1
Eration, Migration, Invasion, Galectin-Backgound In spite of recent advances in the treatment of patients with glioblastoma, the prognosis for those afflicted remains poor. Even when these tumors harbor a favorable gene methylation profile, the newest standard of care, including temozolomide as a chemotherapeutic [1],* Correspondence: Gtoussaint@medicine.tamhsc.edu 1 The Texas Brain and Spine Inst
1
Radial migration assay was performed as previously described [30,31]. In a blinded fashion, various U87Gal-1 clones were analyzed and compared to U87GFP controls and parental U87MG cells. Of each clone, 2500 cells were allowed to sediment through a cooled manifold onto laminin-coated cell culture wells (Creative Scientific Methods, Inc., Phoenix, AZ). The manifold and slide were incubated together
1
That is relatively over-expressed at the tumor periphery. These graphical representations of gene expression data compare the relative expression of galectin-1 from the core and edge of tumors to pooled data from normal mouse brain samples. (Graphics from GeneSpringW).created. To ensure that galectin-1 over-expression would not enhance proliferation of the U87MG line (and hence alter the interpret
1
Ite were true: lowest relative expression value at the tumor edge compared to tumor core and normal brain. The genes meeting this ideal profile were ranked by pvalue (between core and edge).ImmunohistochemistryRaw data from chip hybridization experiments were normalized across chips and across probesets using a fast linear Loess routine [29], known as Fastlo. This normalization routine, in some re
1
Ns were transferred to nitrocellulose and these membranes were incubated with primary antibody for 60 minutes (anti-Gal1 from Research Diagnostics, Flanders, New Jersey, anti-beta actin from Sigma, St. Louis, Missouri). After washing and incubation with secondary antibody (Goat Anti-Mse IgG-HRP, Pierce, Rockford, IL), developing solution was added to the membrane (Supersignal West Femto Substrate,
1
That is relatively over-expressed at the tumor periphery. These graphical representations of gene expression data compare the relative expression of galectin-1 from the core and edge of tumors to pooled data from normal mouse brain samples. (Graphics from GeneSpringW).created. To ensure that galectin-1 over-expression would not enhance proliferation of the U87MG line (and hence alter the interpret
1
By the ample amount of normal mouse brain tissue available for dissection. In spite of species differences, cross-hybridization of mouse genetic material to human probes did prove to be a common occurrence. These data made it possible to control, rather stringently, for the potential contamination of tumor edge samples with mouse brain. Of course, there could still be possible contamination ?react
1
Rbance values of the other wells. Average corrected absorbance was compared between transfectant and parental cells, using a t-test.ECM attachment assaysThe U87MG human glioma cell line was kept in tissue culture in DMEM (Cellgro Mediatech, Inc.), with 10 fetal bovine serum, and penicillin/streptomycin. For transfection, 2.5x106 cells were plated overnight on a 100 mm round dish. Cells were trans