Gentao Liu

Analysis of gene expression and chemoresistance of CD133 + cancer stem cells in glioblastoma

BackgroundRecently, a small population of cancer stem cells in adult and pediatric brain tumors has been identified. Some evidence has suggested that CD133 is a marker for a subset of leukemia and glioblastoma cancer stem cells. Especially, CD133 positive cells isolated from human glioblastoma may initiate tumors and represent novel targets for therapeutics. The gene expression and the drug resistance property of CD133 positive cancer stem cells, however, are still unknown.ResultsIn this study, by FACS analysis we determined the percentage of CD133 positive cells in three primary cultured cell lines established from glioblastoma patients 10.2%, 69.7% and 27.5%, respectively. We also determined the average mRNA levels of markers associated with neural precursors. For example, CD90, CD44, CXCR4, Nestin, Msi1 and MELK mRNA on CD133 positive cells increased to 15.6, 5.7, 337.8, 21.4, 84 and 1351 times, respectively, compared to autologous CD133 negative cells derived from cell line No. 66. Additionally, CD133 positive cells express higher levels of BCRP1 and MGMT mRNA, as well as higher mRNA levels of genes that inhibit apoptosis. Furthermore, CD133 positive cells were significantly resistant to chemotherapeutic agents including temozolomide, carboplatin, paclitaxel (Taxol) and etoposide (VP16) compared to autologous CD133 negative cells. Finally, CD133 expression was significantly higher in recurrent GBM tissue obtained from five patients as compared to their respective newly diagnosed tumors.ConclusionOur study for the first time provided evidence that CD133 positive cancer stem cells display strong capability on tumors resistance to chemotherapy. This resistance is probably contributed by the CD133 positive cell with higher expression of on BCRP1 and MGMT, as well as the anti-apoptosis protein and inhibitors of apoptosis protein families. Future treatment should target this small population of CD133 positive cancer stem cells in tumors to improve the survival of brain tumor patients.

Isolation of cancer stem cells from adult glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common adult primary brain tumor and is comprised of a heterogeneous population of cells. It is unclear which cells within the tumor mass are responsible for tumor initiation and maintenance. In this study, we report that brain tumor stem cells can be identified from adult GBMs. These tumor stem cells form neurospheres, possess the capacity for self-renewal, express genes associated with neural stem cells (NSCs), generate daughter cells of different phenotypes from one mother cell, and differentiate into the phenotypically diverse populations of cells similar to those present in the initial GBM. Having a distinguishing feature from normal NSCs, these tumor stem cells can reform spheres even after the induction of differentiation. Furthermore, only these tumor stem cells were able to form tumors and generate both neurons and glial cells after in vivo implantation into nude mice. The identification of tumor stem cells within adult GBM may represent a major step forward in understanding the origin and maintenance of GBM and lead to the identification and testing of new therapeutic targets.

Vaccination with Tumor Lysate-Pulsed Dendritic Cells Elicits Antigen-Specific, Cytotoxic T-Cells in Patients with Malignant Glioma

The primary goal of this Phase I study was to assess the safety and bioactivity of tumor lysate-pulsed dendritic cell (DC) vaccination to treat patients with glioblastoma multiforme and anaplastic astrocytoma. Adverse events, survival, and cytotoxicity against autologous tumor and tumor-associated antigens were measured. Fourteen patients were thrice vaccinated 2 weeks apart with autologous DCs pulsed with tumor lysate. Peripheral blood mononuclear cells were differentiated into phenotypically and functionally confirmed DCs. Vaccination with tumor lysate-pulsed DCs was safe, and no evidence of autoimmune disease was noted. Ten patients were tested for the development of cytotoxicity through a quantitative PCR-based assay. Six of 10 patients demonstrated robust systemic cytotoxicity as demonstrated by IFN-γ expression by peripheral blood mononuclear cells in response to tumor lysate after vaccination. Using HLA-restricted tetramer staining, we identified a significant expansion in CD8+ antigen-specific T-cell clones against one or more of tumor-associated antigens MAGE-1, gp100, and HER-2 after DC vaccination in four of nine patients. A significant CD8+ T-cell infiltrate was noted intratumorally in three of six patients who underwent reoperation. The median survival for patients with recurrent glioblastoma multiforme in this study (n = 8) was 133 weeks. This Phase I study demonstrated the feasibility, safety, and bioactivity of an autologous tumor lysate-pulsed DC vaccine for patients with malignant glioma. We demonstrate for the first time the ability of an active immunotherapy strategy to generate antigen-specific cytotoxicity in brain tumor patients.

