Hiroaki Wakimoto

Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma

Cancer at single-cell resolution Single-cell sequencing can illuminate the genetic properties of brain cancers and reveal heterogeneity within a tumor. Patel et al. examined the genome sequence of single cells isolated from brain glioblastomas. The findings revealed shared chromosomal changes but also extensive transcription variation, including genes related to signaling, which represent potential therapeutic targets. The authors suggest that the variation in tumor cells reflects neural development and that such variation among cancer cells may prove to have clinical significance. Science, this issue p. 1396 Screening individual cancer cells within a brain tumor may help to guide treatment and predict prognosis. Human cancers are complex ecosystems composed of cells with distinct phenotypes, genotypes, and epigenetic states, but current models do not adequately reflect tumor composition in patients. We used single-cell RNA sequencing (RNA-seq) to profile 430 cells from five primary glioblastomas, which we found to be inherently variable in their expression of diverse transcriptional programs related to oncogenic signaling, proliferation, complement/immune response, and hypoxia. We also observed a continuum of stemness-related expression states that enabled us to identify putative regulators of stemness in vivo. Finally, we show that established glioblastoma subtype classifiers are variably expressed across individual cells within a tumor and demonstrate the potential prognostic implications of such intratumoral heterogeneity. Thus, we reveal previously unappreciated heterogeneity in diverse regulatory programs central to glioblastoma biology, prognosis, and therapy.

Assessment of therapeutic efficacy and fate of engineered human mesenchymal stem cells for cancer therapy

The poor prognosis of patients with aggressive and invasive cancers combined with toxic effects and short half-life of currently available treatments necessitate development of more effective tumor selective therapies. Mesenchymal stem cells (MSCs) are emerging as novel cell-based delivery agents; however, a thorough investigation addressing their therapeutic potential and fate in different cancer models is lacking. In this study, we explored the engineering potential, fate, and therapeutic efficacy of human MSCs in a highly malignant and invasive model of glioblastoma. We show that engineered MSC retain their “stem-like” properties, survive longer in mice with gliomas than in the normal brain, and migrate extensively toward gliomas. We also show that MSCs are resistant to the cytokine tumor necrosis factor apoptosis ligand (TRAIL) and, when engineered to express secreted recombinant TRAIL, induce caspase-mediated apoptosis in established glioma cell lines as well as CD133-positive primary glioma cells in vitro. Using highly malignant and invasive human glioma models and employing real-time imaging with correlative neuropathology, we demonstrate that MSC-delivered recombinant TRAIL has profound anti-tumor effects in vivo. This study demonstrates the efficacy of diagnostic and therapeutic MSC in preclinical glioma models and forms the basis for developing stem cell-based therapies for different cancers.

Oncolytic virus therapy of multiple tumors in the brain requires suppression of innate and elicited antiviral responses.

The occurrence of multiple tumors in an organ heralds a rapidly fatal course. Although intravascular administration may deliver oncolytic viruses/vectors to each of these tumors, its efficiency is impeded by an antiviral activity present in complement-depleted plasma of rodents and humans. Here, this activity was shown to interact with complement in a calcium-dependent fashion, and antibody neutralization studies indicated preimmune IgM has a contributing role. Short-term exposure to cyclophosphamide (CPA) partially suppressed this activity in rodents and humans. At longer time points, cyclophosphamide also abrogated neutralizing antibody responses. Cyclophosphamide treatment of rats with large single or multiple intracerebral tumors substantially increased viral survival and propagation, leading to neoplastic regression.

