkage. Cells labeled with [1-¹³C]glucose and [3-¹³C]glutamine, the principal sources of cellular glutamate, showed that flux to glutamate both from glucose via the tricarboxylic acid cycle and from glutamine were increased following temozolomide treatment. In line with these results, hyperpolarized [5-¹³C]glutamate produced from [2-¹³C]pyruvate and hyperpolarized [1-¹³C]glutamate produced from [1-¹³C]α-ketoglutarate were significantly higher in temozolomide-treated cells compared with controls. Collectively, our findings identify ¹H MRS-detectable elevation of glutamate and hyperpolarized ¹³C MRS-detectable glutamate production from either pyruvate or α-ketoglutarate as potential translatable metabolic biomarkers of response to temozolomide treatment in mutant IDH1 glioma. SIGNIFICANCE: These findings show that glutamate can be used as a noninvasive, imageable metabolic marker for early assessment of tumor response to temozolomide, with the potential to improve treatment strategies for mutant IDH1 patients.