Julian K. Wu

STAT3 Is Required for Proliferation and Maintenance of Multipotency in Glioblastoma Stem Cells

Signal transducer and activator of transcription 3 (STAT3) regulates diverse cellular processes, including cell growth, differentiation, and apoptosis, and is frequently activated during tumorigenesis. Recently, putative glioblastoma stem cells (GBM‐SCs) were isolated and characterized. These cells can self‐renew indefinitely in culture, are highly tumorigenic, and retain the ability to differentiate in culture. We have found that treatment of GBM‐SCs with two chemically distinct small molecule inhibitors of STAT3 DNA‐binding inhibits cell proliferation and the formation of new neurospheres from single cells. Genetic knockdown of STAT3 using a short hairpin RNA also inhibits GBM‐SC proliferation and neurosphere formation, confirming that these effects are specific to STAT3. Although STAT3 inhibition can induce apoptosis in serum‐derived GBM cell lines, this effect was not observed in GBM‐SCs grown in stem cell medium. Markers of neural stem cell multipotency also decrease upon STAT3 inhibition, suggesting that STAT3 is required for maintenance of the stem‐like characteristics of these cells. Strikingly, even a transient inhibition of STAT3 leads to irreversible growth arrest and inhibition of neurosphere formation. These data suggest that STAT3 regulates the growth and self‐renewal of GBM‐SCs and is thus a potential target for cancer stem cell‐directed therapy of glioblastoma multiforme. STEM CELLS 2009;27:2383–2392

Targeting of melanoma brain metastases using engineered neural stem/progenitor cells

Brain metastases are an increasingly frequent and serious clinical problem for cancer patients, especially those with advanced melanoma. Given the extensive tropism of neural stem/progenitor cells (NSPCs) for pathological areas in the central nervous system, we expanded investigations to determine whether NSPCs could also target multiple sites of brain metastases in a syngeneic experimental melanoma model. Using cytosine deaminase-expressing NSPCs (CD-NSPCs) and systemic 5-fluorocytosine (5-FC) pro-drug administration, we explored their potential as a cell-based targeted drug delivery system to disseminated brain metastases. Our results indicate a strong tropism of NSPCs for intracerebral melanoma metastases. Furthermore, in our therapeutic paradigm, animals with established melanoma brain metastasis received intracranial implantation of CD-NSPCs followed by systemic 5-FC treatment, resulting in a significant (71%) reduction in tumor burden. These data provide proof of principle for the use of NSPCs for targeted delivery of therapeutic gene products to melanoma brain metastases.

The bystander effect exerted by tumor cells expressing the herpes simplex virus thymidine kinase (HSVtk) gene is dependent on connexin expression and cell communication via gap junctions

To elucidate the role gap junctions play in the bystander effect, we examined the cytotoxic effect of herpes simplex virus thymidine kinase (HSVtk) modified tumor cells on gap junction communication-deficient tumor cells and their connexin transfectants. Communication competent Walker 256 tumor cells engineered to express the HSVtk gene (Walker-tk+) when cocultured with N2A mouse neuroblastoma and PC12 rat pheochromocytoma cells with absent endogenous junctional conductance showed no bystander cytotoxicity. Transfection of N2A cells with the rat connexin37 gene (5Q) and PC12 cells with the human connexin43 gene rendered them susceptible to bystander cell death. Additionally, communication-deficient N2A cells transfected with the HSVtk gene failed to exert a bystander effect, whereas N2A transfectants coexpressing the connexin37 and HSVtk genes (5Qtk+ cells) exerted bystander cytotoxicity on gap junction communication-competent 5Q but not on communication-deficient N2A cells in vitro. In vivo experiments also showed tumor growth inhibition of communication-competent 5Q but not communication-incompetent N2A cells by 5Qtk+ cells. In conclusion, these results indicate that in several cellular environments the bystander effect is dependent on connexin expression and gap junctional communication between HSVtk-positive and HSVtk-negative cells.

Bystander tumoricidal effect in the treatment of experimental brain tumors.