Vaccination elicits correlated immune and clinical responses in glioblastoma multiforme patients.

Cancer vaccine trials have failed to yield robust immune-correlated clinical improvements as observed in animal models, fueling controversy over the utility of human cancer vaccines. Therapeutic vaccination represents an intriguing additional therapy for glioblastoma multiforme (GBM; grade 4 glioma), which has a dismal prognosis and treatment response, but only early phase I vaccine trial results have been reported. Immune and clinical responses from a phase II GBM vaccine trial are reported here. IFN-gamma responsiveness was quantified in peripheral blood of 32 GBM patients given therapeutic dendritic cell vaccines. Posttreatment times to tumor progression (TTP) and survival (TTS) were compared in vaccine responders and nonresponders and were correlated with immune response magnitudes. GBM patients (53%) exhibited >or=1.5-fold vaccine-enhanced cytokine responses. Endogenous antitumor responses of similar magnitude occurred in 22% of GBM patients before vaccination. Vaccine responders exhibited significantly longer TTS and TTP relative to nonresponders. Immune enhancement in vaccine responders correlated logarithmically with TTS and TTP spanning postvaccine chemotherapy, but not with initial TTP spanning vaccination alone. This is the first report of a progressive correlation between cancer clinical outcome and T-cell responsiveness after therapeutic vaccination in humans and the first tracing of such correlation to therapeutically exploitable tumor alteration. As such, our findings offer unique opportunities to identify cellular and molecular components of clinically meaningful antitumor immunity in humans.

Clinical Responsiveness of Glioblastoma Multiforme to Chemotherapy after Vaccination

Purpose: Although the development of immune-based therapies for various cancers including malignant glioma has been heralded with much hope and optimism, objective clinical improvements in most vaccinated cancer patients have not been realized. To broaden the search for vaccine-induced benefits, we examined synergy of vaccines with conventional chemotherapy. Experimental Design: Survival and progression times were analyzed retrospectively in 25 vaccinated (13 with and 12 without subsequent chemotherapy) and 13 nonvaccinated de novo glioblastoma (GBM) patients receiving chemotherapy. Immune responsiveness and T-cell receptor excision circle (TREC) content within CD8+ T cells (CD8+ TRECs) was determined in vaccinated patients. Results: Vaccinated patients receiving subsequent chemotherapy exhibited significantly longer times to tumor recurrence after chemotherapy relative to their own previous recurrence times, as well as significantly longer postchemotherapy recurrence times and survival relative to patients receiving isolated vaccination or chemotherapy. Patients exhibiting objective (>50%) tumor regression, extremely rare in de novo GBM, were also confined to the vaccine + chemotherapy group. Prior tumor behavior, demographic factors, other treatment variables, distribution of vaccine responders, and patients with high CD8+ TRECs all failed to account for these differences in clinical outcome. Within all GBM patients receiving post-vaccine chemotherapy, however, CD8+ TRECs predicted significantly longer chemotherapeutic responses, revealing a strong link between the predominant T-cell effectors in GBM and tumor chemosensitivity. Conclusions: We propose that therapeutic vaccination synergizes with subsequent chemotherapy to elicit tangible clinical benefits for GBM patients.

