Frame-Based Stereotactic Biopsy of Canine Brain Masses: Technique and Clinical Results in 26 Cases.

This report describes the methodology, diagnostic yield, and adverse events (AE) associated with frame-based stereotactic brain biopsies (FBSB) obtained from 26 dogs with solitary forebrain lesions. Medical records were reviewed from dogs that underwent FBSB using two stereotactic headframes designed for use in small animals and compatible with computed tomographic (CT) and magnetic resonance (MR) imaging. Stereotactic plans were generated from MR and CT images using commercial software, and FBSB performed both with (14/26) and without intraoperative image guidance. Records were reviewed for diagnostic yield, defined as the proportion of biopsies producing a specific neuropathological diagnosis, AE associated with FBSB, and risk factors for the development of AE. Postprocedural AE were evaluated in 19/26 dogs that did not proceed to a therapeutic intervention immediately following biopsy. Biopsy targets included intra-axial telencephalic masses (24/26), one intra-axial diencephalic mass, and one extra-axial parasellar mass. The median target volume was 1.99 cm(3). No differences in patient, lesion, or outcome variables were observed between the two headframe systems used or between FBSB performed with or without intraoperative CT guidance. The diagnostic yield of FBSB was 94.6%. Needle placement error was a significant risk factor associated with procurement of non-diagnostic biopsy specimens. Gliomas were diagnosed in 24/26 dogs, and meningioma and granulomatous meningoencephalitis in 1 dog each. AE directly related to FBSB were observed in a total of 7/26 (27%) of dogs. Biopsy-associated clinical morbidity, manifesting as seizures and transient neurological deterioration, occurred in 3/19 (16%) of dogs. The case fatality rate was 5.2% (1/19 dogs), with death attributable to intracranial hemorrhage. FBSB using the described apparatus was relatively safe and effective at providing neuropathological diagnoses in dogs with focal forebrain lesions.

Selective anti-tumor activity of the novel fluoropyrimidine polymer F10 towards G48a orthotopic GBM tumors.

F10 is a novel anti-tumor agent with minimal systemic toxicity in vivo and which displays strong cytotoxicity towards glioblastoma (GBM) cells in vitro. Here we investigate the cytotoxicity of F10 towards GBM cells and evaluate the anti-tumor activity of locally-administered F10 towards an orthotopic xenograft model of GBM. The effects of F10 on thymidylate synthase (TS) inhibition and Topoisomerase 1 (Top1) cleavage complex formation were evaluated using TS activity assays and in vivo complex of enzyme bioassays. Cytotoxicity of F10 towards normal brain was evaluated using cortices from embryonic (day 18) mice. F10 displays minimal penetrance of the blood-brain barrier and was delivered by intra-cerebral (i.c.) administration and prospective anti-tumor response towards luciferase-expressing G48a human GBM tumors in nude mice was evaluated using IVIS imaging. Histological examination of tumor and normal brain tissue was used to assess the selectivity of anti-tumor activity. F10 is cytotoxic towards G48a, SNB-19, and U-251 MG GBM cells through dual targeting of TS and Top1. F10 is not toxic to murine primary neuronal cultures. F10 is well-tolerated upon i.c. administration and induces significant regression of G48a tumors that is dose-dependent. Histological analysis from F10-treated mice revealed tumors were essentially completely eradicated in F10-treated mice while vehicle-treated mice displayed substantial infiltration into normal tissue. F10 displays strong efficacy for GBM treatment with minimal toxicity upon i.c. administration establishing F10 as a promising drug-candidate for treating GBM in human patients.

New agents for targeting of IL-13RA2 expressed in primary human and canine brain tumors.

