Abstract Introduction: New therapies for glioblastoma (GBM) are urgently needed due to its poor prognosis and chimeric antigen receptor T (CAR-T) cell therapy is one of the promising strategies. CAR-T cell therapy targeting EGFRvIII,HER2, and IL13Rα2 have been tested for GBM, but have limited efficacies. To further develop CAR-T cell therapy, cell surface targets that is highly specific for GBM cells are needed. In this study, we applied this strategy to search for GBM-specific cell surface targets using patient derived tumor spheres. Methods: Patient derived tumor spheres was established from GBM surgical specimens, and immunized to Balb/c mice. The B cells from the mice were fused with myeloma cells, immortalized, and cultured nonclonally to obtain a number of monoclonal antibodies reacted GBM cells. The obtained antibodies were reacted with GBM cells and normal brain cells, and those that specifically react to GBM were selected by flowcytometry to obtain antibody candidates that could be specifically expressed on the surface of GBM cells. Then, we generated CAR-T cells derived from the obtained antibody and confirmed anti-tumor effect of CAR-T cells in vitro by ELISA. Results: Approximately 25,000 antibody-producing strains were generated. Within these candidates, we identified antibody X, which reacted with several GBM samples and does not reacted with several normal brain cells. The antibody X is a reported to be a cell adhesion molecule. The CAR-T cells derived from the antibodies X produced IL-2 and IFNγ significantly in co-culture with GBM cells. Conclusion: In this study, we identified antigen X and analyzed its anti-tumor effect in vitro. The antigen X has been reported to be associated with the progression of solid tumors and is expected to be a tumor-specific antigen. Further studies are necessary to examine whether the CAR-T cells targeting antigen X have anti-tumor effect in vivo.
Abstract For improvement of glioma therapy, advances in treatment after recurrence are essential, but no standard second line therapy has been estimated. Lately, bevacizumab (Bev) and BCNU wafer became available in Japan, we have more therapeutic choices. Since we took part in Kansai molecular diagnosis network for CNS tumors in 2013, we have referred molecular information. We retrospectively examined and report our experiment about recurrent malignant gliomas in Kansai Rossi Hospital. Twenty-two histopathologically proved grade 3 and grade 4 patients who were diagnosed as recurrence between January 2013 and December 2018 were included. We examined treatment and analyzed factors that influenced overall survival (OS) after recurrence. Glioblastoma were 14 cases (IDH wild 12 cases, IDH mutant one case, unknown one case) Median age and KPS at recurrence were 70 years old and 60%, respectively. Ten patients received any anti-cancer treatment and 2 received best supportive care (BSC). Radiation therapy (RT) with Bev were used in 9 patients (5 with gamma knife). Their median OS after first recurrence was 324 days and significantly longer than that of BSC patients (p=0.00174). Grade 3 gliomas were 8 cases (IDH-mutant 3 cases, IDHwild 5 cases, H2F3A mutant 1 case). Median age and KPS at recurrence were 45 years old and 70%, respectively. Five patients were treated with temozolomide (TMZ) and others were observed. In addition to TMZ, Bev were used for 4 patients and RT for 3. Median OS of these patients was significantly longer (p=0.0198). In both grade, patients with better KPS (>60%) statistically lived longer than poor KPS, but methylation status of MGMT promoter and IDH mutation did not influent their OS. Radiation with Bev for good KPS patients might improve prognosis. Further multicenter prospective study must be needed.
Abstract BACKGROUND Malignant brain tumors are frequently resistant to current treatments and have a poor prognosis. Many malignant brain tumors invade the surrounding brain tissue and/or show leptomeningeal metastasis when they recur. Thus, elucidating the mechanisms of invasion and leptomeningeal metastasis is important to improve the prognosis of malignant brain tumors. In this study, we comprehensively analyzed the mechanisms of invasion and leptomeningeal metastasis of malignant brain tumors using mouse xenograft models. METHODS Patient derived glioblastoma cells (GDC519) and breast cancer cell line, MDA-MB-231, were labeled with GFP and luciferase. GDC519 were transplanted into the right cerebral cortex of immunodeficient mice and when the mice got tumors, the right and left cerebrum were dissected separately. We sorted tumor cells by flow cytometry and extracted RNA from tumor cells in right and left cerebrum, respectively. We performed RNA sequencing and analyzed the transcriptome differences. On the other hand, MDA-MB-231 cells were injected into the right ventricle of immunodeficient mice. Tumor cells from cerebrospinal fluid, and spinal cords were sorted and analyzed the transcriptome differences by RNA sequencing. RESULTS In the model in which GDC519 was transplanted, we found significant differences in expressions of extracellular matrix related proteins, cell motility related proteins, and cell adhesion related proteins between tumor cells in right and left cerebrum. In addition, in the model in which MDA-MB-231 was transplanted into the right ventricle, we found significant differences in expressions of extracellular matrix related proteins and PI3K-Akt signaling pathway related proteins between tumor cells from cerebrospinal fluid and spinal cord. CONCLUSION These results suggest that it would be possible to identify molecules involved in the invasion and leptomeningeal metastasis of malignant brain tumors using a mouse xenograft model. Further studies are necessary to examine the functional role of these genes in invasion and leptomeningeal metastasis.
