Expression of calcium-permeable AMPA receptor subunits in GBM BTICs.

<p>GBM BTICs strongly express calcium-permeable subunits of AMPA receptor (GluR1 and GluR4) compared to non-stem tumor cells (Primary GBM Cultures) and established GBM cell line U251 (A). Normal mouse brain homogenate (postive control) expressed both GluR1 and GluR4. To confirm that these calcium-permeable AMPA receptors are expressed on the surface membrane, GBM BTICs were stained with N-terminus GluR1 antibody under non-permeabilized condition (B). Compared to negative controls (C), GluR1 stained the surface membranes of BTICs. α-tubulin was stained after permeabilizing the membrane as a postive control (D).</p

CD97 isoform expression in low grade astrocytoma and glioblastoma.

<p>Quantitative PCR was performed on frozen tumor specimens histologically confirmed as low grade astrocytoma (n = 3) and GBM (n = 3). The mean proportion of EGF(1,2,3,5) in low grade astrocytomas was 15% compared to 17% in GBM (p = 0.46), a difference that was not statistically significant (A). Among in vitro samples, the mean percent of EGF(1,2,3,5) comprising total CD97 in patient-matched GBM and BTIC cell lines was 16% and 14%, respectively (p = 0.83). Normal human astrocytes were found to express 4% EGF(1,2,3,5), which was significantly less than both GBM (p = 0.01) and BTIC (p = 0.02) cell lines (B).</p

CD97 expression in glioblastoma-derived brain tumor initiating cells.

<p>Brain tumor initiating cell lines were derived from three patients with histologically confirmed GBM. These cell lines were found to express the stem cell markers nestin and Sox2, as well as CD97, while normal human brain expressed none of these (A). PCR analysis of these cell lines found them to express the EGF(1,2,5) and EGF(1,2,3,5) isoforms of CD97 as confirmed by sequencing analysis (B). Immunocytochemistry demonstrated localization of CD97 to the cell membrane in BTIC cell line 3 (C).</p

CD97 expression across genetic subtypes of glioblastoma.

<p>Gene expression data from the Cancer Genome Atlas (TCGA) was used to characterize CD97 expression across genetic subtypes of glioblastoma. CD97 upregulation was most commonly found in the classical and mesenchymal subtypes, while downregulation was more common in the neural and proneural subtypes.</p

Characterization of CD97 isoforms in glioblastoma.

<p>RNA was isolated from the GBM cell lines U251 and U87MG, then converted to cDNA. PCR with primers spanning the variably spliced regions of CD97 were used to identify the specific isoforms expressed in these cells. Sequencing analysis confirmed these as the EGF(1,2,5) and EGF(1,2,3,5) isoforms of CD97 (A). A schematic of these gene transcripts is also shown with the signal peptide (SP), RGD domain, and seven-span transmembrane (7TM) segments (B).</p

Intracellular calcium imaging in GBM BTICs in response to AMPA receptor stimulation.

<p>AMPA stimulation results in increased intracellular calcium levels in GBM BTICs. Cell morphology in 1.2 mM Ca⁺² HBSS before and 10 minutes after AMPA and CYTZ stimulation showed no changes in cell morphology (A and B, respectively). Calcium levels 10 minutes after 100 µM AMPA and 100 µM CYTZ stimulation showed increase in intracellular calcium concentrations well exceeding 500 µM, suggesting that surface AMPA receptors on GBM BTICs are functional and conduct calcium currents (C and D, false color calcium images before and 10 minutes after calcium stimulation, respectively). The calcium concentrations color-coding is provided by the color scale on the right.</p

IDH1 mutation status of human GBM samples.

<p>Three frozen GBM specimens used for previous analysis and known to express CD97 were found to express wild-type IDH1, but not mutant IDH1 (R132H) by Western blot.</p

Confirmation of adherent GBM BTIC cultures.

<p>Adherent GBM BTICs were prepared and subjected to (A) immunoassays, (B and C) immunocytochemistry, and (D) plating on non-adherent conditions to confirm their stem cell properties. GBM BTICs strongly expressed neural stem cell markers, nestin and Sox-2, by both (A) immunoblotting and (B and C) immunocytochemistry. BTICs formed neurospheres when plated in non-adherent conditions (D).</p

Expression of CD97 across glioma grades.

<p>Frozen tumor specimens histologically confirmed as low grade astrocytoma (n = 3), anaplastic astrocytoma (n = 3), and GBM (n = 3) were homogenized analyzed by Western blot. CD97 was expressed in the GBM specimens, but not low grade or anaplastic astrocytomas (A). Quantitative PCR was performed using these specimens and demonstrated a significant increase in CD97 expression among GBMs compared to low grade and anaplastic astrocytomas when normalized to normal brain (B). PCR using primers flanking the variably spliced regions of the CD97 transcript demonstrate the EGF(1,2,5) and EGF(1,2,3,5) in all GBM specimens and a single anaplastic astrocytoma, but not in the low grade or remaining anaplastic astrocytomas (C).</p

CD97 expression in GBM is inversely related to survival.

<p>Using The Cancer Genome Atlas database, we performed univariate analysis to assess a potential association between CD97 expression and overall survival. Tumors were classified as possessing intermediate expression, down-regulated expression, or up-regulated expression. Up-regulated tumors were defined as those with a greater than two-fold increase in CD97 transcript, while those with a greater than two-fold decrease were classified as down-regulated. When comparing overall survival among patients with tumors demonstrating up-regulation of CD97 to those with down-regulation of CD97, there was a significant difference in survival (p = 0.0065) demonstrating an association between increased CD97 expression and decreased survival.</p