Interleukin 13 receptor alpha 2 (IL-13RA2) is over-expressed in a vast majority of human patients with high-grade astrocytomas like glioblastoma. Spontaneous astrocytomas in dogs resemble human disease and have been proposed as translational model system for investigation of novel therapeutic strategies for brain tumors. We have generated reagents for both detection and therapeutic targeting of IL-13RA2 in human and canine brain tumors. Peptides from three different regions of IL-13RA2 with 100% sequence identity between human and canine receptors were used as immunogens for generation of monoclonal antibodies. Recombinant canine mutant IL-13 (canIL-13.E13K) and canIL-13.E13K based cytotoxin were also produced. The antibodies were examined for their immunoreactivities in western blots, immunohistochemistry, immunofluorescence and cell binding assays using human and canine tumor specimen sections, tissue lysates and established cell lines; the cytotoxin was tested for specific cell killing. Several isolated MAbs were immunoreactive to IL-13RA2 in western blots of cell and tissue lysates from glioblastomas from both human and canine patients. Human and canine astrocytomas and oligodendrogliomas were also positive for IL-13RA2 to various degrees. Interestingly, both human and canine meningiomas also exhibited strong reactivity. Normal human and canine brain samples were virtually negative for IL-13RA2 using the newly generated MAbs. MAb 1E10B9 uniquely worked on tissue specimens and western blots, bound live cells and was internalized in GBM cells over-expressing IL-13RA2. The canIL-13.E13K cytotoxin was very potent and specific in killing canine GBM cell lines. Thus, we have obtained several monoclonal antibodies against IL-13RA2 cross-reacting with human and canine receptors. In addition to GBM, other brain tumors, such as high grade oligodendrogliomas, meningiomas and canine choroid plexus papillomas, appear to express the receptor at high levels and thus may be appropriate candidates for IL-13RA2-targeted imaging/therapies. Canine spontaneous primary brain tumors represent an excellent translational model for human counterparts.

A novel ligand delivery system to non-invasively visualize and therapeutically exploit the IL13Rα2 tumor-restricted biomarker.

Our objective was to exploit a novel ligand-based delivery system for targeting diagnostic and therapeutic agents to cancers that express interleukin 13 receptor alpha 2 (IL13Rα2), a tumor-restricted plasma membrane receptor overexpressed in glioblastoma multiforme (GBM), meningiomas, peripheral nerve sheath tumors, and other peripheral tumors. On the basis of our prior work, we designed a novel IL13Rα2-targeted quadruple mutant of IL13 (TQM13) to selectively bind the tumor-restricted IL13Rα2 with high affinity but not significantly interact with the physiologically abundant IL13Rα1/IL4Rα heterodimer that is also expressed in normal brain. We then assessed the in vitro binding profile of TQM13 and its potential to deliver diagnostic and therapeutic radioactivity in vivo. Surface plasmon resonance (SPR; Biacore) binding experiments demonstrated that TQM13 bound strongly to recombinant IL13Rα2 (Kd∼5 nM). In addition, radiolabeled TQM13 specifically bound IL13Rα2-expressing GBM cells and specimens but not normal brain. Of importance, TQM13 did not functionally activate IL13Rα1/IL4Rα in cells or bind to it in SPR binding assays, in contrast to wtIL13. Furthermore, in vivo targeting of systemically delivered radiolabeled TQM13 to IL13Rα2-expressing subcutaneous tumors was demonstrated and confirmed non-invasively for the first time with 124I-TQM13 positron emission tomography imaging. In addition, 131I-TQM13 demonstrated in vivo efficacy against subcutaneous IL13Rα2-expressing GBM tumors and in an orthotopic synergeic IL13Rα2-positive murine glioma model, as evidenced by statistically significant survival advantage. Our results demonstrate that we have successfully generated an optimized biomarker-targeted scaffolding that exhibited specific binding activity toward the tumor-associated IL13Rα2 in vitro and potential to deliver diagnostic and therapeutic payloads in vivo.

EphrinA1 is released in three forms from cancer cells by matrix metalloproteases.

EphrinA1 is a glycosylphosphatidylinositol (GPI)-linked ligand for the EphA2 receptor, which is overexpressed in glioblastoma (GBM), among other cancers. Activation of the receptor by ephrinA1 leads to a suppression of oncogenic properties of GBM cells. We documented that a monomeric functional form of ephrinA1 is released from cancer cells and thus explored the mechanism of ephrinA1 release and the primary protein sequence. We demonstrate here that multiple metalloproteases (MMPs) are able to cleave ephrinA1, most notably MMP-1, -2, -9, and -13. The proteolytic cleavage that releases ephrinA1 occurs at three positions near the C terminus, producing three forms ending in valine-175, histidine-177, or serine-178. Moreover, deletion of amino acids 174 to 181 or 175 to 181 yields ephrinA1 that is still GPI linked but not released by proteolysis, underlining the necessity of amino acids 175 to 181 for release from the membrane. Furthermore, recombinant ephrinA1 ending at residue 175 retains activity toward the EphA2 receptor. These findings suggest a mechanism of release and provide evidence for the existence of several forms of monomeric ephrinA1. Moreover, ephrinA1 should be truncated at a minimum at amino acid 175 in fusions or conjugates with other molecules in order to prevent likely proteolysis within physiological and pathobiological environments.