Proteasome inhibitor PS-341 causes cell growth arrest and apoptosis in human glioblastoma multiforme (GBM)

The proteasome plays a pivotal role in controlling cell proliferation, apoptosis, and differentiation in a variety of normal and tumor cells. PS-341, a novel boronic acid dipeptide that inhibits 26S proteasome activity, has prominent effects in vitro and in vivo against several solid tumors. We examined its antiproliferation, proapoptotic effects using three human glioblastoma multiforme (GBM) cell lines and five primary GBM explants. PS-341 markedly inhibited proliferation of GBM cell lines and explants in liquid and soft agar culture. These cells developed a G2/M cell cycle arrest with a concomitant decreased percentage of cells in S phase (≈2-fold), associated with an increased expression of p21WAF1, p27KIP1, as well as cyclin B1 and decreased levels of CDK2, CDK4, and E2F4. About 35–40% of the cells became apoptotic when exposed to PS-341 (10−7 M, 24–48 h) as shown by Annexin V analysis; in concert with these findings, immunobloting showed a C-terminal 85 kDa apoptotic fragment of poly ADP-ribose polymerase (PARP), and a decreased level of Bcl2 and Bcl-xl. PS-341 downregulated the expression of Bcl-2 and Bcl-xl in protein levels at an early time of treatment. These changes occurred irrespective of the p53 mutational status of the cells. PS-341 activated JNK/c-Jun signaling in GBM cells, and the JNK inhibitor SP600125 blocked the JNK signaling to reverse partially the PS-341 growth inhibition. PS-341 (10−7 M, 24 h) decreased nuclear NF-κB levels as shown by Western blot, and reduced transcriptional activity of NF-κB as measured by reporter assays in these transformed cells. Also, PS-341 enhanced TRAIL (TNF-related apoptosis-inducing ligand) and TNFα (tumor necrosis factor alpha) induced cell death and apoptosis (two- to five-fold) in GBM cells. In summary, PS-341 has profound effects on growth and apoptosis of GBM cells, suggesting that PS-341 may be an effective therapy for patients with gliomas.

Levels of Expression of CYR61 and CTGF Are Prognostic for Tumor Progression and Survival of Individuals with Gliomas

The biological properties of CCN proteins include stimulation of cell proliferation, migration, and adhesion, as well as angiogenesis and tumorigenesis. We quantified CYR61, CTGF, WISP-1, and NOV mRNA expression levels in samples from sixty-six primary gliomas and five normal brain samples using quantitative real-time PCR assay. Statistical analysis was performed to explore the links between expression of the CCN genes and clinical and pathological parameters. Overexpression of CYR61, CTGF, WISP-1, and NOV occurred in 48% (32 of 66), 58% (38 of 66), 36% (24 of 66), and 15% (10 of 66) of primary gliomas, respectively. Interestingly, significant associations were found between CYR61 expression versus tumor grade, pathology, gender, and age at diagnosis. Also, a significant correlation existed between CTGF mRNA levels versus tumor grade, gender, and pathology. In contrast to CYR61 and CTGF, no significant association was found between expression of either WISP-1 or NOV versus any of the pathological features. Furthermore, Cox regression analysis showed that CYR61 and CTGF expression had a significant correlation with patient survival. These results suggest that CYR61 and CTGF may play a role in the progression of gliomas; their levels at diagnosis may have prognostic significance; and these proteins might serve as valuable targets for therapeutic intervention.

Induction of a CD4+ T Regulatory Type 1 Response by Cyclooxygenase-2-Overexpressing Glioma

