Transient decrease in tumor oxygenation after intravenous administration of pyruvate

Abstract

MRI using hyperpolarized (13) C-labeled pyruvate is a promising tool to biochemically profile tumors and monitor their response to therapy. This technique requires injection of pyruvate into tumor-bearing animals. Pyruvate is an endogenous entity but the influence of exogenously injected bolus doses of pyruvate on tumor microenvironment is not well understood. In this study, the effect of injecting a bolus of pyruvate on tumor oxygen status was investigated. EPR oxygen imaging revealed that the partial pressure of oxygen (pO(2)) in squamous cell carcinoma implanted in mice decreased significantly 30 min after [1-(13) C]pyruvate injection, but recovered to preinjection levels after 5 h. Dynamic contrast-enhanced-MRI studies showed that, at the dose of pyruvate used, no changes in tumor perfusion were noticed. Immunohistochemical analysis of hypoxic marker pimonidazole independently verified that the squamous cell carcinoma tumor transiently became more hypoxic by pyruvate injection. Efficacy of radiotherapy was suppressed when X-irradiation was delivered during the period of pyruvate-induced transient hypoxia. These results suggest importance of taking into account the transient decrease in tumor pO(2) after pyruvate injection in hyperpolarized (13) C MRI, because tumor oxygen status is an important factor in determining outcomes of therapies.

Figure 1

Comparison of tumor pO₂ between “with and without” hyperpolarized [1-¹³C]pyruvate experiments. (A,B) T₂ weighted anatomical image of a representative SCC tumor–bearing mouse, and the corresponding pO₂ maps. (A) Control tumor which didn't receive [1-¹³C]pyruvate injection. (B) Tumor measured after hyperpolarized [1-¹³C]pyruvate experiments. (C) Median pO₂ and (D) percentage of the hypoxic region in control tumors (n=5) and tumors after [1-¹³C]pyruvate experiments (n = 4). There was no significance between control and after ¹³C-MRI.

Figure 2

Non-invasive monitoring of tumor pO₂ by EPRI and effects of pyruvate injection. (A) T₂-weighted anatomical image of a representative SCC tumor–bearing mouse, and the corresponding pO₂ maps measured before, 30 min, 1 h, and 5 h after [1-¹³C]pyruvate injection. The adjacent center three slices of the 3D images were displayed, and the every slice has 2 mm thickness. (B) Frequency histogram of the tumor pO₂ shown in (A) before (blue) and 30 min after (red) pyruvate injection. (C) Median pO₂ in the tumors, and (D) percentage of the hypoxic region (pO₂ < 10 mmHg) in the tumors. Each value in (C) and (D) indicates the mean ± SEM of 4 mice. * P<0.01, ** P<0.05, compared with before pyruvate injection.

Figure 3

(A) Immunostaining of hypoxia marker pimonidazole of representative tumors of control mice and pyruvate-treated mice (1 h and 5 h after [1-¹³C]pyruvate injection). (B) Pimonidazole positive area in control tumors (n = 5) and tumors 1 h after (n=5) and 5 h after (n=5) [1-¹³C]pyruvate injection.

Figure 4

Tumor perfusion measurements by DCE-MRI. (A) Signal intensity change in T₁-weighted images of SCC tumors by Gd-DTPA injection. Gd-DTPA (234.5 mg/kg body weight) was intravenously injected to the mice 2 min after starting the scan. The control mice (n = 4) didn't receive [1-¹³C]pyruvate injection. The pyruvate group (n = 4) received [1-¹³C]pyruvate injection 30 min before DCE-MRI. (B) Area under the curves. Each value represents the average of 4 mice and error bars represent SEM. There was no significant difference between the control group and the pyruvate group.

Figure 5

Tumor growth curves after X-irradiation. Control group (●, n = 5) was treated neither X-irradiation nor [1-¹³C]pyruvate injection. X-irradiations to the tumors were carried out without (■, n = 5), 30 min after (◆, n = 6), and 5 h after (▲, n = 7) [1-¹³C]pyruvate injection.