Genetic programming of macrophages to perform anti-tumor functions using targeted mRNA nanocarriers.

Tumor-associated macrophages (TAMs) usually express an M2 phenotype, which enables them to perform immunosuppressive and tumor-promoting functions. Reprogramming these TAMs toward an M1 phenotype could thwart their pro-cancer activities and unleash anti-tumor immunity, but efforts to accomplish this are nonspecific and elicit systemic inflammation. Here we describe a targeted nanocarrier that can deliver in vitro-transcribed mRNA encoding M1-polarizing transcription factors to reprogram TAMs without causing systemic toxicity. We demonstrate in models of ovarian cancer, melanoma, and glioblastoma that infusions of nanoparticles formulated with mRNAs encoding interferon regulatory factor 5 in combination with its activating kinase IKKß reverse the immunosuppressive, tumor-supporting state of TAMs and reprogram them to a phenotype that induces anti-tumor immunity and promotes tumor regression. We further establish that these nanoreagents are safe for repeated dosing. Implemented in the clinic, this immunotherapy could enable physicians to obviate suppressive tumors while avoiding systemic treatments that disrupt immune homeostasis.

Optimization of radiation dosing schedules for proneural glioblastoma.

Glioblastomas are the most aggressive primary brain tumor. Despite treatment with surgery, radiation and chemotherapy, these tumors remain uncurable and few significant increases in survival have been observed over the last half-century. We recently employed a combined theoretical and experimental approach to predict the effectiveness of radiation administration schedules, identifying two schedules that led to superior survival in a mouse model of the disease (Leder et al., Cell 156(3):603-616, 2014). Here we extended this approach to consider fractionated schedules to best minimize toxicity arising in early- and late-responding tissues. To this end, we decomposed the problem into two separate solvable optimization tasks: (i) optimization of the amount of radiation per dose, and (ii) optimization of the amount of time that passes between radiation doses. To ensure clinical applicability, we then considered the impact of clinical operating hours by incorporating time constraints consistent with operational schedules of the radiology clinic. We found that there was no significant loss incurred by restricting dosage to an 8:00 a.m. to 5:00 p.m. window. Our flexible approach is also applicable to other tumor types treated with radiotherapy.

TRIM3, a tumor suppressor linked to regulation of p21(Waf1/Cip1.).

The TRIM family of genes is largely studied because of their roles in development, differentiation and host cell antiviral defenses; however, roles in cancer biology are emerging. Loss of heterozygosity of the TRIM3 locus in ∼20% of human glioblastomas raised the possibility that this NHL-domain containing member of the TRIM gene family might be a mammalian tumor suppressor. Consistent with this, reducing TRIM3 expression increased the incidence of and accelerated the development of platelet-derived growth factor -induced glioma in mice. Furthermore, TRIM3 can bind to the cdk inhibitor p21(WAF1/CIP1). Thus, we conclude that TRIM3 is a tumor suppressor mapping to chromosome 11p15.5 and that it might block tumor growth by sequestering p21 and preventing it from facilitating the accumulation of cyclin D1-cdk4.

Standard of care therapy for malignant glioma and its effect on tumor and stromal cells.

Glioblastoma is the most common and deadly of the primary central nervous system tumors. Recent advances in molecular characterization have subdivided these tumors into at least three main groups. In addition, these tumors are cellularly complex with multiple stromal cell types contributing to the biology of the tumor and treatment response. Because essentially all glioma patients are treated with radiation, various chemotherapies and steroids, the tumor that finally kills them has been modified by these treatments. Most of the investigation of the effects of therapy on these tumors has focused on the glioma cells per se. However, despite the importance of the stromal cells in these tumors, little has been done to understand the effects of treatment on stromal cells and their contribution to disease. Understanding how current standard therapy affects the biology of the tumor and the tumor stroma may provide insight into the mechanisms that are important to the inhibition of tumor growth as well as the biology of recurrent tumors.

Oncogenic YAP promotes radioresistance and genomic instability in medulloblastoma through IGF2-mediated Akt activation.

