Qijin Xu

Antigen-Specific T-Cell Response from Dendritic Cell Vaccination Using Cancer Stem-Like Cell-Associated Antigens

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor, with current treatment remaining palliative. Immunotherapies harness the bodys own immune system to target cancers and could overcome the limitations of conventional treatments. One active immunotherapy strategy uses dendritic cell (DC)‐based vaccination to initiate T‐cell‐mediated antitumor immunity. It has been proposed that cancer stem‐like cells (CSCs) may play a key role in cancer initiation, progression, and resistance to current treatments. However, whether using human CSC antigens may improve the antitumor effect of DC vaccination against human cancer is unclear. In this study, we explored the suitability of CSCs as sources of antigens for DC vaccination again human GBM, with the aim of achieving CSC‐targeting and enhanced antitumor immunity. We found that CSCs express high levels of tumor‐associated antigens as well as major histocompatibility complex molecules. Furthermore, DC vaccination using CSC antigens elicited antigen‐specific T‐cell responses against CSCs. DC vaccination‐induced interferon‐γ production is positively correlated with the number of antigen‐specific T cells generated. Finally, using a 9L CSC brain tumor model, we demonstrate that vaccination with DCs loaded with 9L CSCs, but not daughter cells or conventionally cultured 9L cells, induced cytotoxic T lymphocytes (CTLs) against CSCs, and prolonged survival in animals bearing 9L CSC tumors. Understanding how immunization with CSCs generates superior antitumor immunity may accelerate development of CSC‐specific immunotherapies and cancer vaccines. STEM CELLS 2009;27:1734–1740

Hedgehog signaling regulates brain tumor-initiating cell proliferation and portends shorter survival for patients with PTEN-coexpressing glioblastomas.

The identification of brain tumor stem‐like cells (BTSCs) has implicated a role of biological self‐renewal mechanisms in clinical brain tumor initiation and propagation. The molecular mechanisms underlying the tumor‐forming capacity of BTSCs, however, remain unknown. Here, we have generated molecular signatures of glioblastoma multiforme (GBM) using gene expression profiles of BTSCs and have identified both Sonic Hedgehog (SHH) signaling‐dependent and ‐independent BTSCs and their respective glioblastoma surgical specimens. BTSC proliferation could be abrogated in a pathway‐dependent fashion in vitro and in an intracranial tumor model in athymic mice. Both SHH‐dependent and ‐independent brain tumor growth required phosphoinositide 3‐kinase‐mammalian target of rapamycin signaling. In human GBMs, the levels of SHH and PTCH1 expression were significantly higher in PTEN‐expressing tumors than in PTEN‐deficient tumors. In addition, we show that hyperactive SHH‐GLI signaling in PTEN‐coexpressing human GBM is associated with reduced survival time. Thus, distinct proliferation signaling dependence may underpin glioblastoma propagation by BTSCs. Modeling these BTSC proliferation mechanisms may provide a rationale for individualized glioblastoma treatment.

Isolation of tumour stem-like cells from benign tumours.

Background:Cancerous stem-like cells (CSCs) have been implicated as cancer-initiating cells in a range of malignant tumours. Diverse genetic programs regulate CSC behaviours, and CSCs from glioblastoma patients are qualitatively distinct from each other. The intrinsic connection between the presence of CSCs and malignancy is unclear. We set out to test whether tumour stem-like cells can be identified from benign tumours.Methods:Tumour sphere cultures were derived from hormone-positive and -negative pituitary adenomas. Characterisation of tumour stem-like cells in vitro was performed using self-renewal assays, stem cell-associated marker expression analysis, differentiation, and stimulated hormone production assays. The tumour-initiating capability of these tumour stem-like cells was tested in serial brain tumour transplantation experiments using SCID mice.Results:In this study, we isolated sphere-forming, self-renewable, and multipotent stem-like cells from pituitary adenomas, which are benign tumours. We found that pituitary adenoma stem-like cells (PASCs), compared with their differentiated daughter cells, expressed increased levels of stem cell-associated gene products, antiapoptotic proteins, and pituitary progenitor cell markers. Similar to CSCs isolated from glioblastomas, PASCs are more resistant to chemotherapeutics than their differentiated daughter cells. Furthermore, differentiated PASCs responded to stimulation with hypothalamic hormones and produced corresponding pituitary hormones that are reflective of the phenotypes of the primary pituitary tumours. Finally, we demonstrated that PASCs are pituitary tumour-initiating cells in serial transplantation animal experiments.Conclusion:This study for the first time indicates that stem-like cells are present in benign tumours. The conclusions from this study may have applications to understanding pituitary tumour biology and therapies, as well as implications for the notion of tumour-initiating cells in general.

