In vivo observation of intracellular oximetry in perfluorocarbon-labeled glioma cells and chemotherapeutic response in the CNS using fluorine-19 MRI

Abstract

Preclinical development of therapeutic agents against cancer could greatly benefit from noninvasive markers of tumor killing. Potentially, the intracellular partial pressure of oxygen (pO(2) ) can be used as an early marker of antitumor efficacy. Here, the feasibility of measuring intracellular pO(2) of central nervous system glioma cells in vivo using (19) F magnetic resonance techniques is examined. Rat 9L glioma cells were labeled with perfluoro-15-crown-5-ether ex vivo and then implanted into the rat striatum. (19) F MRI was used to visualize tumor location in vivo. The mean (19) F T(1) of the implanted cells was measured using localized, single-voxel spectroscopy. The intracellular pO(2) in tumor cells was determined from an in vitro calibration curve. The basal pO(2) of 9L cells (day 3) was determined to be 45.3 ± 5 mmHg (n = 6). Rats were then treated with a 1 × LD10 dose of bischloroethylnitrosourea intravenously and changes in intracellular pO(2) were monitored. The pO(2) increased significantly (P = 0.042, paired T-test) to 141.8 ± 3 mmHg within 18 h after bischloroethylnitrosourea treatment (day 4) and remained elevated (165 ± 24 mmHg) for at least 72 h (day 6). Intracellular localization of the perfluoro-15-crown-5-ether emulsion in 9L cells before and after bischloroethylnitrosourea treatment was confirmed by histological examination and fluorescence microscopy. Overall, noninvasive (19) F magnetic resonance techniques may provide a valuable preclinical tool for monitoring therapeutic response against central nervous system or other deep-seated tumors.

Fig. 1

In vitro characterization of 9L cell labeling. (a) Confocal microscopy (left panel) of 9L cells expressing GFP and labeled with PCE-BODIPy emulsion. Internalized PCE-BODIPy appears as punctate (red) deposits consistent with vesicular localization within cytoplasm (green). Nuclei (blue) are stained by Hoechst 3342. The right panel shows a white light image of the same 9L cell. (b) ¹⁹F NMR is used to quantify PCE uptake by 9L cell pellets. Shown is the PCE peak (−92 ppm) from a cell pellet and the TFA peak (−76 ppm) that is used as an absolute ¹⁹F reference for quantification. From this spectrum the average uptake of PCE is calculated to be ~2×10^{11 19}F/cell.

Fig. 2

In vivo ¹⁹F MRI and MRS of 9L glioma in rat brain. (a) A composite ¹⁹F/¹H image of the labeled 9L glioma cells stereotaxically injected into the right striatum three days prior. The ¹⁹F is rendered in a hot-iron intensity scale and the ¹H is grayscale. Control, unlabeled 9L cells were injected into the contralateral striatum in the same imaging plane; a small tumor is visible to the right of the asterisk. The white rectangular box encompassing the ¹⁹F signal represents the approximate voxel placement for MRS. Panel (b) shows a typical in vivo ¹⁹F spectrum obtained from the a single voxel using the PRESS MRS sequence.

Fig. 3

Calibration curve for R₁ versus pO₂ at 7 T and 37 °C. The curve was derived from ¹⁹F R₁ measurements of four different concentrations of O₂ in a saturated O₂/N₂ mixture of PFC emulsion in water. A linear least square analysis yields a slope of 0.0036, intercept of 0.542 and R²=0.99. Here, 100% oxygen is equivalent to 760 mm Hg.

Fig. 4

Intracellular pO₂ and BCNU treatment. (a) Typical T₁ recoveries on day 3 (solid circles) and after BCNU treatment (day 4, open squares). T₁ becomes significantly reduced 18 hours following BCNU treatment (day 4). The two recovery curves were normalized to the signal at the initial time point. (b) Summarizes the initial pO₂ changes after BCNU treatment. Data is an average of n=6 rats and shows a significant effect due to BCNU (asterisk, p = 0.042, paired T test).

Fig. 5

Longitudinal pO₂ changes after BCNU treatment. Post-BCNU, the pO₂ increase persists for at least 72 hours (day 6). In controls, pO₂ gradually decreased from day 3 to day 6 (closed circles).

Fig. 6

Histology and fluorescent microscopy of 9L glioma in rat brain. (a) H&E staining of glioma shows reduced cell density in the periphery of the tumor after BCNU treatment (day 4) compared to control, where upper and lower panels are at 200× and 400× magnification, respectively. (b) Fluorescent microscopy of brain tissue before and after BCNU treatment (day 4) confirms the intracellular localization of PCE emulsion, where GFP expressing 9L cells (green) and PCE-BODIPy (red) co-localize (630× magnification).