The invasive nature of GBM has been recognized for over a century. In 1934, Voris reported that nearly 25% of frontal lobe GBMs had bilateral infiltration across the corpus callosum 
. Thirty years later, Matsukado reviewed 100 grade 3 and grade 4 astrocytomas and found that nearly 50% had bilateral extension 
. More recently, disseminated and multifocal GBM have been shown to confer a worse prognosis 
. Microscopic invasion makes truly complete surgical resection of GBM impossible. Although several markers of tumor proliferation and metabolism have been identified in GBM and are used clinically, prognostic markers of invasive capacity are largely lacking. Such markers can offer prognostic value and may identify new targets for therapy. In this study we demonstrate that CD97 expression has a functional effect on tumor migration and invasion in vitro
, and correlate expression with survival in a large cohort of GBM patients. Our data suggests that CD97-mediated tumor invasion may be an important contributing factor to the observed decreased survival associated with CD97 overexpression.
CD97, along with other members of the EGF-TM7 family, possesses a unique structure involving a large extracellular domain linked to a seven-spanning transmembrane subunit. The CD97 transcript is alternatively spliced to produce isoforms with 3–5 EGF-like domains. These isoforms vary in their binding affinity and may confer functionally distinct phenotypes 
. Although once thought to be leukocyte restricted, CD97 is now believed to play an important role in a number of malignancies. In thyroid cancer, CD97 overexpression is associated with increased tumor grade and is a marker of dedifferentiation 
. In colon cancer, its expression has been demonstrated at the leading edge and correlates with poor clinical stage and increased lymphatic invasion 
. Using an animal model of colon cancer, Becker and colleagues showed that CD97 was localized to E-cadherin-based adherens junctions and strengthened lateral cell-cell contact between enterocytes 
. The authors also showed that overexpression of CD97 lead to upregulation of membrane-bound β-catenin, with subsequent activation of Akt. In prostate cancer, CD97 has been found in nearly 60% of clinical samples, across Gleason scores, but is not expressed in normal prostate 
. In the same study, Ward and colleagues found that CD97 induces Gα12/13
-dependnent RHOA activation and that depletion of CD97 reduced bony metastases by over 50%. The authors also showed that CD97 heterodimerizes and positively regulates lysophosphatidic acid receptor 1 (LPAR1) signaling, a well-established mediator of tumorigenesis and metastasis in prostate cancer 
Our findings in this study expand upon an initial report by Chidambaram et al. identifying CD97 expression in GBM 
. The authors demonstrated a decrease in CD97 transcript after suppression of Wilms tumor 1 (WT1) by siRNA. The authors then identified CD97 expression in three GBM cells lines and showed that knockdown of CD97 resulted in decreased cell invasion. The findings presented in our study characterize CD97 in low passage primary GBM cultures and show that CD97 is also associated with cell migration, but not proliferation. Although the difference in migration and invasion after CD97 knockdown were not as significant in the U87MG cell line compared to U251, we believe this is attributable to differences in cell phenotype, since U251 are known to behave more aggressively and exhibit increased invasiveness in vivo
. Most importantly, we provide the first report of an association between CD97 expression and survival in patients with GBM based on pooled analysis of patients from the TCGA. There are limitations to using TCGA data since these results do not control for known prognostic factors including age, Karnofsky Performance Status, and extent of resection. Regardless, this univariate data is compelling and provides impetus for further studies on CD97 in malignant glioma. Furthermore, different CD97 isoforms are known to confer different phenotypes in other malignancies. We are currently investigating these isoforms and their associated phenotypes.
