Amoureux et al. BMC Cancer 2010, 10:91 http://www.biomedcentral.com/1471-2407/10/91 samples, which generated a corresponding PSA-NCAM value by ELISA, similar to non-tumoral adult human brain tissue for GBM1 (Figure 2A) and higher for GBM2 (Figure 2B). It is worth noticing that PSA- NCAM expression was different from one GBM to another: either weak and diffuse in large area of the biopsy in some cases (Figure 2B), or strong but restricted to fewer tumor cells in others (Figure 2A), this last pattern of expression being more frequent. We also included in our study 3 MB known to express high levels of PSA [16]. These MB samples were highly stained by immunohistochemistry (score 360 to 400) (Figure 2C) and displayed high PSA-NCAM concentra- tions by ELISA (150-1030 pg PSA-NCAM/μg of protein, n = 3). In total, a series of 56 GBM samples were ana- lyzed by immunohistochemistry and given a score, and by ELISA. Figure 2D shows the 56 individual values of PSA-NCAM measured by ELISA and immunohisto- chemistry, and on Figure 2E, patients were separated in two groups (ELISA PSA-NCAM <10 pg/μg of protein and PSA-NCAM >10 pg/μg of protein). Spearman cor- relation test shows a statistical positive correlation between the two methods of PSA-NCAM measurement (Rho = 0.439, p = 0.005). PSA-NCAM is expressed in 70% of 56 GBM biopsies We quantified PSA from 56 GBM biopsies. As much as 70% of them expressed PSA-NCAM at levels above 10 pg PSA-NCAM/μg of protein. The median was of 24.73 pg PSA-NCAM/μg of protein. Apart from some very high values as depicted on the histogram (Figure 2F), 50% of the biopsies gathered around the median. It should be noted that 90% of them contained less PSA- NCAM than the MB with the lowest PSA-NCAM con- tent (150 pg PSA-NCAM/μg of protein). PSA-NCAM expression in GBM is correlated to shorter OS and DFS In this series, none of the clinical factors including age, preoperative KPS and extent of surgical removal was correlated to OS or DFS. The patient cohort includes long-term survivors, and surgical resection was not sys- tematically analyzed by MRI. These factors could have prevented to reveal the prognosis value of these classical clinical parameters. However, PSA-NCAM expression was significantly associated to OS [median OS: 20.8 months (CI:14.2-27.4) in patients with PSA-NCAM GBM content ≤ 10 pg/μg of protein versus 12.2 months (CI:11.0-13.5) in the others; p = 0.04, n = 56] (Figure 3A). PSA-NCAM expression was also statistically corre- lated to DFS (median DFS: 11.2 months (CI:7.6-14.8) in patients with PSA-NCAM GBM content ≤ 10 pg/μg of protein versus 6 months (CI:3.4-8.5) in the others; p = 0.017, n = 54] (Figure 3B). Page 6 of 12 On multivariate analysis (age, KPS and extent of surgi- cal excision), PSA-NCAM expression was the unique predictive parameter of OS [hazard ratio (HR): 2.01 for patients suffering from a GBM with PSA-NCAM con- tent >10 pg/μg of protein (CI:1.01-4); p = 0.046, n = 56] and DFS [HR: 2.89 for patients suffering from a GBM with PSA-NCAM content >10 pg/μg of protein (CI:1.34- 6.27); p = 0.007, n = 54]. PSA-NCAM regulates olig2 expression in glioma To further elucidate the role that PSA-NCAM could play in GB, we investigated whether PSA removal using endoN affected the properties of the GBM9 cell line we previously developed and characterized as a highly pro- liferative neurosphere forming cell line [9]. To this end we compared features of endoN treated and non treated cells in different culture conditions. In basal conditions, cells were grown in presence of EGF and FGF [9]. In a representative experiment described on Figure 4, out of two performed, 88% of the GBM9 cells expressed the olig2 transcription factor. Representative pictures taken from at least 5 independent fields are shown on Figure 4A. Under conditions of PSA removal, the percentage of olig2 positive cells decreased to 64% (Figure 4B). If EGF and FGF were removed from the culture medium, 52% of the cells were olig2 positive, and this percentage further decreased to 35% after PSA digestion. The mean of at least 5 fields is represented on Figure 4B. In the two experiments performed, the percentage of cells los- ing olig2 was 30 and 46% in the presence of EGF and FGF, and 17 and 35% in the absence of growth factors, respectively. PSA removal was capable of decreasing sig- nificantly olig2 content in this GB cell line both under proliferative conditions in the presence of EGF and FGF (p = 0.0153 and p = 0.0399 for the 2 experiments, respectively), and in their absence (p = 0.0289 and p = 0.0297 for the 2 experiments, respectively). We found no effect of PSA removal on the percentage of other cell lineages generated (GFAP+ astrocytes, b3-tubulin+ neu- rons or Nestin+ progenitors) neither in presence, nor in absence of growth factors (not shown). To uncover a possible causal relationship between olig2 and PSA expression, we tested whether PSA- NCAM gain of function would affect olig2 expression. We chose C6 cells which displayed undetectable PSA- NCAM by ELISA or Western blot and show many fea- tures of GB tumors after intracranial injection such as an undifferentiated morphology [20], neovascularization [24], diffuse infiltrating borders and invasion of the sur- rounding tissue by isolated cells [25]. Moreover, PSA- NCAM forced expression in these cells has been shown to be associated with their increased migration when transplanted in mice [20]. Therefore, we compared olig2 content in PSA-NCAM negative C6, and C6-PSA-NCAM