An interleukin 13 receptor α 2-specific peptide homes to human Glioblastoma multiforme xenografts.

Interleukin 13 receptor α 2 (IL-13Rα2) is a glioblastoma multiforme (GBM)-associated plasma membrane receptor, a brain tumor of dismal prognosis. Here, we isolated peptide ligands for IL-13Rα2 with use of a cyclic disulphide-constrained heptapeptide phages display library and 2 in vitro biopanning schemes with GBM cells that do (G26-H2 and SnB19-pcDNA cells) or do not (G26-V2 and SnB19-asIL-13Rα2 cells) over-express IL-13Rα2. We identified 3 peptide phages that bind to IL-13Rα2 in cellular and protein assays. One of the 3 peptide phages, termed Pep-1, bound to IL-13Rα2 with the highest specificity, surprisingly, also in a reducing environment. Pep-1 was thus synthesized and further analyzed in both linear and disulphide-constrained forms. The linear peptide bound to IL-13Rα2 more avidly than did the disulphide-constrained form and was efficiently internalized by IL-13Rα2-expressing GBM cells. The native ligand, IL-13, did not compete for the Pep-1 binding to the receptor and vice versa in any of the assays, indicating that the peptide might be binding to a site on the receptor different from the native ligand. Furthermore, we demonstrated by noninvasive near infrared fluorescence imaging in nude mice that Pep-1 binds and homes to both subcutaneous and orthotopic human GBM xenografts expressing IL-13Rα2 when injected by an intravenous route. Thus, we identified a linear heptapeptide specific for the IL-13Rα2 that is capable of crossing the blood-brain tumor barrier and homing to tumors. Pep-1 can be further developed for various applications in cancer and/or inflammatory diseases.

IL-13Rα2-Targeted Therapy Escapees: Biologic and Therapeutic Implications.

Glioblastoma multiforme (GBM) overexpresses interleukin 13 receptor α2 (IL-13Rα2), a tumor-restricted receptor that is not present in normal brain. We and others have created targeted therapies that specifically eradicate tumors expressing this promising tumor-restricted biomarker. As these therapies head toward clinical implementation, it is critical to explore mechanisms of potential resistance. We therefore used a potent IL-13Rα2-targeted bacterial cytotoxin to select for naturally occurring "escapee" cells from three different IL-13Rα2-expressing GBM cell lines. We found that these side populations of escapee cells had significantly decreased IL-13Rα2 expression. We examined clinically relevant biologic characteristics of escapee cell lines compared to their parental cell lines and found that they had similar proliferation rates and equal sensitivity to temozolomide and radiation, the standard therapies given to GBM patients. In contrast, our escapee cell lines were less likely to form colonies in culture and migrated more slowly in wound healing assays. Furthermore, we found that escapee cells formed significantly less neurospheres in vitro, suggesting that IL-13Rα2-targeted therapy preferentially targeted the "stem-like" cell population and possibly indicating decreased tumorigenicity in vivo. We therefore tested escapee cells for in vivo tumorigenicity and found that they were significantly less tumorigenic in both subcutaneous and intracranial mouse models compared to matching parental cells. These data, for the first time, establish and characterize the clinically relevant biologic properties of IL-13Rα2-targeted therapy escapees and suggest that these cells may have less malignant characteristics than parental tumors.

Fos-related antigen 1 (Fra-1) pairing with and transactivation of JunB in GBM cells.

Fos-related antigen 1 (Fra-1) plays an important role in maintenance/progression of various cancers, including glioblastoma multiforme (GBM). In this study, we used both shRNA and siRNA to examine the effect of fra-1 knockdown in GBM cells over-expressing Fra-1. Furthermore, we analyzed both the expression of JunB and its knockdown, a previously identified target for Fra-1, and also examined its potential association with Fra-1. When using fra-1 shRNA and siRNA, we found that GBM cells has Fra-1 levels diminished together with the levels of JunB, but Fra-1 remains unchanged in cells with junB knockdown. This is accompanied by dramatic changes in cell morphology and significant alteration in their migration. We next uncovered that the expression of JunB increased in response to ectopic Fra-1 and also to EGF-induced signaling, similarly to Fra-1. This was associated with an avid pairing between phosphorylated Fra-1 and JunB. Importantly, we found that Fra-1 paired with JunB binds to an AP-1 site in the junB gene promoter. JunB knockdown did not affect Fra-1 and the changes in cell morphology did not fully replicate that seen with Fra-1 knockdown. Thus, Fra-1 takes part in a control of architecture and migratory nature of GBM cells. Moreover, Fra-1 is a phosphorylated factor that transactivates JunB with which it makes effectively AP-1 pairs in GBM cells.

