Assessing Amide Proton Transfer (APT) MRI Contrast Origins in 9 L Gliosarcoma in the Rat Brain Using Proteomic Analysis

Abstract

Purpose: To investigate the biochemical origin of the amide photon transfer (APT)-weighted hyperintensity in brain tumors.

Procedures: Seven 9 L gliosarcoma-bearing rats were imaged at 4.7 T. Tumor and normal brain tissue samples of equal volumes were prepared with a coronal rat brain matrix and a tissue biopsy punch. The total tissue protein and the cytosolic subproteome were extracted from both samples. Protein samples were analyzed using two-dimensional gel electrophoresis, and the proteins with significant abundance changes were identified by mass spectrometry.

Results: There was a significant increase in the cytosolic protein concentration in the tumor, compared to normal brain regions, but the total protein concentrations were comparable. The protein profiles of the tumor and normal brain tissue differed significantly. Six cytosolic proteins, four endoplasmic reticulum proteins, and five secreted proteins were considerably upregulated in the tumor.

Conclusions: Our experiments confirmed an increase in the cytosolic protein concentration in tumors and identified several key proteins that may cause APT-weighted hyperintensity.

Fig. 1

Illustration of the fresh rat brain tissue sample preparation. The brain was placed into a rat brain matrix with intervals of 2 mm (Zivic Instruments, Pittsburgh, PA) and then cut by blades (red lines). Two tissue samples from tumor (red arrow) and contralateral normal (green arrow) regions were punched with a 3-mm-diameter biopsy punch (Zivic Instruments, Pittsburgh, PA). Tissue samples from the tumor (red circle) and contralateral normal (green circle, primarily gray matter) regions of equal volumes (2 mm thickness and 3 mm diameter) were obtained.

Fig. 2

MR images of 9 L gliosarcoma in a representative rat (13 days after implantation). The tumor (red arrows) was visible on T₂-weighted, APT-weighted, and MTR images.

Fig. 3

2D DIGE images of normal brain and tumor regions for four rats (labels A–D). Green spots indicate proteins from the normal brain tissue; red spots indicate proteins from the tumor tissue, and yellow spots represent the equal protein amounts in the normal brain and tumor tissues. Protein profiles showed similarities in both tumor and normal regions from different rats but substantial differences between tumor and normal regions within the same rat.

Fig. 4

2D DIGE image of 16 upregulated (±1.5-fold) proteins in the tumor tissue compared to the contralateral normal brain tissue. Green spots indicate proteins from the normal brain tissue; red spots indicate proteins from the tumor tissue, and yellow spots represent the equal protein amounts in the normal brain and tumor tissues.