Pituispheres Contain Genetic Variants Characteristic to Pituitary Adenoma Tumor Tissue

Abstract

The most common type of pituitary neoplasms is benign pituitary adenoma (PA). Clinically significant PAs affect around 0.1% of the population. Currently, there is no established human PA cell culture available and when PA tumor cells are cultured they form two distinct types depending on culturing conditions either free-floating aggregates also known as pituispheres or cells adhering to the surface of cell plates and displaying mesenchymal stem-like properties. The aim of this study was to trace the origin of sphere-forming and adherent pituitary cell cultures and characterize the potential use of these surgery derived cell lines as PA model. We carried out a paired-end exome sequencing of patients' tumor and germline DNA using Illumina NextSeq followed by characterization of corresponding PA cell cultures. Variation analysis revealed a low amount of somatic mutations (mean = 5.2, range 3-7) in exomes of PAs. Somatic mutations of the primary surgery material can be detected in the exomes of respective pituispheres, but not in exomes of respective mesenchymal stem-like cells. For the first time, we show that the genome of pituispheres represents genome of PA while mesenchymal stem cells derived from the PA tissue do not contain mutations characteristic to PA in their genome, therefore, most likely representing normal cells of pituitary or surrounding tissues. This finding indicates that pituispheres can be used as a human model of PA cells, but combination of cell culturing techniques and NGS needs to be employed to adjust for disability to propagate spheres in culturing conditions.

Keywords: pituispheres; pituitary adenoma; pituitary adenoma cultures; tumor sequencing; whole exome sequencing.

Figure 1

Morphology of pituispheres and MSC isolated from pituitary adenoma samples. Representative phase-contrast images from short term (4 days) cultured pituispheres (A,C) and first passage MSC culture (B,D) isolated from PA03 (A,B) and PA05 (C,D) adenoma samples. 200 × magnification of pituispheres and 400 × magnification of MSC.

Figure 2

Expression of cell markers in pituispheres. Representative immunofluorescence images of (A) PA02, (B) PA03, and (C) PA05 pituispheres. Cells were double-stained for (A,B) CD15 (green) and GFRα2 (red), (C) Pit-1 (green), and SF-1 (red) (B) CD15 (green) and SF-1 (red) and PA02 pituispheres were single stained for (A) Pit-1 (green) and SF-1 (red) and PA05 pituispheres were single stained for (C) Pit-1 (green) and GFRα2 (red). Isotype-matched antibodies were used for negative controls. Cell nuclei were counterstained with DAPI (blue). Scale bar, 13 μm.

Figure 3

Expression of CD90 marker in pituispheres and MSC. Immunofluorescence images of PA02, PA03, and PA05 (A) MSC and (B) pituispheres stained for CD90 (green). Isotype controls were used as negative control. Cell nuclei were counterstained with DAPI (blue). Scale bar, 13 μm.

Figure 4

Overlapping somatic mutations in somatic tumor tissue, pituispheres, MSC, and patients' germline DNA.