Molecular targeting of intracellular compartments specifically in cancer cells.

We have implemented a strategy in which a genetically engineered, single-chain protein specifically recognizes cancer cells and is trafficked to a targeted subcellular compartment, such as the nucleus. The recombinant protein termed IL-13.E13K-D2-NLS has a triple functional property: (1) it binds a cancer-associated receptor, interleukin 13 receptor alpha 2 (IL-13Rα2), using modified IL-13 ligand, IL-13.E13K; (2) it exports its C-terminal portion out of the endosomal compartment using Pseudomonas aeruginosa exotoxin A (PE) translocation domain (D2); and (3) it travels to and accumulates in the nucleus guided by the nuclear localization signal (NLS). Here, we have demonstrated that this protein is transported into the brain tumor cells' nucleus, using 3 different methods of protein conjugation to dyes for the purpose of direct visualization of the protein's intracellular trafficking. IL-13.E13K-D2-NLS, and not the controls such as IL-13.E13K-D2, IL-13.E13K-NLS, or IL-13.E13K, accumulated in nuclei very efficiently, which increased with the time the cells were exposed to the protein. Also, IL-13.E13K-D2-NLS did not exhibit nuclear transport in cells with low expression levels of IL-13Rα2. Thus, it is possible to recognize cancer cells through their specific receptors and deliver a conjugated protein that travels specifically to the nucleus. Hence, our molecular targeting strategy succeeded in generating a single-chain proteinaceous agent capable of delivering drugs/labels needed to be localized to the cells' nuclei or potentially any other subcellular compartment, for their optimal efficacy or ability to exert their specific action.

Reoxygenation of hypoxic glioblastoma multiforme cells potentiates the killing effect of an interleukin-13-based cytotoxin.

PURPOSE: Hypoxia is a cause for resistance to cancer therapies. Molecularly targeted recombinant cytotoxins have shown clinical efficacy in the treatment of patients with primary brain tumors, glioblastoma multiforme, but it is not known whether hypoxia influences their antitumor effect. EXPERIMENTAL DESIGN: We have exposed glioblastoma multiforme cells, such as U-251 MG, U-373 MG, SNB-19, and A-172 MG, to either anoxia or hypoxia and then reoxygenated them while treating with an interleukin (IL)-13-based diphtheria toxin (DT)-containing cytotoxin, DT-IL13QM. We measured the levels of immunoreactive IL-13Ralpha2, a receptor that mediates IL-13-cytotoxin cell killing, and the levels of active form of furin, a protease that activates the bacterial toxin portion in a cytotoxin. RESULTS: We found that anoxia/hypoxia significantly alters the responsiveness of glioblastoma multiforme cells to DT-IL13QM. Interestingly, bringing these cells back to normoxia caused them to become even more susceptible to the cytotoxin than the cells maintained under normoxia. Anoxia/hypoxia caused a highly prominent decrease in the immunoreactive levels of both IL-13R and active forms of furin, and reoxygenation not only restored their levels but also became higher than that in normoxic glioblastoma multiforme cells. CONCLUSIONS: Our results show that a recombinant cytotoxin directed against glioblastoma multiforme cells kills these cells much less efficiently under anoxic/hypoxic conditions. The reoxygenation brings unexpected additional benefit of making glioblastoma multiforme cells even more responsive to the killing effect of a cytotoxin.

Protein- and DNA-based active immunotherapy targeting interleukin-13 receptor alpha2.

