Identification of A2B5+CD133- tumor-initiating cells in adult human gliomas.
ABSTRACT Several studies have shown that human gliomas contain a small population of cells with stem cell-like features. It has been proposed that these "cancer stem cells" may be uniquely responsible for glioma formation and recurrence. However, human gliomas also contain an abundance of cells that closely resemble more differentiated glial progenitors. Animal model studies have shown that these cells also possess the capacity to form malignant gliomas.
To investigate the contributions of stem-like and progenitor-like cells in human gliomas, we used flow cytometry to characterize the expression of a cancer stem cell marker (CD133) and a glial progenitor marker (A2B5) in 25 tumors. We found that human gliomas consistently express A2B5 in a large percentage of cells (61.7 +/- 3.8%, standard error of the mean). In contrast, CD133 expression was less abundant and less consistent (14.8 +/- 3.6%, standard error of the mean), with several glioblastomas containing very few or no detectable CD133+ cells. When present, the CD133+ population was almost entirely contained within the A2B5+ population. Thus, most gliomas could be divided into three distinct populations on the basis of these markers (A2B5+CD133+, A2B5+CD133-, and A2B5-CD133-). To test the tumorigenic potential of these populations, we separated cells from six tumors by fluorescence-activated cell sorting and reinjected them into nude rats.
We found that the capacity for these different populations to form tumors varied depending on the human tumor specimen from which they were isolated. Of the six human gliomas tested, four contained A2B5+/CD133- cells that formed tumors when transplanted into nude rats, three contained A2B5+/CD133+ cells that formed tumors, and only one glioma contained A2B5-/CD133- cells with the capacity to form tumors.
Together, these results demonstrate that human gliomas contain multiple populations of cells with the capacity to form tumors and specifically identify a population of tumorigenic A2B5+ cells that are phenotypically distinct from CD133+ cells.
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ABSTRACT: The interleukin-13 receptor alpha2 (IL13Rα2) is a cell surface receptor that is over-expressed by a subset of high-grade gliomas, but not expressed at significant levels by normal brain tissue. For both malignant and non-malignant cells, IL13Rα2 surface expression is reported to be induced by various cytokines such as IL-4 or IL-13 and tumor necrosis factor (TNF). Our group has developed a therapeutic platform to target IL13Rα2-positive brain tumors by engineering human cytotoxic T lymphocytes (CTLs) to express the IL13-zetakine chimeric antigen receptor. We therefore sought to investigate the potential of cytokine stimulation to induce IL13Rα2 cell surface expression, and thereby increase susceptibility to IL13Rα2-specific T cell killing. In the course of these experiments, we unexpectedly found that the commercially available putative IL13Rα2-specific monoclonal antibody B-D13 recognizes cytokine-induced VCAM-1 on glioblastoma. We provide evidence that the induced receptor is not IL13Rα2, because its expression does not consistently correlate with IL13Rα2 mRNA levels, it does not bind IL-13, and it is not recognized by IL13-zetakine CTL. Instead we demonstrate by immunoprecipitation experiments and mass spectrometry that the antigen recognized by the B-D13 antibody following cytokine stimulation is VCAM-1, and that VCAM-1, but not IL13Rα2, is induced on glioma cells by TNF alone or in combination with IL-13 or IL-4. Further evaluation of several commercial B-D13 antibodies revealed that B-D13 is bi-specific, recognizing both IL13Rα2 and VCAM-1. This binding is non-overlapping based on soluble receptor competition experiments, and mass spectrometry identifies two distinct heavy and light chain species, providing evidence that the B-D13 reagent is di-clonal. PE-conjugation of the B-D13 antibody appears to disrupt IL13Rα2 recognition, while maintaining VCAM-1 specificity. While this work calls into question previous studies that have used the B-D13 antibody to assess IL13Rα2 expression, it also suggests that TNF may have significant effects on glioma biology by up-regulating VCAM-1.PLoS ONE 05/2014; 9(5):e95123. · 3.53 Impact Factor
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ABSTRACT: Glioma is the most common intracranial tumor and has a poor patient prognosis. The presence of brain tumor stem cells was gradually being understood and recognized, which might be beneficial for the treatment of glioma.Neural Regeneration Research 05/2013; 8(15):1431-8. · 0.23 Impact Factor
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ABSTRACT: A2B5+ glioblastoma (GBM) cells have glioma stem-like cell (GSC) properties that are crucial to chemotherapy resistance and GBM relapse. T-cell-based antigens derived from A2B5+ GBM cells provide important information for immunotherapy. Here, we show that HEAT repeat containing 1 (HEATR1) expression in GBM tissues was significantly higher than that in control brain tissues. Furthermore, HEATR1 expression in A2B5+ U87 cells was higher than that in A2B5-U87 cells (P = 0.016). Six peptides of HEATR1 presented by HLA-A∗02 were selected for testing of their ability to induce T-cell responses in patients with GBM. When peripheral blood mononuclear cells from healthy donors (n = 6) and patients with glioma (n = 33) were stimulated with the peptide mixture, eight patients with malignant gliomas had positive reactivity with a significantly increased number of responding T-cells. The peptides HEATR1682-690, HEATR11126-1134, and HEATR1757-765 had high affinity for binding to HLA-A∗02:01 and a strong capacity to induce CTL response. CTLs against HEATR1 peptides were capable of recognizing and lysing GBM cells and GSCs. These data are the first to demonstrate that HEATR1 could induce specific CTL responses targeting both GBM cells and GSCs, implicating that HEATR1 peptide-based immunotherapy could be a novel promising strategy for treating patients with GBM.Research Journal of Immunology 01/2014; 2014:131494.