Keita Saito1, Shingo Matsumoto1,2, Yoichi Takakusagi1,3, Masayuki Matsuo1,4, Hellmut Merkle5, James B Mitchell1, and Murali C Krishna1
1National Cancer Institute, Bethesda, MD, United States, 2Hokkaido University School of Engineering, Sapporo, Japan, 3National Institutes for Quantum and Radiological Science and Technology, Chiba, Japan, 4Gifu University School of Medicine, Gifu, Japan, 5National Institute of Neurological Disorder and Stroke, Bethesda, MD, United States
Synopsis
Effect
of an mTOR inhibitor rapamycin on pyruvate metabolism in squamous cell
carcinoma (SCC) xenografts was investigated using hyperpolarized 13C-MRI.
[1-13C]lactate to [1-13C]pyruvate ratio (Lac/Pyr) in the
SCC tumors increased as tumor grew in non-treated control mice, whereas it
significantly dropped after 2 days of the rapamycin treatment. Inhibition of
mTOR caused a drop of LDH protein level and the activity in the SCC tumor, and
perfusion in the tumor was improved by the rapamycin treatment. Lac/Pyr
monitored using hyperpolarized 13C-MRI would become a useful marker
for tumor response to mTOR inhibitors.
Introduction
Mammalian
target of rapamycin (mTOR) is a protein kinase that is centrally involved in
the control of cancer cell metabolism, growth, and proliferation, and therefore
has become an attractive therapeutic target. Rapamycin is an inhibitor of mTOR,
and inhibition of mTOR by rapamycin causes diverse effects on tumor
microenvironment, such as oxygenation and energy metabolism. Such changes can
serve as markers to assess tumor response to rapamycin therapy. We investigated
effects of rapamycin on pyruvate metabolism to lactate in squamous cell
carcinoma (SCC) xenograft using hyperpolarized 13C-MRI. Lactate dehydrogenase (LDH) is downstream
protein of mTOR and expected to be affected by rapamycin treatments.
Methods
SCC
cells (5x105 cells) were implanted subcutaneously into a right hind
leg of female C3H mice. Treatment with rapamycin and MRI scans were started 8
days after the implantation of the SCC tumor at that time the size of SCC
tumors was around 600 mm3. [1-13C]pyruvate containing 15
mM OX063 was polarized for approximately 1 hour using a hyperpolarizer
(HyperSense, Oxford Instruments), and the hyperpolarized [1-13C]pyruvate
(300 µL of 96 mM solution) was injected intravenously to the tumor-bearing
mice. The MRI scans were carried out using a 4.7 T scanner (Bruker Bio-Spin MRI
GmbH) with a
17 mm home-built 13C solenoid coil placed inside of a saddle coil
for 1H.
Results
To
confirm mTOR inhibition causes a drop of LDH activity in SCC tumor cells, LDH
activity and LDH protein level in SCC cells were assessed with and without
rapamycin (100 nM, 48 h). LDH activity in the cell lysates estimated from NADH
consumption in the presence of pyruvate was significantly smaller in rapamycin
treated cells compared to non-treated control cells (Figure 1). The LDH protein
level in rapamycin treated cells also dropped to 49.1±8.6 % of the non-treated
control SCC cells. Pyruvate metabolism to lactate in SCC tumor xenograft was
evaluated using 13C-MR spectroscopy with hyperpolarized [1-13C]pyruvate.
Signals of [1-13C]pyruvate and [1-13C]lactate were
detected in the SCC tumor immediately after hyperpolarized [1-13C]pyruvate
injection, indicating that exogenously injected pyruvate was quickly converted
to lactate by LDH catalyzed reaction in the SCC tumor. To assess rapamycin
effect on lactate formation, lactate to pyruvate ratio (Lac/Pyr) was calculated
from area under the curve of signal intensity curves of [1-13C]lactate
and [1-13C]pyruvate. In non-treated SCC tumors, Lac/Pyr increased as
tumor grew from 1.62±0.12 on day 8 to 2.26±0.23 on day 10. On the other hand,
Lac/Pyr was significantly dropped by rapamycin treatments from 1.63±0.19 on day
8 (before rapamycin treatments) to 1.07±0.07 on day 10 (after 2 days rapamycin
treatments). Then, chemical shift images in SCC tumors were acquired 30-50 sec
after hyperpolarized [1-13C] injection (Figure 2). Lac/Pyr in the
tumor region calculated from chemical shift images also showed similar behavior
to the 13C-MRS results; lactate formation dropped by rapamycin in a
dose dependent manner (Figure 3). We also carried out DCE-MRI with gadolinium-DTPA
to evaluate changes of perfusion in the SCC tumor by rapamycin treatments. The
Gd-DTPA uptake was significantly higher in the rapamycin treated group compared
to the non-treated control group (Figure 4), indicating tumor perfusion was
improved by the rapamycin treatment.
Conclusion
Pyruvate
metabolism to lactate was dropped by rapamycin treatments in SCC tumor, and both
the drop of LDH activity and the improvement of tumor perfusion contributed to
the Lac/Pyr drop. The results suggest that Lac/Pyr monitored with
hyperpolarized 13C-MRI could become a useful marker for evaluating
early tumor response to mTOR inhibitors.
Acknowledgements
No acknowledgement found.References
No reference found.