Testicular seminoma in the aged rat visualized by phase-contrast X-ray computed tomography.

Spontaneously growing testicular seminoma in the aged rat was imaged by one of the most sensitive imaging modalities, namely, phase-contrast X-ray computed tomography (CT) with crystal X-ray interferometry. Phase-contrast X-ray CT clearly depicted the detailed inner structures of the tumor and provided 20× magnified images compared to light-microscopic images. Phase-contrast X-ray CT images are generated based on density variations in the object, whereas pathological images are based on differentiation of cellular structures, such as the cellular nuclei and cytoplasm. The mechanism of image generation differs between the two techniques: phase-contrast X-ray CT detects even minute differences in the density among pathological structures, depending, for example, on the number and sizes of the nuclei, variations of the cytoplasmic components, and presence/absence of fibrous septa, cystic changes, and hemorrhage. Thus, phase-contrast X-ray CT with a spatial resolution of 26 µm might allow prediction of the morphological characteristics of a tumor even before histopathological processing.

Introduction

The incidence of testicular cancer, the most frequently encountered cancer among younger men, has been increasing steadily over the last 40 years (1). In cancer research, tumor models are mostly generated by implanting the tumor cells into the target organ, because of the rare natural occurrence of these tumors. At our laboratory, while we were engaged in research on the effects of aging, we accidentally detected masses in the testicles of either side of an otherwise healthy two-year-old Wistar rat. In a previous two-year carcinogenicity study involving 1261 male Wistar rats, the incidence of testicular tumors during the study period was reported to be approximately 7.2% (2).

Preclinical tumor imaging in an animal model plays an important role in the evaluation of the growth, progression, metastatic pattern, and therapy of the tumor. In the currently used conventional absorption X-ray imaging technique, image contrast is generated based on attenuation differences among the tissues within an object. Tumors are mostly composed of soft tissues, which show significantly low attenuation, and it is very difficult to distinguish the detailed inner structure of the tumor, even using a contrast agent. Thus, a high contrast resolution imaging technique is indispensable to observe the detailed morphological characteristics of tissues. The phase-contrast X-ray imaging technique enables visualization of soft tissues with high contrast and spatial resolution. In phase-contrast X-ray imaging based on the phase shift of the X-ray, the density resolutions for elements with low atomic numbers, such as H, C, N and O, are approximately 1000 times higher than those in absorption X-ray imaging (3). Although, many phase-contrast X-ray imaging techniques (4–7) have been used for biomedical imaging studies, crystal X-ray interferometry-based phase-contrast X-ray computed tomography (CT) provides the highest sensitivity resolution as compared to other methods and enables fine observation of soft tissues (8–12).

Here, very rare spontaneous growing testicular masses in an aged rat were examined by crystal X-ray interferometry-based phase-contrast X-ray CT imaging, to investigate whether this imaging modality would allow differentiation of the morphological structures in the testicular tumor as clearly as histopathological images.

Case report

Animal

Asymmetrically enlarged testicles were incidentally observed in an otherwise healthy two-year-old Wistar rat during the course of one of our studies. No abnormal lesions were observed in any other organs, including the brain, lung, heart, liver, kidney, or spleen. For phase-contrast X-ray CT imaging, the testes were extracted from the animals after transcardiac perfusion of physiological saline with heparin and fixed using 10% formalin.

Phase-contrast X-ray imaging

The experiment was performed at the vertical wiggler beamline of the Photon Factory, Tsukuba, Japan. A two-crystal X-ray interferometry-based phase-contrast X-ray imaging system was used. The X-ray charge-coupled device camera with a 1300 × 1000-pixel sensor had a pixel size of 13 × 13 µm2. The field of view was 16 × 13 mm2. During the phase-contrast imaging, the excised testicles were placed in a sample cell filled with formalin to prevent drying of the samples. The X-ray energy was set at 35 keV. The exposure time was 5 s for each interference pattern and 250 projections were delivered over 180°. The density and spatial resolution of the phase-contrast X-ray CT system that we used were approximately 0.5 mg/cm3 and 26 µm, respectively (9).

Numerical evaluation of the three-dimensional (3D) image data was conducted using a volume-rendering software (Real INTAGE; KGT Inc., Tokyo, Japan). A gray value of 30% was chosen as the cutoff for viewing the intra-tumoral vessels.

Histopathological staining

After the phase-contrast X-ray imaging, testicular masses were cut 3-µm thick and hematoxylin and eosin (H&E) staining, reticulum staining, and placental alkaline phosphatase (PLAP) immunostaining were carried out. The stained sections were imaged using an optical microscope (Olympus FSX100; Olympus, Tokyo, Japan).

The original H&E color images were converted into 8-bit gray-scale images. The nuclei became dark, whereas the cytoplasm and extracellular matrix showed varying shades of gray. These pathological gray-scale images were compared with the phase-contrast X-ray CT images.

Phase-contrast X-ray CT image and histopathological findings

Phase-contrast X-ray CT images of the testes revealed heterogeneity of multiple lesions embedded in the seminiferous tubules, found to be closely similar to the gray-scale H&E-stained histopathological images (hereinafter, simply referred to as H&E images).

