Rationale and Objectives Cancer cells generate more lactate than normal cells under aerobic and hypoxic conditions – exhibiting the so-called Warburg effect. However, the relationship between the Warburg effect and tumor metastatic potential remains controversial. We intend to investigate whether the higher lactate reflects higher tumor metastatic potential. Materials and Methods We employed hyperpolarized 13C-pyruvate magnetic resonance spectroscopy (MRS) to compare lactate 13C-labeling in vivo in breast cancer mouse xenografts of highly metastatic (MDA-MB-231) and relatively indolent (MCF-7) human cell lines. We obtained the kinetic parameters of the lactate dehydrogenase (LDH)-catalyzed reaction by three methods of analysis including the differential equation (DE) fit, q-ratio (qR) fit, and ratio fit (RF) methods. Results Consistent results from the three methods showed that the highly metastatic breast tumors exhibited a smaller apparent forward rate constant (k+ = 0.060 ± .004 s−1) than the relatively indolent tumors (k+ = 0.097 ± .013 s−1). The RF fit generates the highest statistical significance for the difference (p=0.02). No significant difference is found for the reverse rate constant. Conclusion The result indicates that the less metastatic breast tumors may produce more lactate than the highly metastatic ones from the injected 13C-pyruvate, and supports the viewpoint that breast tumor metastatic risk is not necessarily associated with the high levels of glycolysis and lactate production. More studies are needed to confirm whether and how much the measured apparent rate constants are affected by the membrane transporter activity and whether they are primarily determined by the LDH activity or not.

Conventional methods for analyzing the in vivo hyperpolarized 13C-NMR (HP-MR) data from the lactate dehydrogenase (LDH) reaction usually make assumptions on the stability of rate constants and/or the validity of two-site exchange model. In this study, we developed a framework to test the validity of the assumption of stable reaction rate constants and the two-site exchange model in vivo via ratiometric fitting of the time courses of signal ratio L(t)/P(t). Our analysis provided evidence that the LDH enzymatic kinetics observed by HP-MR is in near-equilibrium and satisfies the two-site exchange model for only a specific time window. Additionally we quantified both the forward and reverse exchange rate constants of the LDH reaction for the transgenic and mouse xenograft models of breast cancer using the ratio fit (RF) method we developed that includes only two modeling parameters and is less sensitive to the influence of instrument settings/protocols such as flip angels, degree of polarization, and tracer dosage. We further compared the RF method with a conventional two-site exchange modeling method, i.e., the differential equations fitting (DEF) method using both the experimental and simulated HP-MR data. The RF method appeared to fit better than the DEF method for the reverse rate constant on the mouse tumor data with less relative errors on average, whereas the DEF method also resulted in a negative reverse rate constant for one tumor. The simulation results indicated that the accuracy of both methods depends on the width of transport function (TF), noise level, and rate constant ratio; one method may be more accurate than the other one based on experimental/biological conditions aforementioned. We were able to categorize our tumor models into specific conditions of the computer simulation and estimate the errors of rate quantification. We also discussed possible approaches to developing more accurate rate-quantification methods for HP-MR.

In vivo imaging/spectroscopic biomarkers for solid tumor aggressiveness are needed in the clinic to facilitate cancer diagnosis and treatment strategies. In mouse models of human melanoma and breast cancer we were able to detect the metabolic differences among tumors of different metastatic potential and between normal and cancer tissues by optical imaging of the mitochondrial redox state of snap-frozen tissue samples. Such metabolic differences indicate that tumors of different aggressiveness have different metabolic homeostasis, which supports that kinetic parameters such as rate constant(s) can also serve as bio-markers for cancer aggressiveness and treatment response. Here we present our preliminary study on the mouse xenografts of the aggressive and indolent human breast cancer cell lines using the hyperpolarized 13C-NMR (HP-NMR) technique. By recording the time courses of 13C-pyruvate tracer and its metabolite signals in vivo, particularly the 13C-lactate signal, the apparent rate constants of both the forward and reverse reactions catalyzed by lactate dehydrogenase (LDH) were extracted via the ratiometric modeling of the two-site exchange reaction that we developed. Data from four breast tumors (MCF-7, MDA-MB-468, and MDA-MB-231 medium and large) with different aggressiveness are included. We demonstrate the feasibility to quantify the apparent rate constants of LDH reactions in breast tumor xenografts.

Purpose: Early detection of lung-residing tumor is of high importance as 1) lung cancer is the number one leading cause of cancer death in the US and early-stage lung cancer was found to be associated with lower mortality than late-stage disease; 2) the lung is one of most common sites of metastasis for various cancers, and metastasis accounts for ∼90% of cancer death. Early confirmation of lung metastasis is expected to significantly change the therapeutic strategies. Currently, the major tools for screening lung-residing tumors in high risk populations include radiography and low dose CT. However, their false positive and over-diagnosis rates are very high (>90%). We aimed to explore the feasibility of employing hyperpolarized-13C NMR (HP-13C-NMR) technique for the early detection of primary lung cancer and lung metastases. HP-13C-NMR is a non-invasive spectroscopy/imaging technique that enhances regular NMR signal by ∼10000 times and has already been demonstrated in both preclinical and clinical cancer studies to quantify cancer enzymatic activities in vivo. Since cancer cells produce more lactate via the Warburg effect, by measuring the kinetics of lactate dehydrogenase, we expect to see the difference between the normal and cancerous lungs. Methods: We used the perfused mouse lungs infested with metastatic breast cancer cells to prove the concept. Nude mice of 7-9 weeks old were injected red fluorescent protein-transfected MDA-MB-231 cell line via tail vein. Tumors were allowed to grow for 4-39 weeks. Lung metastasis was confirmed by optical imaging in vivo. Perfused mouse lungs were placed in a NMR tube (9.4T vertical bore magnet). The lung viability was monitored using 31P spectroscopy before and after HP-13C-pyruvate injection. 8mM [1- 13C] pyruvate polarized via dynamic nuclear polarization (Hypersense, Oxford Instruments) was infused for HP-13C NMR. A series of 13C NMR spectra was acquired (α = 15°) every second for 5 min followed by data processing using custom MATLAB routines to obtain the time courses of pyruvate (Pyr) and lactate (Lac) signals and their ratio, Lac/Pyr. Results: Comparison between the normal lungs (N = 6) and the cancerous lungs (N = 8) shows 70% increase in maximum lactate (p = 0.041) and 94% increase in Lac/Pyr (p = 0.059) due to cancer formation. The standard deviations of the maximum lactate and the ratio in cancerous lung group are much larger than the control group, suggesting a large inter-lung heterogeneity in the cancerous lungs, presumably caused by different amount of metastases. Our next step is to evaluate the amount of metastases in these lungs by H&E staining and quantitatively investigate the correlation between the hyperpolarized measurements and the amount of metastases. Conclusions: Our preliminary results warrant further investigation for the potential of developing a novel HP-NMR technique for the early detection of lung cancer/metastasis in vivo. Citation Format: He N. Xu, Mehrdad Pourfathi, Hoora Shaghaghi, Stephen Kadlececk, Harrilla Profka, Rahim Rizi, Lin Z. Li. Detecting lung metastases by hyperpolarized NMR technique: A pilot study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1501A. doi:10.1158/1538-7445.AM2015-1501A