Is Higher Lactate an Indicator of Tumor Metastatic Risk? A Pilot MRS Study Using Hyperpolarized13 C-Pyruvate

Rationale and Objectives

Cancer cells generate more lactate than normal cells under both 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 used hyperpolarized 13 C-pyruvate magnetic resonance spectroscopy (MRS) to compare lactate 13 C-labeling in vivo in mouse xenografts of the highly metastatic (MDA-MB-231) and the relatively indolent (MCF-7) human breast cancer cell lines. We obtained the kinetic parameters of the lactate dehydrogenase (LDH)–catalyzed reaction by three methods of data analysis including the differential equation fit, q-ratio fit, and ratio fit methods.

Results

Consistent results from the three methods showed that the highly metastatic tumors exhibited a smaller apparent forward rate constant ( k + = 0.060 ± 0.004 s −1 ) than the relatively indolent tumors ( k + = 0.097 ± 0.013 s −1 ). The ratio fit generated the greatest statistical significance for the difference ( P = .02). No significant difference in the reverse rate constant was found between the two tumor lines.

Conclusions

The result indicates that the less metastatic breast tumors may produce more lactate than the highly metastatic ones from the injected 13 C-pyruvate and supports the notion 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.

Metastasis results in about 90% of cancer deaths. However, tumor metastatic risk determination remains one of the greatest clinical challenges because of the lack of reliable biomarkers of metastatic potential . In recent years, cancer metabolism has received increasing research attention, and abnormally high level of glycolysis and lactate production (the Warburg effect) has been recognized as the hallmark of cancer . The Warburg effect has been demonstrated by a variety of techniques in vitro, ex vivo, and in vivo with a large proportion of these studies performed with cell culture models. A few methods have been used to demonstrate the Warburg effect in tissues as well. For example, fluorodeoxyglucose–positron emission tomography (FDG-PET) detects the higher uptake and accumulation of FDG in tumors compared to normal tissue in vivo , and the magnetic resonance spectroscopy (MRS) and magnetic resonance imaging including hyperpolarized 13 C-MR techniques monitor higher 13 C-lactate production from 13 C-labeled hyperpolarized pyruvate in vivo.

It is clear that the Warburg effect discriminates cancer from nonmalignant tissues. However, it is less clear if it also accounts for the differences in cancer metastatic potential. Some studies using bioluminescence imaging of biopsies from human cancers indicate a positive correlation between tumor lactate concentration and incidence of metastasis in head and neck cancer and cervical cancer patients . Lactate levels measured by 1 H-MRS were also shown as strong indicators of tumor grade and poor prognosis in various cancers, such as brain , breast , lung , and liver cancers . In addition, a positive correlation between the hyperpolarized lactate level and prostate tumor histological grades has been observed in transgenic mouse models by hyperpolarized nuclear magnetic resonance (HP-NMR) . The hypothesis that lactate enhances tumor survival, invasiveness, and metastatic potential was supported by evidence that lactate stimulates HIF (hypoxia inducible factor)-1α accumulation and increases CD44 expression levels (associated with invasion) in both stromal fibroblasts and cancer cells . On the other hand, other studies showed that lactate levels did not correlate with the invasive/metastatic potential of the breast cancer cells or histopathologic grade of oligodendrogliomas .

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Materials and methods

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Pyruvate↔k−k+Lactate Pyruvate

↔

−

Lactate

where k + and k − are the apparent forward and reverse rate constants. From the two-site exchange model, we have the following differential equations:

ⅆPⅆt=−ρP−k+P+k−L ⅆ

ⅆ

−

ⅆLⅆt=−ρL+k+P−k−L ⅆ

ⅆ

−

where P and L represent the NMR signals of the hyperpolarized pyruvate and lactate, respectively and ρ is the T 1 relaxation rate (assumed equal for pyruvate and lactate in vivo ).

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q=−k−R+k+ q

−

where R ( t ) = L ( t )/ P ( t ) and q=(ⅆR/ⅆt)/(1+R) q

(

ⅆ

)

(

) . Method 3, the RF analysis, fits the time course of R ( t ). From the two-site exchange model shown by Equation 2 , we solve for P ( t ) and L ( t ), giving:

P(t)=P(0)[k−e−ρt+k+e−(ρ+k−+k+)t]/(k−+k+)+L(0)[k−e−ρt−k+e−(ρ+k−+k+)t]/(k−+k+) P

(

)

(

)

[

−

(

−

)

]

(

−

)

(

)

[

−

(

−

)

]

(

−

)

L(t)=P(0)[k+e−ρt−k+e−(ρ+k−+k+)t]/(k−+k+)+L(0)[k+e−ρt+k−e−(ρ+k−+k+)t]/(k−+k+) L

(

)

