Cells isolated from human glioblastoma multiforme express progesterone-induced blocking factor (PIBF)

Abstract

Glioblastoma multiforme (GBM) is the most common and malignant tumor in the central nervous system. One of the contemporary hypotheses postulates that its pathogenesis is associated with the cancer stem cells (CSCs) which originate from mutations in the normal neural stem cells residing in their specific "niches." Simultaneously with its aggressive development the tumor suppresses the local immune system by different secreted and/or cell expressed factors. Progesterone-induced blocking factor (PIBF) is an immunomodulatory protein with known role in the regulation of the immune response in the reproductive system. Expression of PIBF has been described in some tumors as one of the factors suppressing the anti-tumor immunity. The aim of the present study was to check for the expression of PIBF from cells isolated from six GBMs. To characterize the cultured cells and to study the PIBF expression confocal microscopy, flow cytometry, ELISA, and real-time PCR were used. The results obtained showed expression of markers typical for cancer CSCs and secretion of interleukin 6 by the GBM-derived cultured cells. The results convincingly prove that PIBF is intracellularly expressed by the cultured cells from the all six GBM samples, and this fact is confirmed by three different methods-flow cytometry, confocal microscopy, and real-time PCR. This paper reports for the first time the expression of PIBF by GBM-derived cells cultured in vitro and reveals a new aspect of the immunosuppressive mechanism used by GBM in escaping the immune control.

Fig. 1

Growth of cells isolated from GBM and cultured in vitro. When cultured in the presence of EGF, bFGF, and 10 % FBS the cells grow as a adherent cultures—G1, G2, G3, G5, G6 samples or b as semi-adherent cultures (G4 sample). Growth of cells isolated from G1 and G4 is shown at the figure

Fig. 2

Markers typical for CSC are expressed by GBM-derived cells. Cultured cells are positive for a intracellular localization of Nestin in 97.1 % of the cells, b intracellular expression of Sox-2 in 79.3 % of cells, c surface localization of CD44 in 78.8 % of the cells, and d intracellular localization of GFAP in 71.30 % of the cells. The results shown are for G5 sample and illustrate the general tendency. Surface expression of CD133 is recorded in only two of the GBM samples (G2 and G4) processed in these experiments, and the result shown is for G2 (e)

Fig. 3

Expression of PIBF by GBM-derived cultured cells: a surface expression of PIBF was not detected, b intracellular expression of PIBF was found in the cells isolated from all GBM samples included in the experiments. The result shown is with cells from G2 sample (7.56 % positive cells) and illustrates the general tendency

Fig. 4

Different localization of PIBF. GBM cells were analyzed by confocal microscopy. a Homogeneous cytoplasmic and fine granular nuclear pattern of PIBF (magnification ×400), b granular cytoplasmic with dot-like perinuclear staining and fine granular nuclear patter of PIBF (magnification ×400), c intensive granular nuclear pattern (magnification ×400), d perinuclear pattern of PIBF (magnification ×630), e control cells without anti-PIBF 3A6 antibody (magnification ×400). GBM cells were stained with anti-PIBF Mab 3A6 and anti-mose Alexa Fluor 488 (shown as green). Nuclei were counterstained with Hoechst 33258 (shown as blue) (Color figure online)

Fig. 5

Representative agarose gel shows PCR products from amplified cDNA derived from glioblastoma cells. Real-time PCR was performed followed by analysis of the PCR products in ethidium bromide-stained agarose gel showing the expression of mRNAs encoding PIBF and GFAP. β-actin was used as an internal standard. PCR products consisted of 202-bp fragment (PIBF), a 161-bp fragment (GFAP), and a 661-bp fragment (β-actin). The expression of PIBF and GFAP was confirmed in samples isolated from all six GBM-derived cell cultures