Abstract INTRODUCTION Recently, CAR-T cell (chimertic antigen receptor T-cell) therapy has been attracting and has shown high therapeutic efficacy, especially in the hematological tumors. In the brain, EGFRv3 or HER2 have been used as antigens, but have yet to show positive results. It is necessary to find antibodies that are more specific and have a therapeutic effect. METHODS A primary culture line was prepared from glioblastoma surgical specimens, and immunized to Balb/c mice with it. The B cells from the mice were fused with myeloma cells, immortalized, and cultured nonclonally to obtain a number of monoclonal antibodies reacted glioblastoma cells. The obtained antibodies were reacted with glioblastoma cells and normal brain cells, and those that specifically react to glioblastoma were selected by flowcytometry to obtain antibody candidates that could be specifically expressed on the surface of glioblastoma cells. Then, we generated CAR-T cells derived from the obtained antibody and confirmed anti-tumor effect of CAR-T cells in vitro and in vivo. RESULTS Approximately 25,000 antibody-producing strains were generated. From these, we selected the antibody, which reacted with several glioblastomas and did not react with several normal brain cells. Finally, we identified the antibody as Prostaglandin F2 receptor negative regulator (PTGFRN) by using expression cloning. CAR-T cells derived from PTGFRN produced cytokines and exerted cytotoxicity upon co-culture with tumor cells from patients with GBM. Furthermore, intracranial injection of 5E17-CAR-T cells demonstrated antitumor effects in an orthotopic xenograft murine model with patient-derived GBM cells. DISCUSSION In this study, we identified PTGFRN as a tumor-specific surface antigen and confirmed its antitumor effect in vitro and in vivo. PTGFRN is involved in the control of cell proliferation, migration, invasion, cell cycle, and apoptosis, and is expressed in multiple cancers. Cell surface PTGFRN is a candidate target for intracranial CAR-T-cell therapy for GBM.
Background In the reconstruction of the superior sagittal sinus or transverse sinus, it is desirable to place a large-diameter guiding catheter into the transverse sinus to introduce the stent delivery system smoothly. The utility of an anchoring technique with a percutaneous transluminal angioplasty (PTA) balloon for navigating an 8 F guiding catheter into the transverse sinus is demonstrated. Case Descriptions: Two dural arteriovenous fistula (dAVF) cases (Cognard type II a +b, Borden type II) that underwent sinus stenting are presented. In both cases, when the 8 F guiding catheter was placed in the jugular vein, the stent delivery system could not enter the transverse sinus because it could not pass through the transverse-sigmoid sinus junction. Introduction of an 8 F guiding catheter into the transverse sinus was attempted but failed. An 8-mm or 9-mm PTA balloon was used as a distal anchor, and this technique allowed easier guiding of catheter advancement into the transverse sinus. In both cases, Carotid WALLSTENTS were placed in the sinus easily, with no complications. Conclusion Balloon anchoring in the venous system is useful for achieving large-caliber catheter access across difficult anatomy and is technically feasible.
A case of primary dermoid cyst of the urinary bladder in a 29 years olds was reported.The tumor was pediculated and completely localized within the bladder.Histologically, it was shown to be a dermoid cyst of the urinary bladder composed mainly of skin-like structure with cartilage, sweat gland, adipose gland and hair.Left ovarium adhered to the bladder wall but did not infiltrate.So this tumor was thought to be a primary dermoid cyst of the bladder.The authors collected 59 cases of bladder dermoid cysts from Japanese literatures and analysed them statistically.
Abstract Introduction Ependymoma and medulloblastoma are difficult to diagnose by imaging and histology. Since the treatment strategies differ between the two, an accurate and prompt diagnosis is required. Raman spectroscopy can measure tissue-specific molecular vibrations. It shows extract biomolecular information characteristic of tumor tissue. We observed the tissues of ependymoma and medulloblastoma by Raman spectroscopy, and attempted to establish a new differentiation method. Methods The subjects were 13 patients with ependymoma and 5 patients with medulloblastoma who underwent resection from July 2017 to June 2023 at our institution. Their formalin-fixed paraffin-embedded (FFPE) section were observed by Raman spectroscopy. Raman spectra were collected using a line-illumination Raman microscopy developed at the department of Applied Physics, Osaka University. 532 nm excitation light was focused on the sample by an objective lens (Nikon 25x, NA1.1, CFI75). Excitation intensity and measurement time were 3 (mW/um2 ) and 5 seconds, respectively. Results We observed a Raman peak at 1240 cm-1 (Amide II3) that was significantly different in both ependymoma and medulloblastoma, and a similar tendency was observed for each specimen. It can suggest differences in amino acids, proteins, and lipids within tumor tissue. Discussion and conclusion We investigated whether it is possible to make a differential diagnosis between ependymoma and medulloblastoma using Raman spectroscopy. It was suggested that we can detect the molecular difference between the two using FFPE sections. Raman spectroscopy is a label-free measurement technique that does not require staining of biological samples, allowing simple and rapid evaluation. In the future, we plan to verify the validity of the differential method using specimens collected during surgery and frozen specimens.
One of the merits of recently introduced exoscopes, including ORBEYE, is that they are superior to a conventional microscope in terms of ergonomic features. Taking advantage of it, the retrosigmoid approach can be performed in the supine position using ORBEYE. We report a consecutive series of 14 operations through the retrosigmoid approach in the supine position using ORBEYE. Fourteen consecutive patients who underwent surgery through the retrosigmoid approach for cerebellopontine (CP) angle lesions in the supine position using ORBEYE were targeted, and surgical outcomes and complications were examined. We evaluated the posture of the operator and the surgical field during this approach compared with those using a conventional microscope. In all 14 cases, all operative procedures were accomplished only using the ORBEYE. There were no operative complications due to this approach. Using ORBEYE, even when the angle of the operative visual axis was horizontal, the operators could manipulate in a comfortable posture. They were not forced to be in an uncomfortable posture that extended their arms, as is often the case with a conventional microscope. Therefore, they could use shorter surgical instruments. As the cerebellum shifted downward with gravity even using slight retraction during this approach, the working space of the surgical field was easily secured. Through this approach, the operators can perform stable microsurgery of CP angle lesions in a comfortable posture. This approach can reduce the burden on the operator and the patient, leading to a refined surgical procedure.
