Similarity of In Vivo Lactate T1 and T2 Relaxation Times in Different Preclinical Cancer Models Facilitates Absolute Quantification of Lactate
Ellen Ackerstaff1, Nirilanto Ramamonjisoa1, H. Carl LeKaye1, Kristen L. Zakian1, Ekaterina Moroz1, Inna S. Serganova1, Ronald G. Blasberg1, and Jason A. Koutcher1

1Memorial Sloan Kettering Cancer Center, New York, NY, United States

Synopsis

Aggressive, treatment-resistant tumors have been associated with high tumor lactate. For the absolute quantification of in vivo tumor lactate by the substitution method, it is essential to correct for differences between reference phantom and in vivo lactate T1 and T2 relaxation times (LacT1/T2). The LacT1/T2 acquisition requires specialized MR sequences and is hampered in vivo by long acquisition times and low lactate SNR. Here, we measure LacT1/T2 for various orthotopic breast and subcutaneous prostate cancer models in immune-competent and immune-compromised hosts. Our results indicate that using an average LacT1/T2 correction factor introduces less than 20% error in the lactate quantification.

Purpose

Aggressive, treatment-resistant tumors are often characterized by high tumor lactate1. To image tumor lactate in vivo, lactate-edited MRS is used2, 3. To quantify tumor lactate by the phantom replacement method4-6, it is essential to account for differences between phantom and in vivo lactate T1 and T2 relaxation times (LacT1/T2), affecting lactate signal intensities. However, the measurement of LacT1/T2 requires specialized MRS acquisition techniques with long acquisition times7, is hampered by SNR limitations, and thus, would benefit significantly from limiting the number of measurements to representative tumor types. Here, we evaluated the variability of LacT1/T2 and resulting lactate T1/T2 relaxation time correction factor (CF) in different orthotopic breast and subcutaneous prostate cancer models in immune-competent and immune-compromised hosts, and its impact on absolute lactate quantification.

Methods

All experiments were performed in accordance with institutional animal care and use committee protocols.

Tumor Models: We studied multiple murine prostate cancer (CaP) and breast cancer (CaB) cell lines: Myc-CaP (spontaneously immortalized cells from C-Myc transgenic mouse with CaP, androgen naïve8); RM-1 (CaP of Ras+Myc-transformed C57BL/6 mouse9); E0771 (mammary adenocarcinoma10); 4T1wt (mammary carcinoma11). Cells were grown in Dulbecco’s Modified Essential Medium, supplemented with 10% fetal bovine serum, 100 U/ml Penicillin and 100 μg/ml Streptomycin at 37 °C in 5% CO2. Prostate cancer cells were implanted subcutaneously in the flank of immune-compromised, male Nod/SCID mice, while CaB cells were orthotopically injected into the lower mammary fat pad of immune-competent, female Balb/C (4T1wt) or C57BL/6 (E0771) mice.

In Vivo MR: The MR experiments were performed on anesthetized mice using a custom-built, solenoid 1H MR coil in a Bruker 7T magnet. The animal core temperature was maintained at 34-37°C with a breathing rate at 50-90 breaths/min. After tumor positioning, the 1H MR coil was tuned and matched. The water line width was shimmed to ~30-70 Hz full-width-half-maximum. Single-slice tumor lactate and LacT1/T2 were measured using SelMQC3 and SelMQC-based T1 and T2 acquisition7 sequences with the slice thickness varied according to tumor size. Data were analyzed using either XsOsNMR or MNova. Lactate T1 and T2 relaxation times were fitted using GraphPad Prism. The lactate concentration is directly proportional to the lactate signal intensity ratio of tumor to phantom (ST/SPh). To account for LacT1/T2 differences between phantom and tumor affecting signal intensities, ST/SPh is multiplied with the lactate T1/T2 relaxation time correction factor CF that is calculated as per Equation 1.

CF = exp(TE(1/T2,T-1/T2,Ph)) • (1-exp(-TR/T1,Ph))/(1-exp(-TR/T1,T)) – Equation 1

with 120 ms echo time (TE), 3000 ms relaxation time (TR), and the subscripts Ph and T referring to phantom and tumor respectively.

Results & Discussion

Modeling the lactate T1/T2 correction factor CF for a given TE, TR, T1,Ph, and T2,Ph demonstrates that CF increases with decreasing T2,T and increasing T1,T, potentially modifying lactate values by a factor of up-to 8 fold or more (Fig. 1). To answer the important question of how much CF varies in experimental, preclinical tumor models, we measured in vivo the variation of LacT1/T2 for orthotopic breast cancer in immune-competent hosts and in subcutaneous prostate cancer in an immune-compromised host (Fig. 2). Lactate T1 relaxation times differed significantly only between RM-1 and E0771 (P<0.05), while no significant differences between tumor models were observed for the lactate T2 relaxation times (P>0.99) (Fig. 2A). Averaging LacT1T2 across all tumor models results only in a <10% standard error (SE) (Fig. 2B), indicating the introduction of only a moderate error. Using the same experimental parameters for TE, TR, T1,Ph, and T2,Ph as in Fig. 1, CF varied between 2.5 and 2.9 for the 4 experimental models in this study (Fig. 3A, CF), with an average CF of ~2.7(~±3%SE) (Fig. 3B). In Fig. 3A, CF_All depicts an average CF obtained using the LacT1T2 averaged across all tumor models, which is not significantly different from the average CF in Fig. 3B. The CF at 4.7T and 7T (Fig. 3A, green bars), based on LacT1/T2 from the literature4, 7, 12, 13, Rizwan, Zakian personal comm., resulted in similar or slightly lower CFs than for this study. Our data support that LacT1/T2 variations appear to be to a larger extent due to measurement errors and to a lesser extent to biological variability in tumors without extensive necrosis, as studied here.

Conclusions

We demonstrated the similarity of in vivo lactate T1 and T2 relaxation times in a variety of tumor models. By using an average lactate CF, a less than 20% variation will be introduced in the absolute lactate quantification, thus, simplifying significantly future research into investigating the role of lactate metabolism in tumor development, progression, and treatment response.

Acknowledgements

We acknowledge support by NIH / NCI grants R01 CA163980 (RGB), R01 CA172846 (RGB, JAK), R24 CA083084 (SAI Core), and P30 CA008748 (Cancer Center Support Grant).

We like to thank Ms. Natalia Kruchevsky for technical assistance, and Dr. D.C. Shungu and Ms. X. Mao for the XsOsNMR software package.

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Figures

Figure 1: Variations of CF for LacT1/T2 between 900-2000 ms (T1,T) and 60-350 ms (T2,T), with 120 ms TE, 3000 ms TR, and phantom (10 mM lactate in water, pH 2.8) T1 and T2 relaxation times T1,Ph = 1458±34 ms (mean±SE, n = 4); T2,Ph = 1011±78 (mean±SE, n = 4).

Figure 2: In vivo lactate T1 and T2 relaxation times in various preclinical CaP and CaB models (A), and averaged across all tumor models (B). * P=0.0287, two-way ANOVA with Tukey’s multiple comparison test.

Figure 3: (A) To average LacT1/T2 corresponding CF, for each tumor type separately (CF) and across all tumor models (CF_All), from values shown in Fig. 2 and from the literature4, 7, 12, 13, Rizwan, Zakian personal comm. (Literature). (B) Mean(±SE) CF across tumor models, depicted in (A) by CF (pink bars).



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
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