(

)

[

−

(

−

)

]

(

−

)

(

)

[

−

(

−

)

]

(

−

)

where L (0) and P (0) are the signal intensities at t = 0. Dividing Equation 4b by Equation 4a , we have

R(t)={r[1+R(0)]+[R(0)−r]e−st}/{1+R(0)+[r−R(0)]e−st} R

(

)

{

[

(

)

]

[

(

)

−

]

−

}

{

(

)

[

−

(

)

]

−

}

where r = k + / k − , s = k + + k − . By fitting the ratio time course R ( t ) to Equation 5 , we obtain the fitting parameters k + and k − as well as their ratio k + / k − .

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Results

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Table 1

The Apparent Rate Constants (s −1 ) and Rate Constant Ratios (Mean ± Standard Deviation) Quantified by the Three Analysis Methods

Method ∗ Tumor Phenotype_k_ + (s −1 )k − (s −1 )k + / k − Size (mm 3 ) DE MDA-MB-231 0.059 ± 0.020 0.024 ± 0.012 2.64 ± 1.03 611 ± 711 MCF-7 0.111 ± 0.038 0.027 ± 0.010 4.17 ± 0.67 283 ± 120 Both lines † 0.089 ± 0.040 0.026 ± 0.010 3.51 ± 1.12P value .068 .77 .11 .51 qR MDA-MB-231 0.063 ± 0.013 0.028 ± 0.011 2.63 ± 1.54 MCF-7 0.093 ± 0.006 0.022 ± 0.007 4.58 ± 1.27 Both lines † 0.080 ± 0.018 0.024 ± 0.009 3.75 ± 1.64P value .047 .44 .15 RF MDA-MB-231 0.057 ± 0.009 0.023 ± 0.009 2.83 ± 1.68 MCF-7 0.086 ± 0.005 0.019 ± 0.007 4.88 ± 1.32 Both lines † 0.074 ± 0.017 0.021 ± 0.007 4.00 ± 1.73P value .02 .52 .16

N = 3 for MDA-Mb-231 line and N = 4 for MCF-7 line.

The P values are from the Student t test comparing the results of two tumor lines using the same analysis method.

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Table 2

Comparison of Different Analysis Methods with Various Initial Time Points after the Pyruvate Peak

Method ∗ Tumor First Second Third Fourth_k_ + (s −1 )k − (s −1 )k + (s −1 )k − (s −1 )k + (s −1 )k − (s −1 )k + (s −1 )k − (s −1 ) DE 231 0.061 ± 0.022 0.026 ± 0.011 0.056 ± 0.015 0.022 ± 0.006 0.062 ± 0.012 0.028 ± 0.010 0.069 ± 0.014 0.034 ± 0.016 MCF-7 0.109 ± 0.039 0.025 ± 0.011 0.108 ± 0.042 0.025 ± 0.012 0.101 ± 0.029 0.023 ± 0.011 0.101 ± 0.037 0.023 ± 0.012P value .096 .98 .081 .73 .067 .54 .18 .36 qR 231 0.065 ± 0.014 0.029 ± 0.010 0.062 ± 0.008 0.028 ± 0.012 0.067 ± 0.005 0.033 ± 0.017 0.072 ± 0.008 0.037 ± 0.021 MCF-7 0.093 ± 0.006 0.022 ± 0.007 0.090 ± 0.005 0.021 ± 0.008 0.086 ± 0.010 0.020 ± 0.011 0.087 ± 0.012 0.020 ± 0.012P value .060 .38 .010 .45 .028 .34 .12 .30 RF 231 0.057 ± 0.010 0.024 ± 0.008 0.054 ± 0.008 0.022 ± 0.011 0.058 ± 0.005 0.026 ± 0.015 0.060 ± 0.004 0.028 ± 0.018 MCF-7 0.086 ± 0.004 0.019 ± 0.007 0.084 ± 0.005 0.018 ± 0.007 0.081 ± 0.009 0.018 ± 0.009 0.060 ± 0.042 0.014 ± 0.014P value .029 .47 .012 .62 .0081 .48 .99 .32

The k + and k − refer to the forward and reverse rate constant of the lactate dehydrogenase reaction, respectively.

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Discussion

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Conclusions

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Acknowledgment

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Is Higher Lactate an Indicator of Tumor Metastatic Risk? A Pilot MRS Study Using Hyperpolarized13 C-Pyruvate

Rationale and Objectives

Materials and Methods

Results

Conclusions

Materials and methods

Results

Discussion

Conclusions

Acknowledgment

References

Further Reading

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