Evidence That IGF Binding Protein-5 Functions as a Ligand-Independent Transcriptional Regulator in Vascular Smooth Muscle Cells

Abstract— Insulin-like growth factor binding protein (IGFBP)-5 is a conserved protein synthesized and secreted by vascular smooth muscle cells (VSMCs). IGFBP-5 binds to extracellular IGFs and modulates IGF actions in regulating VSMC proliferation, migration, and survival. IGFBP-5 also stimulates VSMC migration through an IGF-independent mechanism, but the molecular basis underlying this ligand-independent action is unknown. In this study, we show that endogenous IGFBP-5 or transiently expressed IGFBP-5-EGFP, but not IGFBP-4-EGFP, is localized in the nuclei of VSMCs. Using a series of IGFBP-4/5 chimeras and IGFBP-5 points mutants, we demonstrated that the IGFBP-5 C-domain is necessary and sufficient for its nuclear localization, and residues K206, K208, K217, and K218 are particularly critical. Intriguingly, inhibition of protein secretion abolishes IGFBP-5 nuclear localization, suggesting the nuclear IGFBP-5 is derived from the secreted protein. When added exogenously, 125I- or Cy3-labeled IGFBP-5 is capable of cellular entry and nuclear translocation. To identify potential transcriptional factor(s) that interact with IGFBP-5, a human aorta cDNA library was screened by a yeast two-hybrid screening strategy. Although this screen identified many extracellular and cytosolic proteins that are known to interact with IGFBP-5, no known transcription factors were found. Further motif analysis revealed that the IGFBP-5 N-domain contains a putative transactivation domain. When fused to GAL-4 DNA dinging domain and tested, the IGFBP-5 N-domain has strong transactivation activity. Mutation of the IGF binding domain or treatment of cells with IGF-I has little effect on transactivation activity. These results suggest that IGFBP-5 is localized in VSMC nucleus and possesses transcription-regulatory activity that is IGF independent. The full text of this article is available online at http://circres.ahajournals.org.

Near-infrared fluorescence heptamethine carbocyanine dyes mediate imaging and targeted drug delivery for human brain tumor

Brain tumors and brain metastases are among the deadliest malignancies of all human cancers, largely due to the cellular blood-brain and blood-tumor barriers that limit the delivery of imaging and therapeutic agents from the systemic circulation to tumors. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. Here we identified and synthesized a group of near-infrared fluorescence (NIRF) heptamethine carbocyanine dyes and derivative NIRF dye-drug conjugates for effective imaging and therapeutic targeting of brain tumors of either primary or metastatic origin in mice, which is mechanistically mediated by tumor hypoxia and organic anion-transporting polypeptide genes. We also demonstrate that these dyes, when conjugated to chemotherapeutic agents such as gemcitabine, significantly restricted the growth of both intracranial glioma xenografts and prostate tumor brain metastases and prolonged survival in mice. These results show promise in the application of NIRF dyes as novel theranostic agents for the detection and treatment of brain tumors.

Chemokine CXC receptor 4–mediated glioma tumor tracking by bone marrow–derived neural progenitor/stem cells

Malignant gliomas manifest frequent tumor recurrence after surgical resection and/or other treatment because of their nature of invasiveness and dissemination. The recognized brain tumor-tracking property of neural progenitor/stem cells opened the possibility of targeting malignant brain tumors using neural progenitor/stem cells. We and others have previously shown that fetal neural progenitor/stem cells can be used to deliver therapeutic molecules to brain tumors. Our recent work has further shown that gene delivery by bone marrow–derived neural progenitor/stem cells achieves therapeutic effects in a glioma model. In this study, we isolate and characterize bone marrow–derived neural progenitor/stem cells, which also express the chemokine receptor chemokine CXC receptor 4 (CXCR4). We show that CXCR4 is required for their chemotaxis and extracellular matrix invasion against a gradient of glioma soluble factors. Furthermore, β-galactosidase-labeled bone marrow–derived neural progenitor/stem cells implanted in the contralateral side of the brain were shown to track gliomas as early as day 1 and increased through days 3 and 7. Intracranial glioma tracking by bone marrow–derived neural progenitor/stem cells is significantly inhibited by preincubation of bone marrow–derived neural progenitor/stem cells with a blocking anti–CXCR4 antibody, suggesting a CXCR4–dependent tracking mechanism. Glioma tracking bone marrow–derived neural progenitor/stem cells were found to express progenitor/stem cell markers, as well as CXCR4. Although bromodeoxyuridine incorporation assays and proliferating antigen staining indicated that tumor tracking bone marrow–derived neural progenitor/stem cells were mostly nonproliferating, these cells survive in the local tumor environment with little apoptosis. Elucidating the molecular mechanism of brain tumor tracking by adult source stem cells may provide basis for the development of future targeted therapy for malignant brain tumors. [Mol Cancer Ther 2009;8(9):2746–53]

