Hyperpolarized 13C MR spectroscopic imaging can be used to monitor Everolimus treatment in vivo in an orthotopic rodent model of glioblastoma

Abstract

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor in humans. Because the phosphatidylinositol-3-kinase (PI3K) signaling pathway is activated in more than 88% of GBM, new drugs which target this pathway, such as the mTOR inhibitor Everolimus, are currently in clinical trials. Early tumor response to molecularly targeted treatments remains challenging to assess non-invasively, because it is often associated with tumor stasis or slower tumor growth. Innovative neuroimaging methods are therefore critically needed to provide metabolic or functional information that is indicative of targeted therapeutic action at early time points during the course of treatment. In this study, we demonstrated for the first time that hyperpolarized (HP) 13C magnetic resonance spectroscopic imaging (MRSI) can be used on a clinical MR system to monitor early metabolic response of orthotopic GBM tumors to Everolimus treatment through measurement of the HP lactate-to-pyruvate ratios. The study was performed on a highly invasive non-enhancing orthotopic GBM tumor model in rats (GS-2 tumors), which replicates many fundamental features of human GBM tumors. Seven days after initiation of treatment there was a significant drop in the HP lactate-to-pyruvate ratio from the tumor tissue in treated animals relative to day 0 (67%±27% decrease). In the control group, no significant changes in the HP lactate-to-pyruvate ratios were observed. Importantly, at the 7 day time point, conventional MR imaging (MRI) was unable to detect a significant difference in tumor size between control and treated groups. Inhibition of tumor growth by conventional MRI was observed from day 15 of treatment. This implies that the decrease in the HP lactate-to-pyruvate ratio could be detected before any treatment-induced inhibition of tumor growth. Using immunohistochemical staining to further examine tumor response to treatment, we found that the decrease in the HP lactate-to-pyruvate ratio was associated with a drop in expression of lactate dehydrogenase, the enzyme that catalyzes pyruvate to lactate conversion. Also evident was decreased staining for carbonic anhydrase IX (CA-IX), an indicator of hypoxia-inducible factor 1α (HIF-1α) activity, which, in turn, regulates expression of lactate dehydrogenase. To our knowledge, this study is the first report of the use of HP 13C MRSI at a clinical field strength to monitor GBM response to molecularly targeted treatments. It highlights the potential of HP lactate-to-pyruvate ratio as an early biomarker of response, thereby supporting further investigation of this non-invasive imaging approach for eventual clinical application.

Figure 1

(A) Axial T2-weighted images of one control (top) and one Everolimus treated (bottom) animal. The left column corresponds to data acquired prior to treatment initiation (D0), the right column to data obtained at D7. Orthotopic GBM tumors can be seen as hyperintense signals on the images (red dotted lines), illustrating tumor growth. (B) Tumor volume was assessed from anatomical images as a function of days of carrier/Everolimus treatment for both control (plain line) and treated (dotted line) groups, respectively. No changes in tumor volume are noticed between the two groups at D7. Slower tumor growth can be seen in Everolimus treated animals at later time points.

Figure 2

(A) HP ¹³C MRSI grids superimposed to anatomical images acquired from one Everolimus treated animal at D0 (left) and D7 (right). The tumor voxels (>80% of tumor tissue) are presented as bold boxes. (B) Corresponding HP ¹³C MRSI spectra. (C) HP ¹³C spectra from the tumor voxels of interest and corresponding values of (Lactate/Pyruvate)^TUMOR and (Lactate/Pyruvate)^NORMALIZED. A decrease in the (Lactate/Pyruvate)^TUMOR ratio can clearly be observed after 7 days of Everolimus treatment.

Figure 3

(A) Axial T2-weighted images and (B) corresponding heatmaps of the (Lactate/Pyruvate)^NORMALIZED ratios derived from HP ¹³C MRSI datasets of one control (left) and one Everolimus treated (right) animal. For each animal, the left column corresponds to data acquired prior to initiation of treatment (D0), the right column to data obtained at D7. Everolimus treatment induces a decrease of the (Lactate/Pyruvate)^NORMALIZED ratio in the tumor region for the treated animal, whereas an increase of this ratio can be seen in the control animal.

Figure 4

Histogram of the average (Lactate/Pyruvate)^NORMALIZED ratios at D0, D2 and D7 for control (black) and treated (white, dotted) groups. The average ratios were significantly different between control and treated animals at D7, and a significant decrease in this ratio was measured between D0 and D7 for the treated group. *p=0.05

Figure 5

(A) Casp3, (B) MIB-1, (C) CA-IX and (D) LDH-A IHC stains of orthotopic GBM tumors resected after 7 days of carrier (left, control) and Everolimus (right, treated) treatments. No differences are observed between the two samples for both Casp3 and MIB-1, suggesting identical levels of apoptosis and proliferation, respectively. However, a clear drop CA-IX and LDH-A can be seen in the treated tumor.