A Study of Immunotherapy for Pediatric Malignancy : Molecular Identification of Tumor Specific Antigen

小儿恶性肿瘤免疫治疗的研究：肿瘤特异性抗原的分子鉴定

• 批准号: 15591118
• 负责人: HYAKUNA Nobuyuki
• 金额: $ 1.86万
• 依托单位: University of the Ryukyus
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591118/
• 关键词: Pediatric malignant tumor; Tumor antigen; Immunotherapy

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 … More 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

目的：需要获得大量的活肿瘤细胞来鉴定肿瘤抗原。因此，我们开始研究从新鲜切除的肿瘤样本中建立原代细胞培养物。此外，我们尝试从同一样本中扩增肿瘤浸润淋巴细胞。方法：从患者身上获取肿瘤样本用于诊断或治疗。将样品切成1mm立方体，用胶原酶消化，然后在37℃、5％Co_2的加湿培养箱中培养。培养1至2周后，当细胞增殖时，用胰蛋白酶处理传代细胞。培养细胞鉴定如下；对于神经母细胞瘤，：通过流式细胞术检测抗 GD2 抗体阳性，对于尤文肉瘤，：通过免疫组织化学检测抗 MIC2 抗体阳性，以及通过 RT-PCR 检测 EWS-F1i1 嵌合基因的表达。 结果：6 个神经母细胞瘤、2 个尤文肉瘤、1 个肾母细胞瘤样本用于研究。在所有标本中，原代培养均成功，细胞在 1 至 4 个月的时间内传代 3 至 4 次。在测试的 5 个神经母细胞瘤样本中，有 2 个细胞的 GD2 呈阴性，而在尤文肉瘤样本中，细胞的 MIC2 呈阳性。在原代组织中表达EWS-F1i1的尤文肉瘤细胞中未检测到EWS-Flit嵌合基因的表达。对于淋巴细胞培养，细胞仅从一份样本中增殖。将回收的淋巴细胞（肿瘤浸润淋巴细胞）与自体肿瘤细胞共培养，通过ELISA测定上清液中IFN-γ的分泌量。肿瘤浸润淋巴细胞的IFN-γ分泌量不高于对照。讨论：本研究表明从临床获得的肿瘤样本中进行原代细胞培养是成功的。然而，正常基质细胞的污染是不可避免的，并且在某些情况下，肿瘤相关抗原可能在传代过程中丢失。我们不能从肿瘤标本中诱导出任何肿瘤特异性淋巴细胞，这表明肿瘤浸润淋巴细胞可能不会被肿瘤抗原致敏。进一步的实验：在上述研究的基础上，我们处理了已建立的肿瘤细胞系（NB16）。简而言之，来自健康供体外周血的淋巴细胞在 NB16 细胞层上用 IL-2 进行扩增。 NB16细胞刺激3次后，ELISA法测定上清液中IFN-γ的分泌量。同样，IFN-γ 分泌量并不高于对照。这一结果可能是由于供体细胞与 NB16 之间的 HLA 不相容性、肿瘤抗原的低表达或无表达、或抑制供体针对 NB16 的免疫反应的某些未知机制引起的。较少的