Phase-contrast X-ray CT images of the testis containing the tumor revealed three masses of different sizes and densities embedded in the seminiferous tubules (Fig. 1a). The largest mass was mostly vascularized and observed as a high-density lesion, with a few low-density areas. The intermediate-sized mass was visualized as a moderately low-density lesion, with cyst-like low densities encapsulated by high-density septa in some parts of the periphery. The smallest mass was visualized as a low-density lobulated lesion with a central high-density area. Surrounding seminiferous tubules of various sizes and shapes were seen. The H&E images showed three different tumor masses with various degrees of cellularity. The largest mass showed heterogeneous cellularity and a lobulated area with dense septa. The intermediate-sized mass showed relatively homogeneous cellularity with densely capsulated lobules in a part of the periphery. The smallest mass was lobulated and was composed of clear cells, with central lymphocytic infiltration (Fig. 1b and c). In addition, the intra-tumoral vessel network within the rounded lesion surrounded by a blue square (Fig. 1a) was clearly depicted by 3D reconstruction (Fig. 1d and e).

Fig. 1.

Testis with the smaller tumors: (a) phase-contrast X-ray CT image; (b) corresponding histological (H&E) gray-scale image; (c) color image. Masses of three different sizes of heterogeneous densities embedded in seminiferous tubules (red arrow). Areas of interest in the phase-contrast X-ray CT image (a): 1) tumor area 1; 2) tumor area 2; 3) tumor area 3. Three-dimensional reconstruction of the region within the blue square in the phase-contrast X-ray CT image (a) to view the vascular network of the tumor in a different direction (d, e).

In the phase-contrast X-ray CT, absolute densities can be calculated (9) for understanding the mean density variations within the tumors. Regions of interest 1, 2, and 3 within the tumor (Fig. 1a) had different densities as 1.062 ± 0.001 g/cm3, 1.066 ± 0.001 g/cm3, and 1.040 ± 0.006 g/cm3, receptively. These different densities were corresponded to the various types of neoplastic cells seen on the H&E images.

Different types of neoplastic cells were observed, including: (i) cells with large clear cytoplasm and small hyperchromatic nuclei; (ii) densely infiltrating lymphocytes with hyperchromatic nuclei and a small volume of relatively enhancing cytoplasm; and (iii) large, round to polyhedral cells with moderately enhancing large cytoplasm and relatively large hypochromatic nuclei (Fig. 2a). The final pathological diagnosis was determined to be seminoma based on the results of fluorescent immunostaining for placental alkaline phosphatase (13) (Fig. 2b). Reticulum staining yielded a negative result (data are not shown), based on which the diagnosis of testicular lymphoma was excluded.

Fig. 2.

Histopathological sections of the tumor: (a) H&E stained image showing different types of neoplastic cells; (b) immunostained image showing positive staining of the tumor cells for placental alkaline phosphatase, a tumor marker for human seminoma.

Discussion

The testicular tumors in the present study were diagnosed by histopathological examination as germ cell tumors, possibly seminoma (14,15). Interstitial cell tumors are usually known to occur in the rat testis, and germ cell tumors, such as seminoma, are exceedingly rare (16). According to one report, the incidence of seminoma in 31,868 rats of several strains was 0.003% (17).

Phase-contrast X-ray CT clearly depicted the spontaneously growing seminoma in the aged rat without the need for use of any contrast agent, and the images closely resembled 20× magnified gray-scale H&E images. However, the phase-contrast X-ray CT images appeared slightly degraded as compared to the gray-scale H&E images, probably due to the relatively large slice thickness: 26 µm in the phase-contrast images versus 3 µm in the histopathological images. Phase-contrast X-ray CT allowed visualization of various components of the tumors, such as the solid tumor components, cysts, and septa, based on density variations. Phase-contrast X-ray CT images are generated by density differences within the object, whereas gray-scale H&E images show cellular morphological components, such as nuclei of various chromaticities, and cytoplasm sizes. The density differences within the solid components might be mainly generated by the number and sizes of the nuclei per unit area and the amounts of protein within the cytoplasm. Thus, minute differences in the absolute densities within the tumor might predict the characteristics of a tumor and its malignancy grade.

Pathological images provide abundant information about micro-structures; however, deformities or artifacts are occasionally caused during the cutting and staining procedures. Furthermore, phase-contrast X-ray CT allowed discrimination of minute differences of the absolute densities within the tumor and also a 3D volumetric study.

White spots within the tumors visualized on phase-contrast X-ray CT images corresponded to blood clots within vessels visualized on the H&E images. Trans-axial phase-contrast X-ray CT images failed to reveal the vascular network within the tumor, which is a unique vital component of the tumor (18); therefore, 3D image reconstruction was performed. This 3D information about the tumor vessels is useful, because the density of the vascular network is known to be related to the histological malignancy grades of seminomas (19).

In conclusion, phase-contrast X-ray CT allowed clear visualization of the detailed inner structures and the 3D vascular network in the seminomas, a rarely encountered germ cell tumor, in the rat. These images are equivalent to low magnified histopathological images. Thus, phase-contrast X-ray CT with a spatial resolution of 26 µm might allow prediction of the morphological characteristics of a tumor even before histopathological processing.