Preparation and characterization of antioxidant nanospheres from multiple α-lipoic acid-containing compounds

The purpose of this study was to prepare and characterize antioxidant nanospheres composed of multiple alpha-lipoic acid-containing compounds (mALAs). It was found that the nanospheres were remarkably stable under physiologic conditions, maintained the antioxidant property of alpha-lipoic acid, and could be destabilized oxidatively and enzymatically. The preparations were simple and highly reproducible providing a new strategy for the development of nanometer-sized antioxidant biomaterials. The nanospheres may find applications as antioxidant therapeutics and oxidation-responsive antioxidant nanocontainers in drug delivery for pathological conditions characterized by oxidative stress including cancer and neurodegenerative diseases.

Stimuli-responsive antioxidant nanoprodrugs of NSAIDs

A novel group of alpha-lipoic acid-containing hydrophobic prodrugs of non-steroidal anti-inflammatory drugs (NSAIDs) was synthesized and transformed into nanometer-sized prodrugs (nanoprodrugs). Three NSAIDs, indomethacin, ibuprofen and naproxen were linked to alpha-lipoic acid via tetraethylene glycol through hydrolytically degradable ester bonds. The three bifunctional derivatives were dissolved in organic solvents and capable of forming stable nanoprodrugs upon addition of the organic solutions into aqueous phase through the spontaneous emulsification mechanism. Antioxidant property and stimuli-responsiveness of the nanoprodrugs were demonstrated by hypochlorous acid (HOCl) scavenging followed by oxidative destabilization of the nanoprodrugs. The effect of varying NSAIDs on the in vitro hydrolytic prodrug activation catalyzed by porcine liver esterase was investigated by monitoring the rates of NSAIDs hydrolysis from the nanoprodrugs. The remarkable feature of these nanoprodrugs is that despite the highly hydrophobic nature of the derivatives NSAIDs were readily hydrolyzed enzymatically from the nanoprodrugs. Furthermore, the rate of hydrolysis was higher when the nanoprodrugs were oxidized and destabilized upon HOCl scavenging suggesting an enhanced activation of the nanoprodrugs in the oxidative environment.

Glioma stem cell research for the development of immunotherapy.

Malignant gliomas are characterized by its invasiveness and dissemination, resulting in frequent tumor recurrence after surgical resection and/or conventional chemotherapy and radiation therapy. Various strategies of active and passive immunotherapy in developing stages have shown promise to increase patient survival time with little severe side effects. In recent years, glioma stem cells had been isolated from patient tumor specimens. Biochemical and biological characterization of these cancer initiating cells implicated their critical roles in cancer growth, malignancy and resistance to conventional treatments. In this chapter, we review recent research progress in targeting brain cancer using neural stem cells delivered cytotoxic factors and immune regulation factor, dendritic cell based vaccination, with special emphasis on targeting glioma stem cells. We present evidence supporting the notion that glioma stem cells may be preferred therapeutic targets not only for conventional therapies, but also for immunotherapies. Future progress in glioma stem cell research may fundamentally improve the prospect of malignant glioma treatments.

Nanoprodrugs of nsaids inhibit the growth of U87-MG glioma cells

Several recent reports have demonstrated that nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the growth of various malignant cells suggesting their application as anticancer agents. In this study, we prepared six nanometer-sized prodrugs (nanoprodrugs) of NSAIDs, ibuprofen, indomethacin, and naproxen through the spontaneous emulsification mechanism using monomeric and dimeric derivatives of the NSAIDs. We evaluated their effect on the proliferation of U87-MG glioma cells by cell counting, WST-1 cell proliferation reagent, and propidium iodide incorporation. The two ibuprofen nanoprodrugs inhibited the cell growth more potently than the indomethacin nanoprodrugs, whereas the naproxen nanoprodrugs did not show any significant effect. Remarkably, ibuprofen did not show any effect at an equimolar concentration. Approximately, 4.4% of the ibuprofen nanoprodrugs was found in the cell, whereas no ibuprofen could be detected suggesting that the superior effect of the nanoprodrugs can be attributed to the efficient cellular uptake of the nanoprodrugs.