The retrovirus-mediated transfer of the herpes simplex virus-thymidine kinase (HSV-tk) gene into tumor cells renders them sensitive to the cytocidal effect of the antiviral drug ganciclovir. This method has shown promising results as a treatment for experimental brain tumors. These experiments indicate that a major mechanism for the effectiveness of HSV-tk retroviral gene therapy may be the bystander tumoricidal effect. The bystander effect was hypothesized to explain tumor eradication, given that the efficacy of in vivo gene transfer to tumor cells was less than 100%. We demonstrate, in this report, that the bystander tumoricidal effect is a major contributor to the tumoricidal effect of ganciclovir in cell culture experiments using the mouse K1735 C19 cerebral melanoma line, thereby expanding the observation of the bystander phenomenon to a broader range of tumor types. The bystander effect was studied in vitro by coculturing wild-type C19 melanoma cells with HSV-tk-expressing C19 (C19-STK) cells. A maximal tumoricidal effect was seen when only 1 in 10 tumor cells expressed the HSV-tk gene. This suggests that in effect, 1 tumor cell with the HSV-tk gene, when given ganciclovir, will destroy 10 neighboring or bystander cells. The destruction of bystander cells does not appear to be mediated by a soluble factor(s) released into the media but, rather, requires close cell proximity or cell contact. In addition, HSV-tk-expressing C19 cells can exert an antitumoral effect not only on wild-type C19 cells but also on cells from a variety of different tumor cell lines, including a human glioblastoma multiforme cell line, indicating that the bystander effect is not a cell line-specific phenomenon. Finally, we observed that the bystander tumoricidal effect could be harnessed directly without using retrovirus-producing cells to increase survival in the mouse C19 brain tumor model. The potential implications of our findings in treating human brain tumors are discussed.

A non-invasive immobilization system and related quality assurance for dynamic intensity modulated radiation therapy of intracranial and head and neck disease.

PURPOSE To develop and implement a non-invasive immobilization system guided by a dedicated quality assurance (QA) program for dynamic intensity-modulated radiotherapy (IMRT) of intracranial and head and neck disease, with IMRT delivered using the NOMOS Corporations Peacock System and MIMiC collimator. METHODS AND MATERIALS Thermoplastic face masks are combined with cradle-shaped polyurethane foaming agents and a dedicated quality assurance program to create a customized headholder system (CHS). Plastic shrinkage was studied to understand its effect on immobilization. Fiducial points for computerized tomography (CT) are obtained by placing multiple dabs of barium paste on mask surfaces at intersections of laser projections used for patient positioning. Fiducial lines are drawn on the cradle along laser projections aligned with nasal surfaces. Lateral CT topograms are annotated with a crosshair indicating the origin of the treatment planning and delivery coordinate system, and with lines delineating the projections of superior-inferior field borders of the linear accelerators secondary collimators, or with those of the fully open MIMiC. Port films exposed with and without the MIMIC are compared to annotated topograms to measure positional variance (PV) in superior-inferior (SI), right-left (RL), and anterior posterior (AP) directions. MIMiC vane patterns superposed on port films are applied to verify planned patterns. A 12-patient study of PV was performed by analyzing positions of 10 anatomic points on repeat CT topograms, plotting histograms of PV, and determining average PV. RESULTS AND DISCUSSION A 1.5+/-0.3 mm SD shrinkage per 70 cm of thermoplastic was observed over 24 h. Average PV of 1.0+/-0.8, 1.2+/-1.1, and 1.3+/-0.8 mm were measured in SI, AP, and RL directions, respectively. Lateral port films exposed with and without the MIMiC showed PV of 0.2+/-1.3 and 0.8+/-2.2 mm in AP and SI directions. Vane patterns superimposed on port films consistently verified the planned patterns. CONCLUSION The CHS provided adequately reproducible immobilization for dynamic IMRT, and may be applicable to decrease PV for other cranial and head and neck external beam radiation therapy.

Connexins are expressed in primary brain tumors and enhance the bystander effect in gene therapy.

OBJECTIVE Experimental brain tumor gene therapy with the herpes simplex virus thymidine kinase (HSV-tk) gene has demonstrated that not only HSV-tk transduced but surrounding non-HSV-tk transduced cells are killed when given ganciclovir. This so-called bystander effect has recently been shown to be dependent on connexin-mediated intercellular communication. To assess potential susceptibility to the bystander effect, we examined levels of connexin-26 and connexin-43 expression in a series of primary brain tumors. Connexin-26 expression has not previously been studied in primary brain tumors and connexin-43 expression has not been studied in nonastrocytic primary brain tumors. We also attempted to enhance the bystander effect in vitro by overexpressing connexin in tumor cells with high basal levels of connexin expression. METHODS Western blot analysis and immunohistochemistry were used to determine levels of connexin-26 and connexin-43 expression in a series of primary brain tumors. Wild-type 9L gliosarcoma cells were transfected in vitro with the connexin-43 gene and the HSV-tk gene or the HSV-tk gene alone. The bystander effect of each transfectant was then assessed and compared. RESULTS Most of the primary brain tumors tested, including low-grade astrocytomas, anaplastic astrocytomas, glioblastomas, oligodendrogliomas, gangliogliomas, meningiomas, and medulloblastomas, showed connexin-26 and connexin-43 expression. Bystander experiments revealed a significant enhancement of the bystander effect in the gliosarcoma cells transfected with connexin-43 and HSV-tk, as compared with gliosarcoma cells transfected with HSV-tk alone. CONCLUSION Most primary brain tumors express connexin-26 and connexin-43. This suggests that most primary brain tumors may be susceptible to the bystander effect of HSV-tk gene therapy. The bystander effect can be enhanced in vitro by overexpression of connexin-43 in a cell line with a high basal level of connexin-43 expression.

