Detection of tumor response to a vascular disrupting agent by hyperpolarized 13C magnetic resonance spectroscopy

Abstract

Nuclear spin hyperpolarization can dramatically increase the sensitivity of the (13)C magnetic resonance experiment, allowing dynamic measurements of the metabolism of hyperpolarized (13)C-labeled substrates in vivo. Here, we report a preclinical study of the response of lymphoma tumors to the vascular disrupting agent (VDA), combretastatin-A4-phosphate (CA4P), as detected by measuring changes in tumor metabolism of hyperpolarized [1-(13)C]pyruvate and [1,4-(13)C(2)]fumarate. These measurements were compared with dynamic contrast agent-enhanced magnetic resonance imaging (DCE-MRI) measurements of tumor vascular function and diffusion-weighted MRI (DW-MRI) measurements of the tumor cell necrosis that resulted from subsequent loss of tumor perfusion. The rate constant describing flux of hyperpolarized (13)C label between [1-(13)C]pyruvate and lactate was decreased by 34% within 6 hours of CA4P treatment and remained at this lower level at 24 hours. The rate constant describing production of labeled malate from hyperpolarized [1,4-(13)C(2)]fumarate increased 1.6-fold and 2.5-fold at 6 and 24 hours after treatment, respectively, and correlated with the degree of necrosis detected in histologic sections. Although DCE-MRI measurements showed a substantial reduction in perfusion at 6 hours after treatment, which had recovered by 24 hours, DW-MRI showed no change in the apparent diffusion coefficient of tumor water at 6 hours after treatment, although there was a 32% increase at 24 hours (P < 0.02) when regions of extensive necrosis were observed by histology. Measurements of hyperpolarized [1-(13)C]pyruvate and [1,4-(13)C(2)]fumarate metabolism may provide, therefore, a more sustained and sensitive indicator of response to a VDA than DCE-MRI or DW-MRI, respectively.

Figure 1

Structure of Combretastatin-A4, the active form of the prodrug Combretastatin-A4-Phosphate

Figure 2

¹³C MR spectra acquired from tumors following intravenous injection of hyperpolarized [1-¹³C]pyruvate and [1,4-¹³C₂]fumarate. Sequential spectra collected from a 6 mm tumor slice over a period of 160 s (starting ~10 s post injection), showing flux of hyperpolarized ¹³C label between [1-¹³C]pyruvate (172.9p.p.m.) and [1-¹³C]lactate (185.1p.p.m.) in an untreated tumor (A) and 24 h after CA4P treatment (B) and between [1,4-¹³C₂]fumarate (177.2p.p.m.) and [1,4-¹³C₂]malate (182.2, 183.6p.p.m.) in an untreated tumor (C) and 24 hours after CA4P treatment (D). Only every 4^th spectrum is shown for clarity.

Figure 3

Time dependent changes in the concentrations of hyperpolarized ¹³C labeled metabolites in treated and untreated tumors. Data were normalized to the initial signal intensity from the injected metabolite and were averaged over all animals within each cohort (data shown as mean ± S.E.M.). (A) [1-¹³C]pyruvate (B) [1-¹³C]lactate (C) [1,4-¹³C₂]fumarate (D) [1,4-¹³C₂]malate.

Figure 4

Sections of tumors stained with hemotoxylin and eosin; scale bar 280 μm. Untreated tumors (A); tumors 6 h after treatment with CA4P (B) and 24 h after treatment (C). The tumors at 6 h after treatment show small areas of necrosis, whereas there is widespread necrosis at 24 h. The relationship between the apparent rate constant describing malate production and the area of necrosis (%), determined from analysis of histological sections, is shown in (D). Error bars for both axes represent standard error on the mean, taken over all animals in each cohort.

Figure 5

Dynamic contrast agent enhanced magnetic resonance imaging measurements of tumor perfusion. (A) Estimated contrast agent concentration, following i.v. injection at t=0, in untreated tumors and in tumors at 6 h and 24 h after CA4P treatment. The error bars indicate S.D; significant difference between all curves was tested by one-way ANOVA (p<0.001). (B) The area under the uptake curve (AUC) showed a slight reduction in contrast agent uptake at 6 h (p=0.030, paired two-tailed t-test) after CA4P treatment, while at 24 h there was a slight but significant increase in uptake compared to pre-treatment levels (p=0.002, paired two-tailed t-test). Horizontal bars indicate the median value.

Figure 6

Diffusion-weighted imaging shows an increase in the apparent diffusion coefficient of water (ADC) at 24 h after treatment with CA4P but not at 6 h. Average histograms of ADC values over all animals imaged are shown in (A), reflecting increased heterogeneity in the tumors at 24 h after treatment. (B) The ADC measured along the slice-selective direction shows a 32% increase (p<0.05) at 24 h after CA4P treatment but is not sensitive to changes occurring at 6 h after treatment (p=0.53). Each data point is the median pixel value in a region of interest drawn within the tumor boundary on the motion-corrected diffusion weighted image. Representative images before (C), at 6 h (D) and at 24 h (E) after treatment. The tumors are outlined in white.