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 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 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

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

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

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

CD97 does not affect cell proliferation.

<p>U251 and U87MG cells were subjected to siRNA-mediated knockdown of CD97. Cells were quantified using an ATP-based luminescent assay, with luminescence quantified in counts per second (CPS). In both U251 (A) and U87MG (B) cells, there was no difference in proliferation at 24 and 48 hours after confirmed protein knockdown.</p

CD97 confers an invasive phenotype.

<p>U251 and U87MG cells were subjected to siRNA-mediated knockdown of CD97. In both cell lines, knockdown of CD97 resulted in decreased invasion through a Matrigel invasion chamber. In U251 and U87MG cells, invasion was decreased by 70% (p = 0.03) and 28% (p = 0.05), respectively (A, B).</p

CD97 is localized to the cell membrane.

<p>To determine the distribution of CD97 within the cell we performed immunostaining on U87MG cells grown in culture. Compared to α-tubulin, a cytoskeletal marker distributed throughout the cytosolic compartment, CD97 expression was mostly localized to the cell membrane.</p