Although the mechanisms underlying glioma invasion have not been completely characterized, several important mediators have been investigated. Glioma cells are known require certain ECM proteins in order to switch to a migratory phenotype; studies have shown that glioma cells do not move when propagated in serum free media, but become motile when treated with specific ECM components including laminin, fibronectin, and collagen 
. Integrins have also been shown to play an important role in glioma invasion 
. These transmembrane glycoproteins are heterodimers composed of an α and β subunit; they are capable of interacting with elements of the ECM and triggering signal transduction. The β1 subunit in particular has been shown to play an important role in malignant behavior and invasion of gliomas 
. More recently, the integrin αvβ8 has been shown to play a central role in GBM angiogenesis and perivascular tumor invasion 
. The association with integrins and invasion is particularly noteworthy since CD97 has been shown to demonstrate strong interactions with integrin α5β1, which is expressed in GBM and other cancers 
; coengagement of CD97 with both chondroitin sulfate, a component of the ECM, and α5β1 synergistically initiates endothelial cell invasion 
. Integrins are being actively investigated as therapeutic targets for GBM; EMD121974 (Cilengitide), a peptide targeting the RGD-motif of integrins, showed modest efficacy in patients with recurrent GBM and will likely be subjected to future studies in combination with cytotoxic therapies 
Functionally, CD97 has been shown to promote cell migration and invasion, as well as increase expression and secretion of matrix metalloproteinase-2 (MMP2) and MMP9 in the fibrosarcoma cell line HT1080 
. By degrading components of the ECM, MMPs create physical space for glioma cells and may be critically important in promoting glioma invasion. Cathepsin B, a cysteine protease, and MMP-9 have been shown to participate in tumor growth, invasion, and angiogenesis in GBM; RNA interference was effective in suppressing this phenotype, suggesting a potential therapeutic application 
. MMP-1 is expressed in GBM and capable of cleaving elements of the ECM or activating downstream signaling pathways that promote oncogenesis and invasion 
. There is compelling data demonstrating the efficacy of MMP inhibitors in vitro
and in animal models 
; they have been evaluated in clinical trials for patients with glioblastoma, but produced mixed results 
. In addition to interaction with integrins, CD97 may promote glioma cell invasion by increasing secretion of MMPs.
There are challenges to using CD97 as a therapeutic target given its expression on normal leukocytes, but several studies have made attempts to modulate or block its expression in certain pathologic conditions. Kop and colleagues used a systemically administered CD97 neutralizing antibody in a rodent model of arthritis to decrease joint inflammation and clinical signs of disease 
. The authors found no difference in important cytokine profiles, particularly IL-10 and interferon-γ (IFN-γ), suggesting that the antibody was exerting its beneficial effect by interfering with the binding of CD97-positive leukocytes and the ligands chondroitin sulfate, CD55, or integrin α5β1. Using a thyroid cancer cell line, Park et al. showed that troglitazone, a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, can downregulate surface expression of CD97 
. Sodium pyruvate has been shown to decrease CD97 expression in oral squamous cell carcinoma cell lines (OSCC) 
, while retinoid acid causes similar effects in both OSCC and thyroid cancers 
. Taken together, these studies suggest that clinically relevant modalities can be used to modulate the expression of CD97 and that translation of these findings into in vivo
pre-clinical models is possible. GBM is characterized by genetic heterogeneity and expression of CD97 likely varies across tumors. However, CD97 targeted therapies may hold promise as a component of molecularly targeted anti-GBM strategies. Future studies must utilize in vivo
models to more thoroughly characterize the role of CD97 in glioma invasion. Xenograft models represent a first step towards correlating the clinical data we have presented with an observable phenotypic change in vivo
, however given the importance of immune cells in the glioma microenvironment, an immunocompetent model is ideal. Use of a syngeneic tumor model with genetic silencing of CD97, as well as neutralizing antibodies or pharmacologic agents known to decrease CD97 expression, will further improve our understanding of CD97 in glioma invasion and potentially influence future treatment paradigms.
Our data demonstrates, for the first time, an association between CD97 expression and survival in patients with glioblastoma. We believe this difference can be explained by an associated increase in tumor migration and invasion, but not proliferation. Future studies are warranted to determine if blockade or downregulation of CD97 confers a similar survival advantage in vivo.