Radiation therapy remains the standard of care for many cancers, including the malignant pediatric brain tumor medulloblastoma. Radiation leads to long-term side effects, whereas radioresistance contributes to tumor recurrence. Radio-resistant medulloblastoma cells occupy the perivascular niche. They express Yes-associated protein (YAP), a Sonic hedgehog (Shh) target markedly elevated in Shh-driven medulloblastomas. Here we report that YAP accelerates tumor growth and confers radioresistance, promoting ongoing proliferation after radiation. YAP activity enables cells to enter mitosis with un-repaired DNA through driving insulin-like growth factor 2 (IGF2) expression and Akt activation, resulting in ATM/Chk2 inactivation and abrogation of cell cycle checkpoints. Our results establish a central role for YAP in counteracting radiation-based therapies and driving genomic instability, and indicate the YAP/IGF2/Akt axis as a therapeutic target in medulloblastoma.

Akt phosphorylation of La regulates specific mRNA translation in glial progenitors.

The Akt signaling pathway activity increases as normal tissue progresses to malignant transformation, and regulates the translation of specific messenger RNAs (mRNAs) through multiple mechanisms. We have identified one such mechanism of Akt-dependent translation control as involving the lupus autoantigen La. La is an RNA-associated protein that contains multiple trafficking elements to support the interaction with RNAs in different subcellular locations. We show here that the La protein is a direct target of the serine/threonine protein kinase Akt on threonine 301, and La nuclear export in mouse glial progenitors, as well as its association with polysomes is modulated by Akt activity. Using a functional approach to determine the network of genes affected by La in the cytoplasm by microarray analysis of polysome-bound mRNAs, we found that La binds 34% of the polysome bound mRNAs and regulates the expression of a specific pool of mRNAs under KRas/Akt activation. Therefore, La appears to be an important contributor to Akt-mediated translational regulation of these transcripts in murine glial cells.

Computational modelling for cerebral aneurysms: risk evaluation and interventional planning.

Advanced computational techniques offer a new array of capabilities in the healthcare provision for cerebral aneurysms. In this paper information is provided on specific simulation methodologies that address some of the unanswered questions about intracranial aneurysm and their treatment. These include the evaluation of rupture risk, the thrombogenic characteristics of specific lesions and the efficacy assessment of particular interventional techniques and devices (e.g. endovascular coil embolisation and flow diversion using stents). The issues connected with ease-of-use and interactivity of computed simulations is discussed, and it is concluded, that the potential of these techniques to optimise planning of complex and multifaceted interventions is very significant, in spite of the fact that most of the methodologies described are still being developed and perfected.

c-Myc and beta-catenin cooperate with loss of p53 to generate multiple members of the primitive neuroectodermal tumor family in mice.

Primitive neuroectodermal tumors (PNETs) are a family of primary malignant brain tumors that include medulloblastomas. Although genetic models of a subset of medulloblastomas are documented over the past decade, the molecular basis of other subclasses of PNET remains unclear. As elevated c-Myc expression, activation of Wnt/beta-catenin signaling and dysfunction of p53 are seen in human PNETs, we investigated what role these abnormalities have in the formation of PNETs. Incorporating these abnormalities, we generated supratentorial PNET (sPNET) in mice using somatic cell gene transfer. We show that sPNETs arise from GFAP-expressing cells by forced c-Myc expression combined with p53 inactivation. beta-catenin activation promotes tumor progression and induces divergent differentiation. These c-Myc+beta-catenin-induced PNETs are histologically similar to large cell/anaplastic medulloblastomas and can occur in both cerebrum and cerebellum. Furthermore, we have obtained one PNET with marked epithelial differentiation having histological resemblance to choroid plexus carcinoma in this series. Our results in mice suggest that sPNET with varied differentiation and large cell/anaplastic medulloblastomas may be two tumor groups with similar genetic foundations. These data provide insights into the biology and classification of human PNETs and suggest that multiple tumor types or variants can be generated from a fixed set of genetic abnormalities.

Loss of Arf causes tumor progression of PDGFB-induced oligodendroglioma.