Human Glioblastoma–Derived Cancer Stem Cells: Establishment of Invasive Glioma Models and Treatment with Oncolytic Herpes Simplex Virus Vectors

Glioblastoma, the most malignant type of primary brain tumor, is one of the solid cancers where cancer stem cells have been isolated, and studies have suggested resistance of those cells to chemotherapy and radiotherapy. Here, we report the establishment of CSC-enriched cultures derived from human glioblastoma specimens. They grew as neurospheres in serum-free medium with epidermal growth factor and fibroblast growth factor 2, varied in the level of CD133 expression and very efficiently formed highly invasive and/or vascular tumors upon intracerebral implantation into immunodeficient mice. As a novel therapeutic strategy for glioblastoma-derived cancer stem-like cells (GBM-SC), we have tested oncolytic herpes simplex virus (oHSV) vectors. We show that although ICP6 (UL39)-deleted mutants kill GBM-SCs as efficiently as wild-type HSV, the deletion of gamma34.5 significantly attenuated the vectors due to poor replication. However, this was significantly reversed by the additional deletion of alpha47. Infection with oHSV G47Delta (ICP6(-), gamma34.5(-), alpha47(-)) not only killed GBM-SCs but also inhibited their self-renewal as evidenced by the inability of viable cells to form secondary tumor spheres. Importantly, despite the highly invasive nature of the intracerebral tumors generated by GBM-SCs, intratumoral injection of G47Delta significantly prolonged survival. These results for the first time show the efficacy of oHSV against human GBM-SCs, and correlate this cytotoxic property with specific oHSV mutations. This is important for designing new oHSV vectors and clinical trials. Moreover, the new glioma models described in this study provide powerful tools for testing experimental therapeutics and studying invasion and angiogenesis.

Alternative Lengthening of Telomeres Renders Cancer Cells Hypersensitive to ATR Inhibitors

Cancers alternative means to an end To stay alive and proliferating, tumor cells must maintain their telomeres: the DNA sequences at the ends of chromosomes. The majority accomplish this by activating the enzyme telomerase. However, certain tumor types favor a different mechanism called alternative lengthening of telomeres (ALT), which involves DNA recombination. Flynn et al. delineated the molecular events that occur at the telomeres of ALT-proficient tumor cells by studying the function of a protein that is altered by mutation in these tumors. The analysis revealed a specific protein kinase that is essential for ALT, which could in principle be targeted to halt tumor growth. Science, this issue p. 273 A potential therapeutic strategy is identified for tumor cells that maintain their telomeres by an unusual mechanism. Cancer cells rely on telomerase or the alternative lengthening of telomeres (ALT) pathway to overcome replicative mortality. ALT is mediated by recombination and is prevalent in a subset of human cancers, yet whether it can be exploited therapeutically remains unknown. Loss of the chromatin-remodeling protein ATRX associates with ALT in cancers. Here, we show that ATRX loss compromises cell-cycle regulation of the telomeric noncoding RNA TERRA and leads to persistent association of replication protein A (RPA) with telomeres after DNA replication, creating a recombinogenic nucleoprotein structure. Inhibition of the protein kinase ATR, a critical regulator of recombination recruited by RPA, disrupts ALT and triggers chromosome fragmentation and apoptosis in ALT cells. The cell death induced by ATR inhibitors is highly selective for cancer cells that rely on ALT, suggesting that such inhibitors may be useful for treatment of ALT-positive cancers.

Complement Depletion Facilitates the Infection of Multiple Brain Tumors by an Intravascular, Replication-Conditional Herpes Simplex Virus Mutant

