Synopsis

Keywords: Deuterium, Cancer

Aberrant choline phospholipid biosynthesis is a metabolic hallmark of cancer. Choline kinase α (CKα) is the key enzyme in this pathway. Non-invasive methods of imaging CKα have the potential to report on tumor proliferation. Here, using a combination of RNA interference and doxycycline-inducible gene silencing systems, we show that deuterium magnetic resonance spectroscopy following administration of [²H₉]-choline non-invasively reports on CKα activity in patient-derived glioma cells and intracranial tumors. Importantly, [²H₉]-choline provides an early readout of response to chemotherapy in mice bearing intracranial gliomas. Our studies identify a novel agent for metabolic imaging of gliomas and their response to therapy.

Introduction

Elevated phospholipid biosynthesis is a hallmark of cancer¹. Due to the high membrane turnover associated with uncontrolled proliferation, tumor cells upregulate phospholipid biosynthesis¹. Choline is phosphorylated to phosphocholine (PC) and subsequently incorporated into phosphatidylcholine, which is the primary phospholipid in mammalian cells (Fig. 1). Choline kinase α (CKα) is the key enzyme in this pathway¹. Identifying non-invasive methods of imaging CKα activity can enable assessment of tumor burden and response to therapy. Deuterium magnetic resonance spectroscopy (DMRS) following administration of ²H-labeled substrates is a clinically translatable method of imaging metabolic activity in vivo². The goals of the current study were to determine whether [²H₉]-choline tracks CKα activity and to establish the utility of [²H₉]-choline for glioma imaging.

Methods

DMRS in cells: Patient-derived glioblastoma (GBM1) and oligodendroglioma (BT88) cells were cultured as described previously^3-6. CKα expression was silenced by RNA interference^7,8. Non-targeting siRNA was used as control. Concomitantly, cells were incubated in media containing 56mM choline chloride-(trimethyl-d9) ([²H₉]-choline) for 48h. ²H-MR spectra were acquired from live cells on a Varian 14.1T scanner using a 16mm ²H surface coil and a pulse-acquire sequence (TR=260ms, NA=2500, complex points=512, flip angle=64^o, spectral width=2kHz)⁹. Peak integrals were normalized to the natural abundance of semi-heavy water (HDO, 4.75ppm) from a saline-containing vial⁹.
In vivo CKα silencing: To determine whether [²H₉]-choline tracks CKα in vivo, we engineered GBM6 cells to silence CKa in a doxycycline-inducible manner (henceforth referred to as GBM6_doxCKα). GBM6_doxCKα cells were intracranially implanted in SCID mice. Tumor volume was determined by T2-weighted MRI on a 14.1T scanner equipped with a ¹H volume coil and a spin-echo multi-slice sequence¹⁰. Once tumors were ~30mm³, [²H₉]-choline metabolism was evaluated. Following injection of a bolus of [²H₉]-choline (200mg/kg) via a tail-vein catheter, non-localized ²H-MR spectra were acquired over 50min with a pulse-acquire sequence on a Bruker 3T scanner (TR=506.361ms, NA=1000, complex points=256, flip angle=64, spectral width=512.8, temporal resolution = 8min 26s). Mice were then treated with doxycycline (50mg/kg daily) and [²H₉]-choline metabolism assessed. The signal to noise ratio (SNR) of PC was calculated. For spatial localization, 2D chemical shift imaging (CSI; TE/TR=1.04/265ms, FOV=30x30mm2, 8x8, 128 points, 2Hz spectral width, NA=30) was performed on a Bruker 3T scanner.

Response assessment: Patient-derived BT257 astrocytoma cells were intracranially implanted and tumor volume determined by T2-weighted MRI on a 14.1T scanner. Mice were treated with temozolomide (50mg/kg intraperitoneally). Non-localized DMRS was performed to evaluate [²H₉]-choline metabolism before and after treatment on a 14.1T scanner (TR=500ms, NA=500, complex points=512, flip angle=64, spectral width=2kHz, temporal resolution = 4min 10s). Peak integrals were normalized to HDO.

Statistical analysis: Results were expressed as mean±standard deviation. Statistical significance was assessed using an unpaired two-tailed Welch’s t-test with p<0.05 considered significant.

Results

[²H₉]-choline provides a readout of CKα activity: Previous studies indicate that a peak at 3.2 ppm can be observed using DMRS following administration of [²H₉]-choline^11,12. However, since choline, PC and glycerophosphocholine, a degradation product of phosphatidylcholine, all resonate at 3.2 ppm^1,11,12, it is not clear whether this peak reflects choline metabolism to PC via CKα. To determine whether [²H₉]-choline tracks CKα activity, we examined the effect of silencing CKα on [²H₉]-choline metabolism in GBM1 and BT88 cells. We confirmed that CKα activity was significantly reduced in siCKα cells relative to siControl (Fig. 2A). Importantly, silencing CKα resulted in a complete loss of the 3.2 ppm peak following incubation with [²H₉]-choline in both GBM1 and BT88 models, suggesting that this peak is PC (Fig. 2B-2C).

To assess the validity of these results in an in vivo setting, we performed experiments with patient-derived GBM6_doxCKα cells in which CKα was silenced in a doxycycline-inducible manner. We intracranially implanted mice with GBM6_doxCKα cells and examined [²H₉]choline metabolism before and after doxycycline treatment. As shown in Fig. 3A-3B, the peak at 3.2 ppm was completely lost upon CKα silencing in vivo, confirming that the 3.2 ppm peak represents PC. To further validate these results and assess the spatial distribution of [²H₉]choline metabolism, we performed 2D CSI before and after doxycycline treatment in mice bearing intracranial GBM6_doxCKα tumors. As shown in Fig. 4A, metabolic heatmaps showed localization of PC to the tumor vs. normal brain, an effect that was lost following CKα silencing. Collectively these results suggest that [²H₉]choline provides a readout of CKα in vivo.

[²H₉]-choline can be used to non-invasively monitor response to therapy: Next, we examined whether [²H₉]-choline provides a readout of response to temozolomide (TMZ), which is a standard of care for glioma patients¹³. As shown in Fig. 5A, TMZ induced tumor shrinkage in BT257 tumor-bearing mice, an effect that was apparent by day 15. Importantly, PC production from [²H₉]-choline was reduced in BT257 tumor-bearing mice at day 7 (Fig. 5B-5C), when no difference in tumor volume was detectable (Fig. 5A).

Conclusions

Elevated CKα activity is observed in most cancers, including gliomas. Non-invasive methods of assessing CKα activity are needed. Our studies indicate that [²H₉]-choline provides a readout of CKα activity in patient-derived glioma cells and tumors. Importantly, [²H₉]-choline provides an early readout of response to therapy, prior to MRI-detectable volumetric alterations. Clinical translation of [²H₉]-choline has the potential to improve response assessment in glioma patients.

Acknowledgements

This study was supported by NIH R01CA239288, Department of Defense W81XWH201055315 and the UCSF NICO initiative.

References

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