Renuka Sriram1, Celine Baligand1, Hecong Qin1, Justin DeLos Santos1, Robert Bok1, John Kurhanewicz1, and Zhen Jane Wang1
1Radiology and Biomedical Imaging, University of California San Francisco, San Francisco, CA, United States
Synopsis
The incidence of renal cell
carcinomas (RCCs) has increased significantly over time due to the widespread use of cross-sectional imaging with incidental cancer detection.
RCCs vary widely in histological grade and risk of metastasis. However, current
imaging techniques cannot reliably differentiate low grade, indolent RCCs from
localized but potentially aggressive RCCs, resulting in the over-treatment of
many indolent cancers. Increasing evidence has shown that increased glycolysis
with lactate production is a dominant metabolic feature of RCCs. In particular,
lactate dehydrogenase expression is positively correlated with RCC grade,
and a strong predictor of tumor progression and poor prognosis. Hyperpolarized 13C MR allows real time investigation of cellular metabolism, and
provides time-resolved metabolic kinetics that reflects flux through enzyme-catalyzed reactions. The purpose of this study is
to investigate whether hypeprolarized 13C pyruvate MR can inform on the LDH
activity in orthotopic RCC tumor models.
Purpose
The incidence of renal cell carcinomas (RCCs) has
increased significantly over time due to the widespread use of cross-sectional
imaging with incidental cancer detection. RCCs vary widely in histological
grade and risk of metastasis. However, current imaging techniques cannot
reliably differentiate low grade, indolent RCCs from localized but potentially
aggressive RCCs, resulting in the over-treatment of many indolent cancers. Increasing
evidence has shown that increased glycolysis with lactate production is a
dominant metabolic feature of RCCs. In particular, lactate
dehydrogenase expression is positively correlated with RCC grade, and a strong
predictor of tumor progression and poor prognosis. Hyperpolarized (HP) 13C magnetic
resonance (MR) allows real time
investigation of cellular metabolism, and provides time-resolved metabolic
kinetics that reflects flux through enzyme-catalyzed reactions. The purpose of
this study is to investigate whether HP 13C pyruvate MR can inform on lactate dehydrogenase (LDH) activity in orthotopic RCC tumor models. Methods
Three RCC
cell lines with varying LDHA expression were chosen to create the orthotopic
tumors in mice. Figure.1 shows the RNA expression levels determined using RT-qPCR
of cells grown in monolayers in tissue culture flasks. A498 (n=2) and 786-O (n=2) were
both derived from clear cell RCCs, and UOK262 (n=3) was derived from a type 2
papillary RCC. Five to ten million cells were injected under the renal capsule
of immune deficient male mice (4-6 weeks old). Tumors were detected in 6-8
weeks post implantation and were imaged using HP 13C pyruvate MR when
they reached a volume of at least 0.2 cc. 350µl of 80mM of hyperpolarized
[1-13C]pyruvate was injected via a tail vein catheter over 10-12 sec. Dynamic,
spectrally and spatially selective RF pulses were used to acquire
hyperpolarized 13C pyruvate and lactate images on a 14T vertical
bore Agilent spectrometer equipped with a dual tune (M2M, Australia) 40mm coil,
with a repetition time of 3s and a total of 20 images. Dynamic 13C lactate
images were acquired using a 90° pulse, while 13C pyruvate
images were acquired using a variable flip angle (from 3-90°). Data were corrected
for the RF flip angle and processed using Matlab (Mathworks, Boston, USA), and
represented as area under the curve of 13C lactate to maximal pyruvate signal
(AUC lac/max
pyr). T2
weighted and diffusion weighted proton images were also acquired for anatomic reference
and proton ADC map generation, respectively. At the end of the imaging, the
animals were euthanized and the tumor tissue was quickly harvested for
immunohistochemical (IHC) staining and biochemical assays. Tissue staining,
including H&E, Ki67 and Hu70 for morphology, proliferation and human cell
identification was performed on all tumors.
LDHA expression as well
as LDH activity from the harvested tumor tissue were also measured. Results
Figure.2
shows a representative T2-weighted image of an orthotopic RCC tumor (UOK262),
with overlaid 13C lactate (15s post infusion) and 13C pyruvate (maximal signal at ~12s)
images. The graph shows the corresponding dynamic lactate and pyruvate signal
in the tumor. Figure 3 shows the mean ratio of HP 13C AUC lac/max pyr, normalized to the tumor proton ADC value of the 3 RCC
tumors. The A498 tumors showed the highest lactate signal, followed by UOK262
tumors, and lastly the 786O tumors, though small sample size precludes
statistical testing. Figure 4 shows the LDH activity and expression of the harvested tumor
tissue, with trends that correspond to the LDH expression level in cell culture.
The HP 13C AUC lac/max pyr ratios of the tumors in vivo correlate with the LDH activity and expression measured from the tumor tissue. Of note, the hyperpolarized lactate data were normalized
to the proton ADC values of the tumors to account for the different tumor cell
density in vivo. The varying tumor cell density is evident from the IHC stain
(fig.5) which is inversely related to measured mean 1H ADC of the in vivo tumors as seen in RCC clinically1.Discussion and Conclusion
Our
initial data suggest that HP 13C pyruvate to lactate conversion can
inform on the LDH enzyme activity in RCC tumors. Studies are ongoing to confirm
the finding in additional mice. As LDH has been shown to correlate with RCC
grade and patient prognosis, this technique may provide a noninvasive means to
differentiate indolent from potentially aggressive RCCs. Acknowledgements
We would like to acknowledge Sukumar Subramaniam, Romelyn DeLos Santos, Jessie Lee, Jinny Sun and Dave Korenchan for their help with experiments.
Grants: NIH
P41EB013598 (JK), Department of Defense Peer Reviewed Visionary Postdoctoral
Fellowship (RS), Department of Defense Peer Reviewed Cancer Research Concept
Award (ZJW), Radiological
Society of North America Scholar grant (ZJW).
References
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