Intravascular routes of administration can provide a means to target gene- and virus-based therapies to multiple tumor foci located within an organ, such as the brain. However, we demonstrate here that rodent plasma inhibits cell transduction by replication-conditional (oncolytic) herpes simplex viruses (HSV), replication-defective HSV, and adenovirus vectors. In vitro depletion of complement with mild heat treatment or in vivo depletion by treatment of athymic rats with cobra venom factor (CVF) partially reverses this effect. Without CVF, inhibition of cell infection by HSV is observed at plasma dilution as high as 1:32, while plasma from CVF-treated animals displays anti-HSV activity at lower dilutions (1:8). When applied to the therapy of intracerebral brain tumors, in vivo complement depletion facilitates the initial infection (assayed at the 2-day time point) by an intra-arterial replication-conditional HSV of tumor cells, located within three separate and distinct human glioma masses. However, at the 4-day time point, no propagation of HSV from initially infected tumor cells could be observed. Previously, we have shown that the immunosuppressive agent, cyclophosphamide (CPA), facilitates the in vivo propagation of an oncolytic HSV, delivered intravascularly, within infected multiple intracerebral masses, by inhibition of both innate and elicited anti-HSV neutralizing antibody response (K. Ikeda et al., Nat. Med. 5:881-889, 1999). In this study, we thus show that the addition of CPA to the CVF treatment results in a significant increase in viral propagation within infected tumors, measured at the 4-day time period. The concerted action of CVF and CPA significantly increases the life span of athymic rodents harboring three separate and large glioma xenografts after treatment with intravascular, oncolytic HSV. Southern analysis of viral genomes analyzed by PCR reveals the presence of the oncolytic virus in the brains, livers, spleens, kidneys, and intestine of treated animals, although none of these tissues displays evidence of HSV-mediated gene expression. In light of clinical trials of oncolytic HSV for malignant brain tumors, these findings suggest that antitumor efficacy may be limited by the host innate and elicited humoral responses.

Maintenance of primary tumor phenotype and genotype in glioblastoma stem cells

The clinicopathological heterogeneity of glioblastoma (GBM) and the various genetic and phenotypic subtypes in GBM stem cells (GSCs) are well described. However, the relationship between GSCs and the corresponding primary tumor from which they were isolated is poorly understood. We have established GSC-enriched neurosphere cultures from 15 newly diagnosed GBM specimens and examined the relationship between the histopathological and genomic features of GSC-derived orthotopic xenografts and those of the respective patient tumors. GSC-initiated xenografts recapitulate the distinctive cytological hallmarks and diverse histological variants associated with the corresponding patient GBM, including giant cell and gemistocytic GBM, and primitive neuroectodermal tumor (PNET)-like components. This indicates that GSCs generate tumors that preserve patient-specific disease phenotypes. The majority of GSC-derived intracerebral xenografts (11 of 15) demonstrated a highly invasive behavior crossing the midline, whereas the remainder formed discrete nodular and vascular masses. In some cases, GSC invasiveness correlated with preoperative MRI, but not with the status of PI3-kinase/Akt pathways or O(6)-methylguanine methyltransferase expression. Genome-wide screening by array comparative genomic hybridization and fluorescence in situ hybridization revealed that GSCs harbor unique genetic copy number aberrations. GSCs acquiring amplifications of the myc family genes represent only a minority of tumor cells within the original patient tumors. Thus, GSCs are a genetically distinct subpopulation of neoplastic cells within a GBM. These studies highlight the value of GSCs for preclinical modeling of clinically relevant, patient-specific GBM and, thus, pave the way for testing novel anti-GSC/GBM agents for personalized therapy.

Altered expression of antiviral cytokine mRNAs associated with cyclophosphamide's enhancement of viral oncolysis

Oncolytic viruses (OVs) are being used as anticancer agents in preclinical and clinical trials. Propagation of OVs inside infected tumors is critical to their efficacy and is mediated by the productive generation of progeny OVs within infected tumor cells. In turn, this progeny can spread the infection to other tumor cells in successive rounds of oncolysis. Previously, we had found that, in rats, cyclophosphamide (CPA) pretreatment increased infection of brain tumors by an intra-arterially administered herpes simplex virus type 1 OV, because it inhibited activation of complement responses, mediated by innate IgM. We also have previously shown that other pharmacologic inhibitors of complement, such as cobra venom factor (CVF), allowed for increased infection. However, in these studies, further inhibition of complement responses by CVF did not result in additional infection of brain tumor cells or in propagation of OV to surrounding tumor cells. In this study, we sought to determine if CPA did lead to increased infection/propagation from initially infected tumor cells. Unlike our results with CVF, we find that CPA administration does result in a time-dependent increase in infection of tumor cells, suggestive of increased propagation, in both syngeneic and athymic models of brain tumors. This increase was due to increased survival of OV within infected tumors and brain surrounding tumors. CPAs effect was not due to a direct enhancement of viral replication in tumor cells, rather was associated with its immunosuppressive effects. RT-PCR analysis revealed that CPA administration resulted in impaired mRNA production by peripheral blood mononuclear cells (PBMCs) of several cytokines (interferons α/β, interferon γ, TNFα, IL-15, and IL-18) with anti-HSV function. These findings suggest that the CPA-mediated facilitation of OV intraneoplastic propagation is associated with a general decrease of antiviral cytokines mRNAs in PBMCs. These findings not only suggest a potential benefit for the addition of transient immunosuppression in clinical applications of oncolytic HSV therapy, but also suggest that innate immunomodulatory pathways may be amenable to manipulation, in order to increase OV propagation and survival within infected tumors.

