Nestin expression in osteosarcomas and derivation of nestin/CD133 positive osteosarcoma cell lines

Eighteen samples of primary untreated high-grade osteosarcoma of bone (17 conventional osteosarcomas: 15 osteoblastic and 2 chondroblastic, and 1 telangiectatic osteosarcoma; 9 males, 9 females; age range: 8–57 years old, mean 21 years old) were included in this study. Formalin-fixed and paraffin-embedded surgical samples of neoplastic tissues were retrieved from the files of the Department of Pathology, University Hospital Brno, Czech Republic, and of the 1^st Department of Pathologic Anatomy, St. Anne's University Hospital, Brno, Czech Republic. The histologic sections stained with H-E were reviewed by three pathologists in total and each individual tumor sample by two of them (MH and IZ; or MH and KV), and representative tissue blocks were selected for immunohistochemical analysis. Fifteen samples were not ossified and were not subjected to decalcification and three cases were decalcified using 8% hydrochloride acid-ferric chloride solution, as indicated in the Table 1. To obtain cell cultures, biopsy samples were taken from 4 patients surgically treated for osteosarcoma. Written informed consent was obtained from each participant before entering into this study. The samples for cell cultures were coded and processed in the laboratory in an anonymous manner. The Research Ethics Committee of the University Hospital Brno approved the study protocol.

Immunohistochemical detection of nestin was performed on 4 μm thick tissue sections applied to positively-charged slides. The sections were deparaffinized in xylene and rehydrated through a graded alcohol series. Antigen retrieval was performed in the lab microwave (Milestone) by heating the sections in citrate buffer at pH 6.0 for 20 min at 98°C. Endogenous peroxidase activity was quenched in 3% hydrogen peroxide in methanol for 10 minutes. Tissue sections were incubated overnight at 4°C with a mouse monoclonal antibody to nestin (clone 10C2, dilution 1:200, Millipore, Billerica, MA, USA). A streptavidin-biotin peroxidase detection system was used in accordance with the manufacturer's instructions (Vectastain Elite Kit, Vector Laboratories, Burlingame, CA, USA); 3,3'-diaminobenzidine was used as the chromogen (DAB, Fluca, USA). Slides were counterstained with Gill's hematoxylin. Tissue sections of glioblastoma multiforme served as external positive controls; nestin-positive endothelial cells in osteosarcoma tissue samples were used as internal positive controls. Negative controls were prepared by incubating samples without the primary antibody. Evaluation of immunohistochemical results was performed using a uniform microscope and camera setting (Olympus BX51, DP70).

For nestin, cytoplasmic immunostaining was considered to be positive. The percentage of Nes+ tumor cells (TC) was counted and categorized into four levels: +/- (<2 % Nes+ TC), + (2–10 % Nes+ TC), ++ (11–50 % Nes+ TC), and +++ (51–100 % Nes+ TC). The intensity of immunostaining was classified as very weak (+/-), weak (+), medium (++), and strong (+++). The intensity of immunostaining was also evaluated in endothelial cells, which were used as an internal positive control. The slides were evaluated with a light microscope at ×400 magnification. At least 5 discrete foci of neoplastic infiltration were analyzed, and the average staining intensity and the percentage of nestin positive cells of the entire covered area were determined.

Starting with primary cultures, fresh specimens of tumor tissue were processed as described previously [34]. The primary cultures were maintained in DMEM supplemented with 20% fetal calf serum, 2 mM glutamine, and antibiotics: 100 IU/ml of penicillin and 100 μg/ml of streptomycin (all purchased from PAA Laboratories, Linz, Austria) and cultivated under standard conditions at 37°C in an atmosphere of 95% air : 5% CO₂. Once the specimen pieces had attached, the volume of the medium was gradually increased to 5 ml over the next 48 hours. As soon as the outgrowing cells covered about 60% of the surface, they were trypsinized, diluted, and transferred into a new flask. A similar procedure was used for further subcultivations of all cell lines that were derived from the primary cultures. Altogether, 4 successfully derived osteosarcoma cell lines (OSA-1, OSA-2, OSA-3, and OSA-5) were included in this study. The basic description (gender and age) of the patients from whom the respective samples were taken is given in the Table 1. The established Saos-2 osteosarcoma cell line (ATCC No. HTB-85) and the GM-7 glioblastoma cell line [34] were used as control cell lines in this study.