In a subset of gliomas, the platelet-derived growth factor (PDGF) signaling pathway is perturbed. This is usually an early event occurring in low-grade tumors. In high-grade gliomas, the subsequent loss of the INK4a-ARF locus is one of the most common mutations. Here, we dissected the separate roles of Ink4a and Arf in PDGFB-induced oligodendroglioma development in mice. We found that there were differential functions of the two tumor suppressor genes. In tumors induced from astrocytes, both Ink4a-loss and Arf-loss caused a significantly increased incidence compared to wild-type mice. In tumors induced from glial progenitor cells there was a slight increase in tumor incidence in Ink4a-/- mice and Ink4a-Arf-/- mice compared to wild-type mice. In both progenitor cells and astrocytes, Arf-loss caused a pronounced increase in tumor malignancy compared to Ink4a-loss. Hence, Ink4a-loss contributed to tumor initiation from astrocytes and Arf-loss caused tumor progression from both glial progenitor cells and astrocytes. Results from in vitro studies on primary brain cell cultures suggested that the PDGFB-induced activation of the mitogen-activated protein kinase pathway via extracellular signal-regulated kinase was involved in the initiation of low-grade oligodendrogliomas and that the additional loss of Arf may contribute to tumor progression through increased levels of cyclin D1 and a phosphoinositide 3-kinase-dependent activation of p70 ribosomal S6 kinase causing a strong proliferative response of tumor cells.

In vivo multiple-mouse imaging at 1.5 T.

A multiple-mouse solenoidal MR coil was developed for in vivo imaging of up to 13 mice simultaneously to screen for tumors on a 1.5 T clinical scanner. For the coil to be effective as a screening tool, it should permit acquisition of MRIs in which orthotopic tumors with diameters >2 mm are detectable in a reasonable period of time (<1 hr magnet time) and their sizes accurately measured. Using a spin echo sequence, we demonstrated that this coil provides sufficient sensitivity for moderately high resolution images (156-176 microm in plane-resolution, 1.5 mm slice thickness). This spatial resolution permitted detection of primary brain tumors in transgenic/knockout mice and orthotopic xenografts. Brain tumor size as measured by MRI was correlated with size measured by histopathology (P < 0.001). Metastatic tumors in the mouse lung were also successfully imaged in a screening setting. The multiple mouse coil is simple in construction and may be implemented without any significant modification to the hardware or software on a clinical scanner.

Progenitor cells and glioma formation.

The gliomas are a collection of tumors that arise within the central nervous system and have characteristics similar to astrocytes, oligodendrocytes, or their precursors. Whether or not the glial characteristics of these tumors mean that they arise from the differentiated glia that they resemble or their precursors has been debated. Even under normal circumstances the cells within the central nervous system of an adult can trans-differentiate to other cell types. In addition, mutations found in gliomas further destabilize the differentiation status of these cells making a determination of what cell type gives rise to a given tumor histology difficult. Lineage tracing studies in animals can be used to correlate some specific cell characteristics with the histology of gliomas that arise from these cells. From these experiments it appears that undifferentiated cells are more sensitive to the oncogenic effects of certain signaling abnormalities than are differentiated cells, but that with the appropriate genetic abnormalities differentiated astrocytes can act as the cell-of-origin for gliomas. These data imply that small molecules that promote differentiation may be a rational component of glioma therapy in combination with other drugs aimed at specific molecular signaling targets.

Modeling gliomagenesis with somatic cell gene transfer using retroviral vectors.

Modeling brain tumor formation in experimental animals with somatic cell gene transfer has a long history. In the early experiments naturally occurring retroviruses were used to induce primary brain tumors in a variety of test animals. The subsequent identification of the v-srconcogene within RSV and other cellular proto-oncogenes encoded by other retroviruses moved the field of oncology into the molecular age. Recombinant retroviruses were originally used to infect cells in vitro followed by transplantation. Since then, retroviral vectors have been used to generate glioma formation in vivo via intracerebral injection of neonatal mice. In the most recent models, primary brain tumors can be induced by injecting avian recombinant retroviruses containing different oncogenes, individually or in combination, into transgenic mice genetically engineered for susceptibility to retroviral gene transfer targeting specific cell types in the brain. Animal modeling experiments have contributed substantially to the understanding of the etiology leading to gliomagenesis. The current models provide tumors, which are genetically and histologically similar to their human counterparts, making them attractive to use in drug discovery for treatment of gliomas.

Surgical resection of intrinsic insular tumors: complication avoidance.