Screening for loss of heterozygosity and microsatellite instability in oligodendrogliomas

To assess the frequency of loss of heterozygosity (LOH) and microsatellite instability (MI) in oligodendrogliomas, we performed an extensive screening of 16 oligodendrogliomas and nine anaplastic oligodendrogliomas by using 132 microsatellite markers on chromosomes 1 through 12 and 15 through 21. In total, 3,135 loci were examined in 25 tumor samples. Only 33/1,965 (1.7%) of oligodendroglioma (low‐grade) and 11/1,070 (1.0%) of anaplastic oligodendroglioma (high‐grade) loci exhibited MI. High‐frequency LOH regions were identified on chromosome arms 1p (31–73% for oligodendrogliomas and 60–100% for anaplastic oligodendrogliomas) and 19q (23–69% for oligodendrogliomas and 100% for anaplastic oligodendrogliomas). In addition, regions on chromosomes 4, 6, and 11 were found to be lost in 30–80% of both oligodendrogliomas and anaplastic oligodendrogliomas. Increased LOH frequency of chromosome 17 (38–40%) was found only in high‐grade oligodendrogliomas. The differences in LOH frequencies between low‐grade and high‐grade oligodendrogliomas in all loci combined and at three loci (D1S447, D1S226, and D1S252) on chromosome arm 1p were determined to be statistically significant [χ2(1) = 20.2, P < 0.0001, and Fishers exact test respective P values: 0.01, 0.03, and 0.02]. Our results provide evidence that microsatellite instability does not play an important role in the development of oligodendrogliomas. Furthermore, high LOH frequency on chromosomes 6 and 11 in addition to that identified previously on chromosomes 1, 19, and 4 suggests that multiple candidate tumor suppressor genes on these chromosomes may underlie the processes of initiation and/or progression in oligodendroglioma tumorigenesis. Genes Chromosomes Cancer 21:207–216, 1998.

The anterior cervical approach to the cervicothoracic junction.

To reach the upper thoracic vertebrae, a number of extensive approaches have been proposed combining thoracotomy, sternotomy, or clavicle resection with anterior dissection into the superior mediastinum. We present a simple anterior cervical approach for patients with disease limited to one vertebral level, in which midline ventral decompression is the goal of surgery. Regardless of the anterior approach used, the caudal extent of exposure is limited to T3 by the great vessels of the mediastinum, whereas the angle of the approach to the cervicothoracic junction is dictated by the manubrium. In the anterior cervical approach, lateral exposure to the uncovertebral joints is easily achieved. Five patients are reviewed in whom this anterior cervical approach was used at the first or second thoracic level. Decompression and instrumentation resulting in neurological improvement and axial stability were achieved in all five patients. The surgical anatomy of the cervicothoracic junction is reviewed with attention to the recurrent laryngeal nerves and the thoracic duct as they relate to the side of approach chosen.

Frequency of p53 Tumor Suppressor Gene Mutations in Human Primary Brain Tumors

Mutations in the p53 tumor suppressor gene are the most common genetic alterations found in diverse types of human cancer, including the primary malignant brain tumor, glioblastoma multiforme. To estimate the frequency of p53 mutations in human brain tumors, we screened 120 human primary brain tumors (59 astrocytic; 61 nonastrocytic) by the polymerase chain reaction-single-strand conformation polymorphism technique. Six astrocytic tumors (one anaplastic astrocytoma and five glioblastoma multiforme) were found to have putative p53 mutations. Direct sequencing of polymerase chain reaction-amplified deoxyribonucleic acid from these six tumors confirmed the presence of different point mutations in the conserved regions of the p53 gene. Allelic losses on chromosome 17p were detected in four (67%) of the six tumors with p53 mutations. p53 mutations were not detected in any of the 61 nonastrocytic brain tumors. Also, polymerase chain reaction-single-strand conformation polymorphism analysis of 74 leukocyte deoxyribonucleic acid samples from patients with astrocytic and nonastrocytic brain tumors failed to detect any germ-line p53 mutations. We conclude from these findings that p53 gene mutations in brain neoplasms are primarily limited to tumors of astrocytic origin and that the p53 gene mutations in sporadic astrocytomas are somatic in origin (i.e., nonprenatally determined).

Intermittent downbeat nystagmus due to vertebral artery compression

Downbeat nystagmus (DBN) uncommonly occurs as a transient phenomenon, and it rarely occurs in patients with cerebrovascular disease. We observed a patient with intermittent DBN and lightheadedness due to transient obstruction of his dominant vertebral artery when he turned his head to his left side. Surgical removal of an osteophyte at the site of the angiographically demonstrated lesion relieved his symptoms