|Budget Amount *help
¥2,900,000 (Direct Cost : ¥2,900,000)
Fiscal Year 2004 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 2003 : ¥1,900,000 (Direct Cost : ¥1,900,000)
Purpose : It is needed to obtain a significant number of live tumor cells to identify the tumor antigens. Thus we started the study to establish primary cell culture from freshly excised tumor samples. Additionally, we try to expand tumor infiltrating lymphocytes from the same samples.
Method : Tumor samples were obtained from the patients for diagnosis or treatment. Sample specimens were cut into 1 mm cubic, and digested with collagenase, then they were cultured in humidified incubator with 5% Co_2 at 37℃ After 1 to 2 weeks culture when cells were proliferating, cells were passaged with trypsine treatment. Cultured cells were identified as follows ; for neroblastoma, : positivity for anti-GD2 antibody by flowcytometry, for Ewing sarcoma, : positivity for anti-MIC2 antibody by immunohistochemistry and expression of EWS-F1i1 chmeric gene by RT-PCR.
Results : Six neuroblastoma, 2 Ewing sarcoma, 1 Wilms tumor samples were subjected for the study. In all specimens primary culture was success
ful and cells were passaged 3 to 4 times for the period of 1 to 4 months. Cells were negative for GD2 in 2 out of 5 neuroblastoma samples tested, and cells were positive for MIC2 in Ewing sarcoma sample. Expression of EWS-Flit chimeric gene was not detected in in one Ewing sarcoma cells which expressed EWS-F1i1 in the primary tissue. For lymphocytes culture, cells proliferated from only one specimen. The recovered lymphocytes (tumor infiltrating lymphocytes) were co-cultured with autologous tumor cells and IFN-γ secretion in the supernatant was measured by ELISA The IFN-γ secretion by tumor infiltrating lymphocytes was not higher than control.
Discussion : This study showed that primary cell culture from clinically obtained tumor samples is successful. However, contamination of normal stroma cells was inevitable and in some cases tumor related antigen might be lost while on passage. We could not induce any tumor specific lymphocytes from tumor specimens, indicating tumor infiltrating lymphocytes might not be sensitized by tumor antigens.
Further experiments : On the basis of the above study, we dealt with established tumor cell lines (NB16). In brief, lymphocytes from healthy donor peripheral blood are expanded with IL-2 on the NB16 cell layer. After 3 times stimulation by NB16 cells, IFN-γ secretion in the supernatant was measured by ELISA. Again, the IFN-γ secretion was not higher than control. This results might be induced by HLA incompatibility between donor cells and NB16, low or no expression of tumor antigens, or some unknown mechanism that suppress the immune responses of donor against NB16. Less