Accumulation of CD133-positive glioma cells after high-dose irradiation by Gamma Knife surgery plus external beam radiation

OBJECT Recent evidence suggests that a glioma stem cell subpopulation might contribute to radioresistance in malignant gliomas. To investigate this hypothesis, the authors examined recurrent malignant gliomas for histopathological changes after high-dose irradiation with Gamma Knife surgery (GKS) and external beam radiation therapy (EBRT). METHODS Thirty-two patients with malignant gliomas (Grade 3 in 8 patients, Grade 4 in 24) underwent GKS in combination with EBRT. Serial MR and L-[methyl-(11)C] methionine PET images were employed to assess remnant or recurrent tumors after GKS. Twelve patients underwent surgical removal after GKS and EBRT. Histological sections were subjected to immunohistochemistry for MIB-1, factor VIII, and stem cell markers, nestin and CD133. RESULTS The site of GKS treatment failure was local in 16 (76.2%) of 21 patients with glioblastomas showing progression; in 9 of these 16 patients, the recurrence clearly arose within the target lesion of GKS. Histopathological examination after GKS and EBRT showed variable mixtures of viable tumor tissues and necrosis. Viable tumor tissues exhibited high MIB-1 indices but reduced numbers of tumor blood vessels. There was marked accumulation of CD133-positive glioma cells, particularly in remnant tumors within the necrotic areas, in sections obtained after GKS plus EBRT, whereas CD133-positive cells appeared very infrequently in primary sections prior to adjuvant treatment. CONCLUSIONS The results indicate that CD133-positive glioma stemlike cells can survive high-dose irradiation, leading to recurrence, despite prolonged damage to tumor blood vessels. This could be an essential factor limiting the effectiveness of GKS plus EBRT for malignant gliomas.

A Dual PI3K/mTOR Inhibitor, PI-103, Cooperates with Stem Cell–Delivered TRAIL in Experimental Glioma Models

The resistance of glioma cells to a number of antitumor agents and the highly invasive nature of glioma cells that escape the primary tumor mass are key impediments to the eradication of tumors in glioma patients. In this study, we evaluated the therapeutic efficacy of a novel PI3-kinase/mTOR inhibitor, PI-103, in established glioma lines and primary CD133(+) glioma-initiating cells and explored the potential of combining PI-103 with stem cell-delivered secretable tumor necrosis factor apoptosis-inducing ligand (S-TRAIL) both in vitro and in orthotopic mouse models of gliomas. We show that PI-103 inhibits proliferation and invasion, causes G(0)-G(1) arrest in cell cycle, and results in significant attenuation of orthotopic tumor growth in vivo. Establishing cocultures of neural stem cells (NSC) and glioma cells, we show that PI-103 augments the response of glioma cells to stem cell-delivered S-TRAIL. Using bimodal optical imaging, we show that when different regimens of systemic PI-103 delivery are combined with NSC-derived S-TRAIL, a significant reduction in tumor volumes is observed compared with PI-103 treatment alone. To our knowledge, this is the first study that reveals the antitumor effect of PI-103 in intracranial gliomas. Our findings offer a preclinical rationale for application of mechanism-based systemically delivered antiproliferative agents and novel stem cell-based proapoptotic therapies to improve treatment of malignant gliomas.