OBJECT: Surgical resection of tumors located in the insular region is challenging for neurosurgeons, and few have published their surgical results. The authors report their experience with intrinsic tumors of the insula, with an emphasis on an objective determination of the extent of resection and neurological complications and on an analysis of the anatomical characteristics that can lead to suboptimal outcomes. METHODS: Twenty-two patients who underwent surgical resection of intrinsic insular tumors were retrospectively identified. Eight tumors (36%) were purely insular, eight (36%) extended into the temporal pole, and six (27%) extended into the frontal operculum. A transsylvian surgical approach, combined with a frontal opercular resection or temporal lobectomy when necessary, was used in all cases. Five of 13 patients with tumors located in the dominant hemisphere underwent craniotomies while awake. The extent of tumor resection was determined using volumetric analyses. In 10 patients, more than 90% of the tumor was resected; in six patients, 75 to 90% was resected; and in six patients, less than 75% was resected. No patient died within 30 days after surgery. During the immediate postoperative period, the neurological conditions of 14 patients (64%) either improved or were unchanged, and in eight patients (36%) they worsened. Deficits included either motor or speech dysfunction. At the 3-month follow-up examination, only two patients (9%) displayed permanent deficits. Speech and motor dysfunction appeared to result most often from excessive opercular retraction and manipulation of the middle cerebral artery (MCA), interruption of the lateral lenticulostriate arteries (LLAs), interruption of the long perforating vessels of the second segment of the MCA (M2), or violation of the corona radiata at the superior aspect of the tumor. Specific methods used to avoid complications included widely splitting the sylvian fissure and identifying the bases of the periinsular sulci to define the superior and inferior resection planes, identifying early the most lateral LLA to define the medial resection plane, dissecting the MCA before tumor resection, removing the tumor subpially with preservation of all large perforating arteries arising from posterior M2 branches, and performing craniotomy with brain stimulation while the patient was awake. CONCLUSIONS: A good understanding of the surgical anatomy and an awareness of potential pitfalls can help reduce neurological complications and maximize surgical resection of insular tumors.

Glioma models.

Gliomas are primary central nervous system tumors that arise from astrocytes, oligodendrocytes or their precursors. Gliomas can be classified into several groups according to their histologic characteristics, the most malignant of the gliomas is glioblastoma multiforme. In contrast to the long-standing and well-defined histopathology, the underlying molecular and genetic bases for gliomas are only just emerging. Many genetic alterations have been identified in human gliomas, however, establishing unequivocal correlation between these genetic alterations and gliomagenesis requires accurate animal models for this disease. Here we are reviewing the existing animal models for gliomas with different strategies and our current knowledge on the important issues about this disease, such as activation of signal transduction pathways, disruption of cell cycle arrest pathways, cell-of-origin of gliomas, and therapeutic strategies.

PDGF autocrine stimulation dedifferentiates cultured astrocytes and induces oligodendrogliomas and oligoastrocytomas from neural progenitors and astrocytes in vivo.

We present evidence that some low-grade oligodendrogliomas may be comprised of proliferating glial progenitor cells that are blocked in their ability to differentiate, whereas malignant gliomas have additionally acquired other mutations such as disruption of cell cycle arrest pathways by loss of Ink4a-Arf. We have modeled these effects in cell culture and in mice by generating autocrine stimulation of glia through the platelet-derived growth factor receptor (PDGFR). In cell culture, PDGF signaling induces proliferation of glial precursors and blocks their differentiation into oligodendrocytes and astrocytes. In addition, coexpression of PDGF and PDGF receptors has been demonstrated in human gliomas, implying that autocrine stimulation may be involved in glioma formation. In this study, using somatic cell type-specific gene transfer we investigated the functions of PDGF autocrine signaling in gliomagenesis by transferring the overexpression of PDGF-B into either nestin-expressing neural progenitors or glial fibrillary acidic protein (GFAP)-expressing astrocytes both in cell culture and in vivo. In cultured astrocytes, overexpression of PDGF-B caused significant increase in proliferation rate of both astrocytes and neural progenitors. Furthermore, PDGF gene transfer converted cultured astrocytes into cells with morphologic and gene expression characteristics of glial precursors. In vivo, gene transfer of PDGF to neural progenitors induced the formation of oligodendrogliomas in about 60% of mice by 12 wk of age; PDGF transfer to astrocytes induced the formation of either oligodendrogliomas or mixed oligoastrocytomas in about 40% of mice in the same time period. Loss of Ink4a-Arf, a mutation frequently found in high-grade human gliomas, resulted in shortened latency and enhanced malignancy of gliomas. The highest percentage of PDGF-induced malignant gliomas arose from of Ink4a-Arf null progenitor cells. These data suggest that chronic autocrine PDGF signaling can promote a proliferating population of glial precursors and is potentially sufficient to induce gliomagenesis. Loss of Ink4a-Arf is not required for PDGF-induced glioma formation but promotes tumor progression toward a more malignant phenotype.

