Immunoreactive IL-13RA2 in human and canine brain tumor specimens/cells by purified MAb’s of Peptide 1

<p><b><i>A</i></b>, Human glioblastoma (G), <b><i>B</i></b>, oligodendroglioma (O), astrocytoma (A), normal brain (NB), and G14 human GBM tumor lysate; meningioma (M), <b><i>C</i></b>, tissue lysates immunoreactivity using Western blots. Canine astrocytoma, glioblastoma and normal brain, <b><i>D</i></b>; oligodendroglioma, gliosarcoma (GSO) and mixed astro-oligo (AO), <b><i>E</i></b>; and choroid plexus papilloma (CPP) and meningioma, <b><i>F</i></b> tissue lysates immunoreactivity using western blots. Western blot of cell lines and parent tumor tissue obtained from dogs with spontaneous GBM, <b><i>G</i></b>. Immunoprecipitation of IL-13RA2 from U-251 MG cell lysate using either MAb 2G12C3, MAb 2G12E2 or a polyclonal antibody (R&D Systems #AF146); the polyclonal antibody was used for the receptor detection after immunoprecipitation<b><i>, H</i></b>.</p

Imunoreactivity of MAbs obtained with immunization using Peptide 2.

<p>Reactivity of MAbs raised against Peptide 2 in ELISA using recombinant IL-13RA2-Fc, <b><i>A</i></b>; and Peptide 2, <b><i>B</i></b>. Detection of recombinant IL-13RA2, but not of IL-13RA1-Fc with MAbs 6D3E9, 4G9G3 and 3D4G10, The proteins were loaded at 0.5 µg/lane. <b><i>C</i></b>. Immunofluorescence in G48a cells using MAb 6D3E9, <b><i>D</i></b>.</p

MAb 1E10B9 binds to live GBM cells.

<p>Flow cytometry on human U-251 MG cells, canine GBM G06-A cells, and human T98G cells using MAb 1E10B9, <b><i>A.</i></b> Internalization of MAb 1E10B9 by G48a human GBM cells, <b><i>B.</i></b> Internalization of MAb 1E10B9 by U-251, G06-A, and T98G cells after 4-hr incubation, <b><i>C</i></b>.</p

Quantitative TaqMan RT-PCR comparing expression of IL-13RA2 in canine primary brain tumors.

<p>Elevated expression, relative to normal canine brain cortex, is seen predominantly in high grade glial tumors, essentially mirroring protein expression determined by western blotting. Off scale values are marked with an asterisk and value. MEN – meningioma; AST – astrocytoma; GBM – glioblastoma multiforme; OLIGO – oligodendroglioma.</p

Immunoreactivity of monoclonal antibodies induced by Peptide 1 and recognition of synthetic and recombinant immunogens.

<p><b><i>A</i></b>, Western blot of U-251 MG and T98G human GBM cell lysates using media of 3G12C3 hybridoma cells. ELISA was conducted using either recombinant IL-13RA2-Fc, <b><i>B</i></b> or the synthetic Peptide 1, <b><i>C</i></b>. </p

Imunoreactivity of MAbs obtained with immunization using Peptide 3.

<p>Reactivity of MAbs raised against Peptide 3 in ELISA using recombinant IL-13RA2-Fc, <b><i>A</i></b>; and the synthetic Peptide 3, <b><i>B</i></b>. Detection of IL-13RA2, but not of IL-13RA1-Fc with MAbs 1E10B9 and 1E10F9, <b><i>C</i></b>. Immunoreactive IL-13RA2 in Western blot of U-251 MG and T98G cell lysates using MAb 1E10B9, <b><i>D</i></b>. Expression of IL-13RA2 detected by immunohistochemistry using MAb 1E10B9 in human GBM specimens and normal brain, <b><i>E</i></b>, and canine GBMs and normal brain, <b><i>F</i></b>.</p

Alignment of human and canine sequences of IL-13RA2.

<p><b><i>A</i></b>, The sequences were obtained from the NCBI database. The regions of complete sequence identity between the two species that were utilized as immunogens are boxed. <b><i>B</i></b>, Ribbon structure of IL-13RA2 in contact with its natural ligand, IL-13. The three immunogenic peptides used for raising monoclonal antibodies are shown in green. Peptide 1 is located in the extracellular domain of the receptor, Peptide 2 in the vicinity of the ligand binding to the receptor and Peptide 3 is within the extracellular, near transmembrane domain.</p

Production and testing of canIL-13.E13K and canIL-13.E13K based cytotoxin.

<p>Superimposition of canIL-13 and huIL-13 molecules, (3D reconstruction using JMol), <b><i>A.</i></b> Purified canIL-13.E13K and canIL-13.E13K cytotoxin, (10% SDS-PAGE), <b><i>B</i><i> and </i><i>C</i></b>. Activation of TF-1 cells proliferation by cytokines, <b><i>D</i></b>. Cytotoxicity of canIL-13 cytotoxin and its neutralization on BTCOE 4795 human GBM cells, <b><i>E</i></b>. P<0.015 and <0.007 for differences between the cytokines (in <b><i>D</i></b>) and the cytotoxin killing vs. neutralization with canIL-13.E13K alone (in <b><i>E</i></b>) using an unpaired t-test. Cytotoxicity of canIL-13.E13K cytotoxin on canine GBM G06-A cells, <b><i>F</i></b>. Cytotoxicity of canIL-13.E13K cytotoxin on human GBM established (U-251 MG), <b><i>G</i></b>, and low passage human GBM cells (BTCOE 4795), <b><i>H</i></b>. CTL – control. Vertical bars represent SEM and if not seen, they are smaller than the points.</p

Supplemental Materials and Methods from Truncated Glioma-Associated Oncogene Homolog 1 (tGLI1) Mediates Mesenchymal Glioblastoma via Transcriptional Activation of CD44

This file contains supplemental materials and methods</p

Suppmental Data from Truncated Glioma-Associated Oncogene Homolog 1 (tGLI1) Mediates Mesenchymal Glioblastoma via Transcriptional Activation of CD44

This file contains Supplemental Figure 1 and Supplemental Tables I and II.</p