Yoshihiro Numa

Morphological and flow cytometric analysis of cell infiltration in glioblastoma: a comparison of autopsy brain and neuroimaging

Even when we successfully perform a total extirpation of glioblastoma macroscopically, we often encounter tumor recurrence. We examined seven autopsy brains, focusing on tumor cell infiltration in the peripheral zone of a tumor, and compared our findings with the MR images. There has so far been no report regarding mapping of tumor cell infiltration and DNA histogram by flow cytometry, comparing the neuroimaging findings with the autopsy brain findings. The autopsy brain was cut in 10-mm-thick slices, in parallel with the OM line. Tissue samples were obtained from several parts in the peripheral zone (the outer area adjacent to the tumor edge as defined by postcontrast MRI) and then were examined by H&E, GFAP, and VEGF staining. We defined three infiltrating patterns based on number of infiltrated cells as follows: A zone, 100%–60% of the cells infiltrated tumor cells compared with tumor cell density of the tumor mass; B zone, 60%–20%; C zone, 20%–0%. In the autopsy brain, the tumor was easily identified macroscopically. We found that (1) the tumor cells infiltrated the peritumoral area; and (2) tumor cell infiltration was detected over an area measuring from 6 to 14 mm from the tumor border in the A zone. When performing surgery on glioblastoma, a macroscopic total extirpation of the tumor as defined by the contrast-enhanced area in MRI is therefore considered to be insufficient for successfully reducing tumor recurrence.

Flow cytometric analysis of antineoplastic effects of interferon-α, β and γ labelled with fluorescein isothiocyanate on cultured brain tumors

SummaryAntineoplastic effects of interferons (IFNs) on brain tumors have often been reported in the literature, however, so far as we know, there are no reports of the study on the antineoplastic effect of IFNs (α, β, and γ) labelled with fluorescein isothiocyanate (FITC) using flow cytometry (FCM). Three established glioma cell lines and 11 cultured cells of brain tumor from surgical specimens were exposed to IFN-α, β, and γ at the concentrations of 102–105 IU/ml for 24 h, respectively. Using FCM, the viability of the cells was evaluated with fluorescein diacetate stain and the cell cycle was analyzed from the DNA-histogram with propidium iodide stain. Furthermore, FITC-labelled IFN-α, β and γ were also contacted with each cell to calculate respective positive cells. The viability decreased about 60% on day 1 and day 3, indicating the effect of IFN-α and β on U373MG cells and on some cultured glioma cells from surgical materials, whereas, IFN-γ had no effects. Antineoplastic effect of each IFN well correlated with FITC-positive rates, demonstrating S phase block in the cell cycle. IFN-γ had no antineoplastic effects, whereas IFN-α and β showed antineoplastic effects, which fact suggested that IFN-γ receptor be different from those of IFN-α and β. The method of FITC-labelling for IFNs with the aid of FCM has the advantages as follows: 1) Antineoplasticity of IFN can be simply evaluated with FCM; 2) It is easy to analyze the action mechanism of IFN; 3) Information on the receptor is obtainable; and 4) Sensitivity can be evaluated prior to administration of IFN, suggesting possibilities of clinical application of this method.

Abstracts of Oral Presentations

S OF ORAL PRESENTATIONS

Flow cytometric study on cell kinetics of brain tumours and their cultured cells

SummaryWith the aid of flow cytometry (FCM), distribution of DNA content in 40 cases of brain tumour, primary culture cell, and secondary culture cell can be determined and chronological change after subculture is studied from the analysis of their cell cycle. In most primary cultures, proliferating index (PI) is likely to decrease, which suggests that environmental change might affect the growth activity. In comparison with that of the original sample, DNA-histogram of the secondary culture can be divided into the following 3 types: the type recovering to the original pattern (“adapting type”), in which astrocytoma, ependymoma, glioblastoma and medulloblastoma are included, 2) the type increasing more at G2 + M phase than the original (“proliferating type”), in which meningioma and some of glioblastoma are included, and 3) the type decreasing so far as to induce degeneration or death (“degenerating type”), in which pituitary adenoma and neurinoma are included. FCM is of great usefulness for the study of cell kinetics of a tumour cell undergoing culture and the present method will be available for the respective study of biological characteristics of the cultured cell, established cell line or sensitivity test for antineoplastic agents.

Flow cytometric analysis for the mechanism of the new antineoplastic agent temozolomide in glioma cells

Temozolomide (TMZ) has been accepted as a standard antitumor drug for glioma worldwide. Regarding its mechanism of action, there are quite a few analyses. In the present study, we investigated the cell-killing effect and mechanism of action of TMZ with flow cytometry using glioblastoma cell lines. Each cell line was divided into three groups: a control group, a low-dose TMZ group, and a highdose TMZ group. On day 1, TMZ was added to each cell line. Then, we counted the numbers of cells on days 2, 3, 4, and 5; in U87MG, we counted the number of cells on days 8 and 9. Simultaneously, we performed flow cytometric analysis with single- and double-staining methods. Although results varied slightly depending on the cell line, with flow cytometric analysis we identified the G0G1-, S-phase block on days 2 through 4, at the beginning of TMZ administration. After that we identified the deviation of the G2M block over days 3 to 5. Dominant morphological changes observed in U87MG were confined to the nuclei, with positive TUNEL staining. Early S-phase block and then a G2M block were observed; consecutively, we could analyze these blocks with a double-staining method more clearly. The flow cytometric method is very effective in the analysis of the antitumor mechanism of each agent. On the basis of our analysis, more effective combined chemotherapy may be expected.

