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In vitro and animal experiments have demonstrated the potential efficacy of using the bystander effect alone in the treatment of brain tumors. A known problem in some in vitro and in vivo experiments is that a fraction of cells engineered to express the herpes simplex virus thymidine kinase (HSV-tk) gene survive ganciclovir (GCV) treatment and undergo cell division. To prevent the recurrent growth of HSV-tk+ cells in the presence of GCV we examined the potential use of lethal or sublethal irradiation of Walker 256 carcinosarcoma cells selected for expression of the HSV-tk gene (Walker-tk+). Western blot analysis of Walter-tk+ cells showed similar levels of HSV-tk protein expression at 0, 1, 3, 6 and 9 days after lethal gamma-irradiation. In vitro, there was no difference in the bystander effect exerted by non-irradiated, sublethally irradiated or lethally irradiated Walker-tk+ cells on wild-type Walker cells in the presence of GCV. In vivo experiments demonstrated long-term survival (100 days) in rats implanted intrathecally with sublethally or lethally irradiated Walker-tk+ cells with GCV treatments. Intrathecal implantation of irradiated Walker-tk+ cells either pre-mixed with Walker cells or used in in situ treatment of established Walker tumors resulted in prolonged animal survival compared to controls (p < 0.05). These experiments suggest that the bystander tumoricidal effect is preserved despite gamma-irradiation of the HSV-tk modified tumor cells and that irradiation could be an effective method to prevent long-term resistance to GCV in HSV-tk+ tumor cells.

Objective: Benign tumors involving cavernous sinus, trigeminal nerve, and middle cranial fossa occasionally extend to the infratemporal fossa (ITF). In this study, we describe the microsurgical anatomy and dissection of the ITF, as viewed laterally and superiorly. We also describe a new bypass graft to the supraclinoid internal carotid artery using the internal maxillary artery (IMA), which is found in the ITF., Methods: Twelve cadaver specimens were used. Dissection required zygomatic arch osteotomy, downward displacement of the temporalis muscle, extensive subtemporal craniectomy, and mild elevation of the temporal lobe together with the dura., Results: The anatomic relationships between the lateral and medial pterygoid muscles and the neurovascular bundle of the ITF are demonstrated. The neurovascular bundle contains the IMA, which runs horizontally, and the main branches of the mandibular nerve, which run vertically. The course and anatomic variations of the IMA and inferior alveolar, lingual, auriculotemporal, and buccal nerves are shown. The distal IMA was quite tortuous and, when the artery straightened, we were able to perform a tension-free in situ IMA graft to the supraclinoid carotid artery in 9 of 12 specimens., Conclusions: Knowledge of the anatomy of the ITF is a prerequisite for tumor resection in this area. The IMA may serve as a bypass graft to the supraclinoid internal carotid artery if the cavernous or petrous carotid artery is involved by tumor and needs to be sacrificed.

A promising strategy in the treatment of neoplastic meningitis involves the use of herpes simplex virus-thymidine kinase (HSV-tk)-modified cells. In these experiments the authors used cells expressing HSV-tk to treat meningeal carcinomatosis in the rat Walker 256 model. Intrathecal injection of 2 x 10(5) Walker cells resulted in a median survival time of 15 days. Up to 80% of animals implanted with HSV-tk-modified Walker cells (Walker-tk+) and treated with ganciclovir showed long-term survival (120 days or more), whereas the remaining animals died from tumor growth between 37 and 44 days after implantation. Tumor cells from an animal in which the treatment failed were cultured in vitro and were shown to be still sensitive to ganciclovir. However, continuous ganciclovir administration for 6 weeks rather than 2 weeks did not improve survival. Histopathological studies confirmed leptomeningeal infiltration in the untreated Walker or Walker-tk+ animals. Walker-tk+ cells were mixed with Walker cells in 1:1, 10:1, or 50:1 ratios, respectively, and implanted intrathecally; the animals were treated with ganciclovir. All groups of treated animals had long-term survivors, with 40% of the rats in the 10:1 and 50:1 groups demonstrating long-term survival and absence of microscopic tumors in the brain or spinal cord. Similarly, murine fibroblast HSV-tk virus-producer cells improved survival. Walker-tk+ cells were better than fibroblast-producer cells in improving the survival of animals with Walker tumors at low (1:1) but not at high (10:1) effector-to-target cell ratios. Repeated intrathecal administration of Walker-tk+ cells resulted in inhibition of established Walker tumors. The authors conclude that Walker-tk+ cells are at least as effective as murine virus-producer cells and could be used in the treatment of meningeal neoplasia.

Retrovirus-mediated herpes simplex virus-thymidine kinase (HSV-tk) gene therapy is a promising approach in the treatment of brain tumors. Previous in vitro and in vivo studies have demonstrated a bystander effect in which nonmodified tumor cells in proximity to HSV-tk-modified tumor cells are killed with the modified cells in the presence of ganciclovir. In the present study the authors assessed the contribution of infectious HSV-tk retrovirus made by producer cells to the bystander cytocidal effect in tissue culture using Walker 256 rat breast carcinosarcoma cells, which represent an established model for carcinomatous meningitis. The authors observed ganciclovir-dependent growth inhibition even when only one HSV-tk-positive Walker cell was mixed with 1000 HSV-tk-negative Walker cells and showed that the bystander cytocidal effect is not mediated by toxic cell lysis products. Walker cells engineered to produce HSV-tk retrovirus with titers ranging from 10(3) to 10(5) colony-forming units/ml exert no greater cytocidal effect than nonviral producer HSV-tk-positive Walker cells in vitro. Murine fibroblast-producer cells with viral titers ranging from 10(6) to 10(7) colony-forming units/ml exerted a stronger cytocidal effect than nonviral producer HSV-tk-positive murine fibroblasts. Despite the high viral titers of fibroblast producer cells, HSV-tk-modified Walker cells performed better than fibroblast producer cells in their cytotoxic effect on wild-type Walker tumor cells. Given that HSV-tk-modified tumor cells can become ganciclovir resistant, we tested gamma-irradiation as a means to overcome resistance. Lethal gamma-irradiation of the HSV-tk-positive Walker cells did not abolish their bystander effect on Walker HSV-tk-negative cells. One can infer from these results that HSV-tk-modified tumor cells, irradiated or not, may be a better alternative to murine fibroblast producer cells in the treatment of central nervous system neoplasia.

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.