High-grade astrocytoma (HGA) is an invariably fatal malignancy with a mean survival of 14 months despite surgery, radiation, and chemotherapy. We have found that a restricted receptor for interleukin-13 (IL-13), IL-13 receptor alpha 2 (IL13Ralpha2), is abundantly overexpressed in the vast majority of HGAs but is not appreciably expressed in normal tissue, with the exception of the testes. Therefore, IL-13Ralpha2 is a very attractive target for anti-HGA immunotherapy. In order to test protein and genetic vaccines that target IL13Ralpha2, we developed a G26-IL13Ralpha2-expressing syngeneic immunocompetent murine glioma model. Using this glioma model, mice were immunized with recombinant extracellular IL13Ralpha2 protein (IL13Ralpha2ex) or a DNA expression vector containing the gene for IL13Ralpha2 and were subsequently challenged with IL13Ralpha2( + ) G26 tumors. Mice immunized with either recombinant or genetic IL13Ralpha2, but not mock-immunized controls, demonstrated complete protection against IL13Ralpha2( + ) glioma growth and mortality. Of interest, only the recombinant-protein-based vaccines generated detectable anti-IL13Ralpha2 antibodies. These studies demonstrate the in vivo efficiency of protein- and DNA-based immunotherapy strategies that target IL13Ralpha2 that may play a clinical role to eradicate the residual microscopic HGA cells that inevitably cause disease recurrence and mortality.

Interleukin-13 receptor alpha 2, EphA2, and Fos-related antigen 1 as molecular denominators of high-grade astrocytomas and specific targets for combinatorial therapy.

PURPOSE: We investigated the expression of interleukin-13 receptor alpha2 (IL-13R alpha 2), EphA2, and Fos-related antigen 1 (Fra-1) in astrocytomas and normal brain. We sought to document whether the expression of the three factors changed with progression to higher grade malignancy and whether two or three targets in combination might be sufficient to target all patients with high-grade astrocytomas. EXPERIMENTAL DESIGN: Immunohistochemistry was done for IL-13R alpha 2, EphA2, and Fra-1 using human brain tumor tissue microarrays containing 30 specimens of WHO grades II and III astrocytomas, 46 glioblastoma multiformes (GBM), and 9 normal brain samples. Sections were scored based on frequency and intensity of expression. Western blotting was done for all three markers using GBM tumor specimens and xenograft cell lines. Two cytotoxins, IL-13.E13K.PE38QQR and ephrinA1-PE38QQR, which target IL-13R alpha 2 or EphA2, respectively, were tested for cytotoxicity against human GBM primary explant cells and established cells. RESULTS: Expression of all three proteins was significantly higher in GBM compared with normal brain, low-grade, and anaplastic astrocytomas. Greater than 95% of GBM overexpressed at least two of the three markers. Importantly, every GBM overexpressed at least one marker. Human GBM primary explant cells and cell lines were potently killed by IL-13.E13K.PE38QQR and ephrinA1-PE38QQR, in accordance with their level of expression of IL-13R alpha 2 and EphA2, respectively. CONCLUSIONS: IL-13R alpha 2, EphA2, and Fra-1 are attractive therapeutic targets representing molecular denominators of high-grade astrocytomas. One hundred percent of GBM tumors overexpress at least one of these proteins, providing the basis for rational combinatorial targeted therapies/diagnostics suitable for all patients with this disease.

A novel, potent, and specific ephrinA1-based cytotoxin against EphA2 receptor expressing tumor cells.

We have previously shown that the EphA2 receptor tyrosine kinase is overexpressed in glioblastoma multiforme (GBM) and represents a novel, attractive therapeutic target for the treatment of brain tumors. Here, we have developed an EphA2-targeted agent, ephrinA1-PE38QQR, a novel cytotoxin composed of ephrinA1, a ligand for EphA2, and PE38QQR, a mutated form of Pseudomonas aeruginosa exotoxin A. EphrinA1-PE38QQR showed potent and dose-dependent killing of GBM cells overexpressing the EphA2 receptor in cell viability and clonogenic survival assays, with an average IC(50) of approximately 10(-11) mol/L. The conjugate was also highly effective in killing breast and prostate cancer cells overexpressing EphA2. The cytotoxic effect of ephrinA1-PE38QQR was specific, as it was neutralized by an excess of EphA2 ligands. Moreover, normal human endothelial cells and breast cancer cells that do not overexpress EphA2, as well as GBM cells that have down-regulated EphA2, were not susceptible to the cytotoxin. EphrinA1-PE38QQR-mediated cytotoxicity induced caspase-dependent apoptosis, which was, however, not responsible for cell death in response to the conjugate. In addition, the conjugate elicited no changes in the activity of survival pathways such as phosphoinositide 3-kinase, measured by AKT phosphorylation. This is the first attempt to create a cytotoxic therapy using any of the ephrin ligands of either class (A or B) conjugated to a bacterial toxin. EphrinA1-PE38QQR is very potent and specific, produces cell death that is caspase independent, and forms the basis for the further development of clinically applicable EphA2-targeted cytotoxins.