Frontozygomatic approach to intraorbital tumors.

We removed 12 intraorbital tumors (5 schwannomas, 3 meningiomas, 2 cavernomas, 1 pleomorphic adenoma, and 1 neuroblastoma) using the frontozygomatic approach. No patients died. Postoperatively, 1 patient developed transient ptosis, and 3 patients had mild enophthalmos. Two patients with a meningioma developed transient worsening of their visual acuity and visual field. The frontozygomatic approach for surgical treatment of intraorbital tumors provides a wide visual field exposing the entire optic nerve. This approach is indicated for large intraorbital tumors, tumors affecting the optic nerve or orbital apex, intraorbital tumors that have extended into the intracranial cavity, and intracranial tumors that have extended into the orbit. The operative procedure for intraorbital tumor is determined by the location of the lesion and by the direction of its growth. The procedure is applicable to all intraorbital tumors. It reduces discomfort for surgeons while providing a relatively wide surgical field.

Reversible Porencephaly Associated with Shunt Malfunction

A patient is presented who was surgically treated for lumbosacral meningocele found at birth, followed by ventriculoperitoneal (V-P) shunting. At age 10 years, she was noticed to have a swelling on the scalp. Subsequent MR imaging revealed a cavity along the ventricular catheter which was thought to be porencephaly. No neurological symptoms appeared, but the V-P shunting was revised because of suspected shunting malfunction. During serial observation of her clinical state, the paraventricular cystic cavity was radiologically confirmed to be reduced in size. Different from the irreversible porencephalic cyst associated with ventricular puncture or ventricular drainage, the present cystic cavity was supposed to have been induced by an accumulation of cerebrospinal fluid (CSF) resulting from CSF leakage from the ventricular side along the catheter due to malfunctioning shunting, and, therefore, was considered to be reversible. MR imaging was helpful for the analysis of the process of disease in the present patient. (Shoni no Noshinkei 14: 365–368, 1989)

Sterile sorting of Leu-lla-positive cells (NK cells) using flow cytometry and the antineoplastic effects on brain tumors

Though we recently found some reports on sterile sorting of natural killer cells (NK cells) by flow cytometer (FCM), which manifested cytotoxicity, no reports have concerned antineoplasticity of sterile NK cells on glioma cell lines and cultured surgical materials of brain tumor so far as we know. Monocytes were sampled from the peripheral blood of healthy adults, stained with fluorescein isothiocyanate (FITC)-labeled antihuman monoclonal antibodies, and sorted by an FCM, which had been sterilized with 0.5% Hibitane alcohol solution. Over 95% of the cells obtained were NK cells, and their viability was disclosed to be 97% by the FDA staining. Using thus obtained NK cells, the antineoplastic effects were evaluated in 3 kinds of glioma cell lines and 5 surgical specimens by the microcytotoxicity test. The effects varied widely from 9 to 74% (E/T ratio 40) for glioma cell lines, and from 1 to 66% (E/T ratio 10-40) for surgical specimens. It is expected in the future to apply NK cells clinically using this sterile sorting technique.

Interleukin-5 and interleukin-10 are produced in central nervous system tumor cysts

Interleukin-5 and interleukin-10, as important mediators of vascular permeability, contribute to the development of various pathologic effusions. However, little is known regarding the involvement of these two cytokines in the formation of cysts associated with central nervous system (CNS) tumors. Twenty-eight patients with various cystic CNS tumors were investigated for expression of interleukin-5 and interleukin-10 in cyst fluid and their matched cytokine receptors in tumor tissue. Interleukin-5 and interleukin-10 were detected in cyst fluid, and interleukin-5 concentration was significantly correlated with interleukin-10 concentration (r=0.508, p=0.006). Moreover, both receptors were also detectable in the tumor tissue specimens and high levels of expression were also found in perivascular cells. Therefore, the local production of interleukin-5 and interleukin-10 might be implicated in some types of cyst formation.

Research the Mechanism of Various Antineoplastic Agents with Use of Flow Cytometry in Vitro Glioma Cells

Nowadays, steady progress like in endovascular, microsurgical, neuroendoscopic fields, affect deeply neurosurgergical field, too. But in spite of arising so many innovations, average survival time in glioma is a year and five year survival rate is 8%. These results have not slightly changed for 30 years.(*1) For us to continue research glioma in future, our purpose discloses what we should be going to do for improve prognosis and needs to analyze data about tumor cells in the various views. In our laboratory, we research brain tumor with flow cytometory. In this chapter, we describe how to analyze the mechanism of various antineoplastic agents for tumor cells centered glioma and research results with flow cytometry.