To perform immunostaining of intracellular and cell surface antigens in these cell lines, cell suspensions at a concentration of 10⁴ cells per ml were seeded on glass coverslips and grown under standard conditions for 24 h. Cells were then washed in PBS, fixed with 3% para-formaldehyde (Sigma) in PBS for 20 min at RT, and permeabilized with 0.2% Triton X-100 (INC Biomedicals) in PBS for 1 min at RT. For detection of the CD133 cell surface molecule, this procedure was performed without permeabilization. The cells were subsequently washed in PBS and incubated for 10 min with 2% BSA (PAA) to block nonspecific binding of the secondary antibodies. All intracellular and cell surface antigens were visualized by indirect immunofluorescence. Mouse monoclonal human-specific anti-nestin antibody (clone 10C2, dilution 1:200, Millipore), mouse monoclonal anti-vimentin antibody (clone LN-6, dilution 1:100, Sigma Chemical Co., St. Louis, MO, USA), mouse monoclonal anti-desmin antibody (clone DE-U-10, dilution 1:40, Sigma), rabbit polyclonal anti-S100 antibody (No. S2644, working concentration 20 μg.ml^-1, Sigma), and rabbit polyclonal anti-CD133 antibody (No. ab19898, dilution 1:100, Abcam, Cambridge, UK) were used as primary antibodies. The cells were treated with primary antibodies at 37°C for 1 h and then washed three times in PBS. Corresponding secondary antibodies, i.e. anti-mouse antibodies conjugated with FITC or TRITC (Sigma) or anti-rabbit antibody conjugated with TRITC (Sigma), were applied under the same conditions. Finally, the cells were mounted onto glass slides in Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA). The cells were observed using an Olympus BX-61 fluorescence microscope. Micrographs were captured with a CCD camera COHU 4910 and analyzed using software Lucia 4.80 – KARYO/FISH/CGH (Laboratory Imaging, Prague, Czech Republic).

The results of the immunohistochemical detection of nestin expression in osteosarcomas are summarized in Table 1. Nes+ tumor cells were found in all examined tumor samples taken from the 18 patients of both gender and various age (Figure 1a–c). Nevertheless, the frequency of these Nes+ cells ranged from the strong positivity in a significant proportion of tumor cells (Table 1, Figure 1a), through a dispersed distribution (Table 1, Figure 1b), to a sporadic occurrence of these cells (Table 1, Figure 1c). Similarly, the intensity of immunostaining for nestin varied from strong to very weak (Table 1, Figure 1a–c). All 18 tumor samples undoubtedly showed Nes+ endothelial cells.

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Immunoreactivity for nestin was examined in four newly derived osteosarcoma cell lines as described above. In the way of other intracellular markers, all of these cell lines were positive for vimentin (Figure 2a), desmin (Figure 2b), and S100 protein (Figure 2c). Results concerning the immunoreactivity for nestin, CD133 and other intracellular proteins are summarized in Table 2. Nestin was detected in three (OSA-1, OSA-2, and OSA-3) of the four examined cell lines; in the OSA-5 cell line no signal for nestin was observed (Table 2, Figure 2k–l). The OSA-3 cell line displayed a strong, even positivity for nestin and a distinct network of Nes+ filaments in the cytoplasm of individual cells (Figure 2h–i), while OSA-1 and OSA-2 cell lines showed medium intensity of fluorescence, and only the diffuse signal in the cytoplasm was observable. All newly derived cell lines also showed a strong positivity for the CD133 membrane antigen (Figure 2g,i), including the nestin-negative OSA-5 cell line (Figure 2j,l). Double-labeling of both of these antigens confirmed their appearance in the same cells (Figure 2g–i). Control Saos-2 osteosarcoma cell line also clearly showed both of nestin (Figure 2e) and CD133 (Figure 2f) positivity.

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