EphA2 as a novel molecular marker and target in glioblastoma multiforme.

We investigated the presence of EphA2, and its ligand, ephrinA1, in glioblastoma multiforme (GBM), a malignant neoplasm of glial cells, and normal brain. We also initially examined the functional importance of the interaction between EphA2 and ephrinA1 in glioma cells. Expression and localization of EphA2 and ephrinA1 in human GBM and normal brain were examined using Western blotting, immunofluorescence, and immunohistochemistry. A functional role for EphA2 was investigated by assessing the activation status of the receptor and the effect of ephrinA1 on the anchorage-independent growth and invasiveness of GBM cells. We found EphA2 to be elevated in approximately 90% of GBM specimens and cell lines but not in normal brain, whereas ephrinA1 was present at consistently low levels in both GBM and normal brain. EphA2 was activated and phosphorylated by ephrinA1 in GBM cells. Furthermore, ephrinA1 induced a prominent, dose-dependent inhibitory effect on the anchorage-independent growth and invasiveness of GBM cells highly overexpressing EphA2, which was not seen in cells expressing low levels of the receptor. Thus, EphA2 is both specifically overexpressed in GBM and expressed differentially with respect to its ligand, ephrinA1, which may reflect on the oncogenic processes of malignant glioma cells. EphA2 seems to be functionally important in GBM cells and thus may play an important role in GBM pathogenesis. Hence, EphA2 represents a new marker and novel target for the development of molecular therapeutics against GBM.

Fos-related antigen 1 modulates malignant features of glioma cells.

Malignant gliomas, and high-grade gliomas (HGG) in particular, are nonmetastasizing but locally infiltrating, hypervascularized brain tumors of poor prognosis. We found previously that a c-fos-inducible vascular endothelial growth factor D is ubiquitously up-regulated in HGG grade IV, glioblastoma multiforme, and that glioblastoma multiforme overexpress Fos-related antigen 1 (Fra-1) rather than the c-Fos. We have thus become interested in the role Fra-1 may play in malignant glioma progression/maintenance, because Fra-1 has the capacity to modulate transcription of a variety of target genes. In this work, we have analyzed the biological effects of ectopic Fra-1 expression or Fra-1 knockdown in malignant glioma cells. Ectopic Fra-1 induced prominent phenotypic changes in all three malignant glioma cell lines examined: H4, U-87 MG, and A-172 MG. These changes were reflected in cells becoming more elongated with larger number of cellular processes. Furthermore, Fra-1 transgene caused H4 cells, which do not form tumor xenografts, to regain tumorigenic capacity. The genotype of these cells changed too, because 50 of 1,056 genes examined became either up-regulated or down-regulated. Conversely, Fra-1 knockdown altered prominently the morphology, anchorage-independent growth, tumorigenic potential, and Fra-1 effector expression, such as vascular endothelial growth factor D, in HGG cells. For example, cells transfected with antisense fra-1 showed shorter cellular processes than the control cells that did not grow in agar, and their tumorigenic potential was significantly diminished. Thus, Fra-1 may likely play an important role in the maintenance/progression of malignant gliomas and potentially represents a new target for therapeutic interventions.

Molecular targeting with recombinant cytotoxins of interleukin-13 receptor alpha2-expressing glioma.

