Molecular imaging HDACs class IIa expression-activity and pharmacologic inhibition in intracerebral glioma models in rats using PET/CT/(MRI) with [18F]TFAHA

Abstract

HDAC class IIa enzymes (HDAC4, 5, 7, 9) are important for glioma progression, invasion, responses to TMZ and radiotherapy, and prognosis. In this study, we demonstrated the efficacy of PET/CT/(MRI) with [¹⁸F]TFAHA for non-invasive and quantitative imaging of HDAC class IIa expression-activity in intracerebral 9L and U87-MG gliomas in rats. Increased accumulation of [¹⁸F]TFAHA in 9L and U87-MG tumors was observed at 20 min post radiotracer administration with SUV of 1.45 ± 0.05 and 1.08 ± 0.05, respectively, and tumor-to-cortex SUV ratios of 1.74 ± 0.07 and 1.44 ± 0.03, respectively. [¹⁸F]TFAHA accumulation was also observed in normal brain structures known to overexpress HDACs class IIa: hippocampus, n.accumbens, PAG, and cerebellum. These results were confirmed by immunohistochemical staining of brain tissue sections revealing the upregulation of HDACs 4, 5, and 9, and HIF-1α, hypoacetylation of H2AK5ac, H2BK5ac, H3K9ac, H4K8ac, and downregulation of KLF4. Significant reduction in [¹⁸F]TFAHA accumulation in 9L tumors was observed after administration of HDACs class IIa specific inhibitor MC1568, but not the SIRT1 specific inhibitor EX-527. Thus, PET/CT/(MRI) with [¹⁸F]TFAHA can facilitate studies to elucidate the roles of HDAC class IIa enzymes in gliomagenesis and progression and to optimize therapeutic doses of novel HDACs class IIa inhibitors in gliomas.

Figure 1

PET/CT/(MRI) of HDACs class IIa expression in intracerebral glioma models in rats. Representative series of coronal images of the rat brain bearing intracerebral U87-MG glioma (A) and 9L gliosarcoma (B). The position of sections relative to bregma is indicated in mm on T2-weighted MR images. [¹⁸F]TFAHA PET/CT images were obtained at 20 minutes post injection of radiotracer and co-registered with T2-weighted MR images. The levels of [¹⁸F]TFAHA accumulation in tumors and different structures of the brain were measured in SUV (C) and SUV ratio normalized by the SUV of the contralateral cortex (D) for 9L (N = 10) and U87-MG (N = 9) gliomas. PET/CT images are color-coded to standard uptake values (SUV). Data - mean ± SEM. Statistical significance was determined via one-way ANOVA, *denotes p < 0.05, **denotes p < 0.01, ***denotes p < 0.001.

Figure 2

Comparative analysis of [¹⁸F]TFAHA PET/CT/(MRI) and immunohistochemical (IHC) staining in 9L glioma and in the contralateral hippocampus. The left column: (top) axial T2-weighted MR image; (middle) corresponding axial PET/CT/(MRI) fusion image; (bottom) corresponding axial PET/CT image. Macroscopic (top row) and microscopic images (x200) of axial brain sections stained with H&E and by IHC to visualize the levels of expression of HDACs 4, 5, 9, and acetylation of histones H2AK5ac, H2BK5ac, H3K9ac, and H4K8ac in 9L tumor (middle row) and in contralateral hippocampal CA2 (bottom row). PET/CT images are color-coded to standard uptake values (SUV).

Figure 3

Comparative analysis of [¹⁸F]TFAHA PET/CT/(MRI) and immunohistochemical (IHC) staining in U87-MG glioma and in the contralateral hippocampus. The left column: (top) coronal T2-weighted MR image; (middle) corresponding coronal PET/CT/(MRI) fusion image; (bottom) corresponding coronal PET/CT image. Macroscopic (top row) and microscopic images (x200) of axial brain sections stained with H&E and by IHC to visualize the levels of expression of HDACs 4, 5, 9, and acetylation of histones H2AK5ac, H2BK5ac, H3K9ac, and H4K8ac in U87-MG tumor (middle row) and in contralateral hippocampal CA2 (bottom row). PET/CT images are color-coded to standard uptake values (SUV).

Figure 4

Immunohistochemical staining (brown) for HIF-1α and KLF4 in 9L, U87 gliomas, and in contralateral cortex; counterstaining with hematoxylin (blue).

Figure 5

Monitoring pharmacologic inhibition of HDACs class IIa with [¹⁸F]TFAHA PET/CT/(MRI). (A) Representative coronal T2-weighted MRI and fused with [¹⁸F]TFAHA PET/CT/(MRI) images depicting different regions of the rat brain with intracerebral 9L tumor (shown by a dotted line on a side-view of a 3D rendered image): through the area of n.accumbens (NA), in the middle of 9L tumor (9L), and through the cerebellum (Cb). [¹⁸F]TFAHA PET/CT/(MRI) were obtained at 20 min post radiotracer administration before (baseline) and after treatment with either MC1568 (HDAC class IIa- selective inhibitor) or EX-527 (SIRT1-selective inhibitor). PET/CT images are color-coded to standard uptake values (SUV). The levels [¹⁸F]TFAHA-derived radioactivity are expressed as (B) standard uptake values (SUV) or (C) distribution volumes (DV) in 9L tumors and different brain structures at baseline (N = 4) and after therapy with MC1568 (N = 3) or EX-527 (N = 3). Data – mean ± SEM. Statistical significance was determined using paired t-test; ***indicates p < 0.005.