PGE2, synthesized by cyclooxygenase-2 (COX-2)-overexpressing tumor, is known to contribute to cellular immune suppression in cancer patients, but the mechanism remains unclear. We report the mechanism of a CD4+ T regulatory type 1 (Tr1) induction by CD11c+ mature dendritic cells (DCs) that phagocytose allogeneic and autologous COX-2-overexpressing glioma. A human glioma cell line, U-87MG, and primary cultured glioblastoma cells (MG-377) overexpressed COX-2. We did not detect IL-10Rα expression in these gliomas, and rIL-10 did not suppress their COX-2 expression. Exposure to COX-2-overexpressing glioma induced mature DCs to overexpress IL-10 and decreased IL-12p70 production. These DCs induced a Tr1 response, which is characterized by robust secretion of IL-10 and TGF-β with negligible IL-4 secretion by CD4+ T cells, and an inhibitory effect on admixed lymphocytes. Peripheral CD4+ T cell populations isolated from an MG-377 patient also predominantly demonstrated a Tr1 response against MG-377 cells. Selective COX-2 inhibition in COX-2-overexpressing gliomas at the time of phagocytic uptake by DCs abrogated this regulatory response and instead elicited Th1 activity. COX-2 stable transfectants in LN-18 (LN-18-COX2) also induced a Tr1 response. The effect of a COX-2 inhibition in LN-18-COX2 is reversible after administration of PGE2. Taken together, robust levels of PGE2 from COX-2-overexpressing glioma, which is unresponsive to IL-10 within the local microenvironment, may cause DCs to secrete high levels of IL-10. These results indicate that COX-2-overexpressing tumors induce a Tr1 response, which is mediated by tumor-exposed, IL-10-enhanced DCs.

Glioma Tropic Neural Stem Cells Consist of Astrocytic Precursors and Their Migratory Capacity Is Mediated by CXCR4

Malignant gliomas spawn disseminated microsatellites, which are largely refractory to currently employed therapies, resulting in eventual tumor recurrence and death. The use of tumor-tropic neural stem cells (NSCs) as delivery vehicles for therapeutic gene products represents an attractive strategy specifically focused at treating these residual neoplastic foci. We wished to elucidate the biological cues governing NSC tropism for glioma. In this context, we describe that tumor-tropic NSCs comprise largely of astrocytic progenitors expressing chemokine receptor 4 (CXCR4). Blocking of CXCR4 significantly inhibits NSC migration toward the tumor. These findings define specific characteristics associated with the cell populations within transplanted NSCs that demonstrate glioma-tracking behavior.

HER-2, gp100, and MAGE-1 Are Expressed in Human Glioblastoma and Recognized by Cytotoxic T Cells

It has recently been demonstrated that malignant glioma cells express certain known tumor-associated antigens, such as HER-2, gp100, and MAGE-1. To further determine the possible utilization of these antigens for glioma immunotherapy and as surrogate markers for specific tumor antigen cytotoxicity, we characterized the presence of mRNA and protein expression in 43 primary glioblastoma multiforme (GBM) cell lines and 7 established human GBM cell lines. HER-2, gp100, and MAGE-1 mRNA expression was detected in 81.4%, 46.5%, and 39.5% of the GBM primary cell lines, respectively. Using immunoreactive staining analysis by flow cytometry, HER-2, gp100, and MAGE-1 protein expression was detected in 76%, 45%, and 38% of the GBM primary cell lines, respectively. HLA-A1-restricted epitope specific for MAGE-1 peptide (EADPTGHSY) CTL clone B07 and HLA-A2-restricted epitope specific for HER-2 peptide (KIFGSLAFL) CTL clone A05 and gp100 peptide (ITDQVPFSV) CTL clone CK3H6 were used in this study. The specificity of CTL clone was verified by HLA/peptide tetramer staining. Three CTL clones could efficiently recognize GBM tumor cells in an antigen-specific and MHC class I-restricted manner. IFN-γ treatment can dramatically increase MHC class I expression of GBM tumor cells and significantly increase CTL recognition of tumor cells. Treatment with the DNA hypomethylating agent 5-aza-2′-deoxycytidine induced and up-regulated the mRNA expression of MAGE-1 and epitope presentation by autologous MHC. These data indicate that HER-2, gp100, and MAGE-1 could be used as tumor antigen targets for surrogate assays for antigen-specific CTLs or to develop antigen-specific active immunotherapy strategies for glioma patients.