Animal models of cell cycle dysregulation and the pathogenesis of gliomas.

Mutations in gliomas, for the most part, fall into two main categories. The first category of mutations affects genes that produce proteins which activate signal transduction pathways downstream of tyrosine kinase receptors; the second category disrupts the pathways leading to cell cycle arrest. Cell cycle arrest pathways normally maintain cells in the G1 phase of the cell cycle, preventing inappropriate proliferation. The role of disregulation of these pathways in tumor formation is currently the focus of many investigations. Studies carried out with astrocytes and other cell types indicate that these pathways may also function in maintenance of appropriate chromosome number and differentiated phenotype, and in acquisition of senescence. Genetically defined mouse models of gliomagenesis have been helpful in increasing our understanding of how cell cycle arrest pathways cooperate with alterations in signal transduction pathways to provoke tumor formation in many cell types, including glial cells. Various strategies for experimental cell cycle arrest disruption show minimal or no formation of gliomas. In contrast, gliomas are generated with a number of strategies that enhance signal transduction downstream of tyrosine kinase receptors. Experimental disruption of the cell cycle arrest pathways is required for gliomagenesis in some of these models, but not in others. Furthermore in some cases, although not required for gliomagenesis, disruption of the cell cycle arrest pathways appears to enhance glioma formation. The results of these mouse model experiments imply a potentially complex role for cell cycle arrest disruption in human gliomagenesis.

Brain tumor animal models: importance and progress.

Recent experiments indicate that some of the genetic abnormalities found in human brain tumors can induce tumors in mice with similar histologic characteristics to their human counterparts. Such studies help unravel the biology of tumorigenesis and indicate that some of the mutations and alterations in gene expression found in human central nervous system tumors may actually contribute to the etiology of these diseases. In addition, these mouse-modeling experiments may identify essential targets for therapy and provide test animals for preclinical trials of mechanistically designed therapeutics.

Gliomagenesis: genetic alterations and mouse models.

Glioblastoma multiforme is the most malignant of the primary brain tumours and is almost always fatal. The treatment strategies for this disease have not changed appreciably for many years and most are based on a limited understanding of the biology of the disease. However, in the past decade, characteristic genetic alterations have been identified in gliomas that might underlie the initiation or progression of the disease. Recent modelling experiments in mice are helping to delineate the molecular aetiology of this disease and are providing systems to identify and test novel and rational therapeutic strategies.

Identification of a multipotent astrocytic stem cell in the immature and adult mouse brain.

The mammalian brain contains a population of neural stem cells (NSC) that can both self-renew and generate progeny along the three lineage pathways of the central nervous system (CNS), but the in vivo identification and localization of NSC in the postnatal CNS has proved elusive. Recently, separate studies have implicated ciliated ependymal (CE) cells, and special subependymal zone (SEZ) astrocytes as candidates for NSC in the adult brain. In the present study, we have examined the potential of these two NSC candidates to form multipotent spherical clones-neurospheres-in vitro. We conclude that CE cells are unipotent and give rise only to cells within the glia cell lineage, although they are capable of forming spherical clones when cultured in isolation. In contrast, astrocyte monolayers from the cerebral cortex, cerebellum, spinal cord, and SEZ can form neurospheres that give rise both to neurons and glia. However, the ability to form neurospheres is restricted to astrocyte monolayers derived during the first 2 postnatal wk, except for SEZ astrocytes, which retain this capacity in the mature forebrain. We conclude that environmental factors, simulated by certain in vitro conditions, transiently confer NSC-like attributes on astrocytes during a critical period in CNS development.