Two biologic characteristics contribute to the lethality of many human brain tumors: 1) their uncontrolled proliferation in the restricted cranial space and 2) the highly dispersive nature of tumors such as glioblastomas [1
]. The rapid and extensive dispersal of GBMs results in the formation of secondary masses throughout the brain, some of which occur in inoperable regions. This, combined with an increased tumor load, leads to rapid mortality [6
]. MRI is limited in its ability to detect these small clusters of glioblastoma cells that can form secondary masses [7
]. Thus, MRI-guided surgical resections are likely to overlook GBM cells that have dispersed away from the main tumor mass, leading to poor prognosis. Clearly, the development of specific molecular diagnostics is critical for the detection and eradication of GBM. In this article, we report the identification of an extracellular fragment of PTPµ that can be molecularly imaged in vivo
and used as a unique biomarker of GBM cells. Importantly, the peptides described in this article offer novel molecular diagnostic tools that are able to cross the compromised bloodbrain barrier to label GBM tumors.
GBM cell dispersal occurs along characteristic pathways of anatomic structures in the brain that are rich in cell adhesion molecules and extracellular matrix molecules that serve as permissive substrates for cell migration [29,30
]. GBM cell dispersal requires the production of proteolytic enzymes [31
], which gives the cell the ability to move through its environment [29
]. GBM cells overexpress growth factor receptor protein tyrosine kinases and their ligands, an important prerequisite for tumor growth and dispersal [32
]. The activity of the receptor tyrosine kinases is normally kept in check by the opposing activity of RPTPs such as PTPµ, which are important regulators of adhesion-dependent signals [8–10
Human tissue samples of GBM and noncancerous “normal” brain from epileptic foci were examined by immunoblot analyses. These studies revealed a dramatic increase of a 55-kDa N-terminal extracellular fragment of PTPµ in dispersive GBMs when compared with normal brain. Using peptides that specifically recognize this PTPµ extracellular fragment, it was determined that the PTPµ fragment is common to high-grade glioblastomas. The PTPµ extracellular fragment is present in human tumor “edge” samples, and the peptides are able to demarcate tumor cells in tissue sections, suggesting that the peptides could be used diagnostically for molecular imaging of dispersive brain tumors or the tumor margin in vivo. To assess whether this is the case, experiments were performed using a rodent flank human GBM tumor model. The proof-of-principle experiments used fluorescently labeled peptide probes to image the tumor cells in live rodents using the Maestro FLEX In Vivo Imaging System. Systemic introduction of the peptide probes resulted in rapid and specific labeling of the flank tumors within minutes. Labeling occurred primarily within the tumor and at the tumor margin, indicating that the extracellular PTPµ fragment remains associated with the tumor. Most importantly, the PTPµ peptides crossed the compromised blood-brain barrier to specifically label GBM tumor cells in the brain. Together, these data demonstrate that the PTPµ peptide probes could be used as molecular indicators of highgrade glioblastoma.
The development of the PTPµ peptide probes was based on a large body of structural and functional data. The sites required for PTPµ-mediated homophilic adhesion have been well characterized by our laboratory and others [11,13–20,33
]. The crystal structure of PTPµ provided valuable information regarding which regions of each functional domain are likely to be exposed to the extracellular environment and therefore available for homophilic binding and detection by a peptide probe. Of the four peptides generated, we found two that are compelling as specific markers of high-grade GBM (SBK2 and SBK4). A point of interest is that these peptides recognize two different domains of PTPµ (SBK2: the MAM domain; SBK4: the Ig domain) that are folded in a close conformation [19
], and are both required for efficient cell-cell adhesion [34
We previously determined that PTPµ is cleaved at three different sites to generate both a mature protein and, ultimately, an intracellular fragment that is found in both the cytoplasm and the nucleus [21
]. In that study, we determined that cleavage of the extracellular domain of PTPµ through a metalloprotease (a matrix metalloproteinase or A Disintegrin And Metalloprotease [ADAM]) was necessary for further processing [21
]. Although we cannot identify the site of cleavage because metalloproteases lack a specific amino acid recognition sequence, it may be that such an enzyme is functioning to yield the extracellular fragment of PTPµ. Another type IIb RPTP, PTPκ is also cleaved by an ADAM to release an extracellular fragment [35
]; thus, the cleavage we observe here may be a common phenomenon. It is likely that cleavage of other cell surface receptors also occurs in GBMs and many other tumors. In support of this, metalloproteases [36
] and metalloprotease cleavage of adhesion molecules are linked to tumor progression [37
]. A similar molecular detection strategy could be used with any other homophilic or heterophilic-binding cell surface protein whose ligand-binding site is known. A large variety of cell surface proteins, including other phosphatases, are cleaved at the cell surface [35,38–40
]. These proteins represent additional targets for the development of novel molecular diagnostics [41
]. Furthermore, the PTPµ peptides could be used as a starting point to develop higher affinity small molecules with similar ligand-binding capabilities.
In the context of this study, a question remains: What is the biologic significance of the cleavage of PTPµ and shedding of the extracellular fragment? Recently, we examined whether the absence of PTPµ influences the migratory behavior of glioblastoma cells in the complex environment of the brain [12
]. We found that full-length PTPµ influences contact-dependent signaling by negatively regulating migration of glial cells [12
]. Therefore, the loss of PTPµ protein expression through proteolysis and fragment generation may be advantageous to GBM during tumor progression. It is unclear whether the association of the PTPµ fragment with dispersing GBM cells has an additional functional role to modulate cell-cell adhesion or interaction with endothelial cells, which also express PTPµ [24–28
]. Alternatively, the extracellular fragment may activate intracellular signaling cascades by binding to cell surface PTPµ on other cells in the tumor microenvironment. Because the extracellular fragment contains all the domains of PTPµ required for efficient homophilic binding and it maintains adhesive activity, it is intriguing to speculate that the PTPµ shedding generates a “highway” for tumor cell migration in the microenvironment.
Neurosurgeons routinely use stereotactic techniques and intraoperative MRI in surgical resections. This allows them to identify and sample tissue from distinct regions of the tumor such as the tumor edge or center. Frequently, they also sample regions of brain on the tumor margin that are outside the tumor edge that appear to be grossly normal but are infiltrated by dispersing tumor cells on histologic examination. Such surgical techniques have been instrumental in characterizing various molecules associated with the invasive phenotype [42,43
].With the discovery of molecular diagnostics for GBM cells, intraoperative imaging techniques could guide neurosurgical resection and eliminate the “educated guess” of the location of the tumor margin by the neurosurgeon. Previous studies have determined that more extensive surgical resection improves patient survival [5,44,45
]. Thus, PTPµ peptide probes that function as diagnostic molecular imaging agents have the potential to increase patient survival. Currently available intraoperative imaging devices could be retrofitted to visualize these PTPµ peptide probes. Alternatively, the PTPµ peptides could be labeled with other agents to identify the best imaging modality for visualizing small numbers of dispersing GBM tumor cells. Such an approach has clear translational applications and may lead to improved outcomes for patients with this devastating disease.