13C-MR Spectroscopic Imaging with Hyperpolarized [1-13C]pyruvate Detects Early Response to Radiotherapy in SCC Tumors and HT-29 Tumors

Abstract

Purpose: X-ray irradiation of tumors causes diverse effects on the tumor microenvironment, including metabolism. Recent developments of hyperpolarized (13)C-MRI enabled detecting metabolic changes in tumors using a tracer [1-(13)C]pyruvate, which participates in important bioenergetic processes that are altered in cancers. Here, we investigated the effects of X-ray irradiation on pyruvate metabolism in squamous cell carcinoma (SCCVII) and colon cancer (HT-29) using hyperpolarized (13)C-MRI.

Experimental design: SCCVII and HT-29 tumors were grown by injecting tumor cells into the hind legs of mice. [1-(13)C]pyruvate was hyperpolarized and injected intravenously into tumor-bearing mice, and (13)C-MR signals were acquired using a 4.7 T scanner.

Results: [1-(13)C]pyruvate and [1-(13)C]lactate were detected in the tumor-bearing legs immediately after hyperpolarized [1-(13)C]pyruvate administration. The [1-(13)C]lactate to [1-(13)C]pyruvate ratio (Lac/Pyr) increased as the tumors grew in nonirradiated SCCVII tumors. The increase in Lac/Pyr was suppressed modestly with a single 10 Gy of irradiation, but it significantly decreased by further irradiation (10 Gy × 3). Similar results were obtained in HT-29; Lac/Pyr significantly dropped with fractionated 30 Gy irradiation. Independent ex vivo measurements revealed that the lactate dehydrogenase (LDH) activity and protein level were significantly smaller in the irradiated SCCVII tumors compared with the nonirradiated tumors, indicating that a decrease in LDH activity was one of the main factors responsible for the decrease of Lac/Pyr observed on (13)C-MRI.

Conclusions: Robust changes of Lac/Pyr observed in the HT-29 after the radiation suggested that lactate conversion from pyruvate monitored with hyperpolarized (13)C-MRI could be useful for the evaluation of early response to radiotherapy. See related commentary by Lai et al., p. 4996.

Figure 1.

(A) pO₂ maps in a SCCVII tumor and a HT-29 tumor. (B) Percentage of hypoxic fraction (pO₂ < 10 mmHg) in SCCVII tumors (n=5) and HT-29 tumors (n=4). (C,D) Growth curves of SCCVII (n=7 for each control and irradiated group) and HT-29 (n=7 for each control and irradiated group) with (●) and without (○) X-irradiation.

Figure 2.

(A) T₂-weighted anatomical image of a SCCVII tumor-bearing leg (3 adjacent slices). White dashed lines indicate the regions of the ¹³C-MRS measurements. (B) ¹³C-MR spectra and (C) signal intensity curves of [1-¹³C]pyruvate and [1-¹³C]lactate detected in the SCCVII tumor after hyperpolarized [1-¹³C]pyruvate injection. Representative signal intensity curves measured in a HT-29 tumor after hyperpolarized [1-¹³C]pyruvate injection was also displayed.

Figure 3.

(A) The [1-¹³C]lactate to [1-¹³C]pyruvate ratio in the SCCVII tumors with (●, n=6) and without (○, n=4) X-ray irradiation, calculated from the area under the curve of the signal intensity curves. * p<0.05 compared with control Day 8. ** p<0.05 and 0.005 compared with X-ray Day 9 and control Day 11, respectively. (B) The [1-¹³C]lactate to [1-¹³C]pyruvate ratio in the HT-29 tumors with (●, n=5) and without (○, n=5) X-ray irradiation. * p<0.05 compared with X-ray Day 10 and control Day 13.

Figure 4.

Representative chemical shift images of a SCC tumor (A) and a HT29 tumor (D) obtained before irradiation, at 1 day after 10 Gy of irradiation, and at 1 day after fractionated 30 Gy irradiation. The images were acquired at 30 to 50 sec after hyperpolarized [1-¹³C]pyruvate injection. The left peak is [1-¹³]lactate, and the right peak is [1-¹³C]pyruvate, which are detected in each voxel. The voxel size is 2 mm × 2 mm × 8 mm. Total ¹³C maps (B,E) and images of the [1-¹³C]lactate to [1-¹³C]pyruvate ratio (C,F) in the SCC tumor (B,C) and the HT-29 tumor (E,F). (G) The [1-¹³C]lactate to [1-¹³C]pyruvate ratio in SCCVII tumors (n=5) and HT-29 tumors (n=5), calculated from the chemical shift images. * p < 0.05 compared with the 10 Gy radiation.

Figure 5.

(A, B) Western blot analysis of LDH-A protein expression and (C) LDH activity in SCCVII tumors (n=3) and HT-29 tumors (n=3), measured using tumor homogenate. The tumors were excised 1 day after fractionated 30 Gy of irradiation and homogenized, and the proteins were extracted. LDH activity in the extraction was assessed from NADH oxidation in the presence of pyruvate. Control was day-matched non-irradiated tumors. (D) The colony-forming ability of non-irradiated tumor cells (n=3) and X-ray irradiated tumor cells (n=3). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control.