A restricted receptor for interleukin 13 (IL-13R alpha2) is over-expressed in high-grade astrocytoma (HGA), but not in normal organs. In order to design and examine new anti-HGA therapies, which are molecularly directed against IL-13R alpha2, we established an IL-13R alpha2-expressing syngeneic immunocompetent murine model of HGA. The model was obtained by transfecting G-26 murine glioma cells with IL-13R alpha2. G-26-IL-13R alpha2(+) cells, but not mock-transfected cells, became susceptible to IL-13 mutant-based cytotoxic proteins that kill human HGA cells. G-26-IL-13R alpha2(+) cells maintained their tumorigenicity in immunocompetent C57BL/J6 mice and preserved their expression of IL-13R alpha2 in vivo. These characteristics of the G-26-IL-13R alpha2(+) tumors allowed us to test molecularly defined anti-glioma passive immunotherapy. A targeted recombinant chimera cytotoxin composed of multiply mutated IL-13 (IL-13.E13Y/R66D/S69D) and a derivative of Pseudomonas exotoxin (PE), PE1E, IL-13.E13Y/R66D/S69D-PE1E, was used in anti-tumor experiments. G-26-IL-13R alpha2(+) cells were killed by IL-13.E13Y/R66D/S69D-PE1E in an IL-4-independent fashion. To test the cytotoxin in vivo, G-26-IL-13R alpha2(+) tumors were established in C57BL/J6 mice and when the tumors reached a size of at least 50 mm3, the mice were treated with IL-13.E13Y/R66D/S69D-PE1E. In the mice treated with the targeted fusion cytotoxin, the tumors regressed and 80% of the animals were cured. This study documents the establishment of an IL-13R alpha2-positive model of HGA in immunocompetent rodents. Furthermore, the effectiveness and safety of the targeted IL-13-based cytotoxin against IL-13R alpha2-expressing tumors in a more clinically relevant in vivo HGA model is promising with regard to the future clinical utility of the cytotoxin.

Epigenetics in high-grade astrocytomas: opportunities for prevention and detection of brain tumors.

Human high-grade astrocytomas (HGA) are the most prevalent incurable brain tumors. We found that the vast majority of HGA patients overexpress a restricted receptor for an immune regulatory cytokine, interleukin 13 (IL-13). Interestingly, the HGA-associated restricted receptor protein IL-13Ralpha2 is expressed in the testes, and its gene is localized to chromosome X. These mirror the expression pattern and genomic localization of cancer/testes tumor antigens (CTA). Hypothetical considerations and now experimental evidence are beginning to point towards epigenetics, and DNA methylation alterations in particular, as being responsible for the appearance in cancer of CTA, including IL-13Ralpha2. In line with our interest in the X chromosome and oncogenesis, we have identified a new ubiquitous angiogenic factor in HGA, a vascular endothelial growth factor-D (VEGF-D). We have also demonstrated that the activating protein-1 (AP-1) family of transcription factors play a potentially critical role in the progression of gliomas by eliciting uncontrolled upregulation of VEGF-D and other compounds essential for cancer cell proliferation, tumorigenesis, and infiltration. The possibility exists that an unopposed constitutive increase in AP-1 activity in HGA is related to epigenetic silencing of the inhibitors of AP-1 activity. These phenomena offer potential targets for exploitation in either prevention or early detection of brain tumors. For example, anticancer vaccines against shared CTA could help in prevention of HGA development. Furthermore, drugs with anti-AP-1 activity could be effective in preventing formation/progression of HGA, or progression from less malignant lower grade gliomas to HGA. Also, circulating antibodies against CTA and factors that are AP-1 regulated may provide a useful tool in early detection of brain tumors or for monitoring their progression following initial treatment.

IL-13Ralpha2 is a glioma-restricted receptor for interleukin-13.

We have found that binding sites for interleukin-13 (IL-13) are overexpressed in a vast majority of high-grade astrocytomas (HGAs). These binding sites for IL-13 are distinct from the physiological receptor in that it does not bind IL-4. We also demonstrated that IL-13 receptor alpha 2 protein chain (IL-13Ralpha2), an IL-4-independent receptor for IL-13, is abundant among HGAs, but not in normal organs. To examine if IL-13Ralpha2 is the tumor-associated site for IL-13, we stably transfected normal Chinese hamster ovary (CHO) cells and glioma G-26 cells to express either human (h) or murine (m) IL-13Ralpha2. CHO-hIL-13Ralpha2(+) cells and G-26-h/mIL-13Ralpha2(+) cells, and not CHO and G-26 parental or mock-transfected cells, specifically bound IL-13 in an IL-4-independent manner. The IL-13Ralpha2(+) cells also became highly susceptible to the killing by an IL-13-based cytotoxic fusion protein. In loss of function studies, a HGA cell line, SNB-19, was transfected with antisense (as) hIL-13Ralpha2. as-SNB-19-hIL-13Ralpha2(+) cells lost their natural affinity towards IL-13 and became resistant to IL-13-based cytotoxins. The fact, that IL-13Ralpha2-positive cells bind IL-13 independent of IL-4, become susceptible to IL-13 cytotoxins, and cells deprived of IL-13Ralpha2 receptor lose these features, demonstrates that IL-13Ralpha2 is the brain tumor-associated receptor for IL-13.