Increase in tumor size following intratumoral injection of immunostimulatory CpG-containing oligonucleotides in a rat glioma model
Increase in tumor size following intratumoral injection of immunostimulatory CpG-containing oligonucleotides in a rat glioma model
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DOI：
10.1007/s00262-009-0771-y
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发表时间：
2010-04-01
影响因子：
5.8
通讯作者：
Hamel, Wolfgang
中科院分区：
文献类型：
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作者：
Ginzkey, Christian;Eicker, Sven O.;Hamel, Wolfgang
恶性胶质瘤的免疫抑制环境可能抑制浸润性小胶质细胞和淋巴细胞的抗肿瘤活性。巨噬细胞和小胶质细胞可能被含有未甲基化cpg基序的寡核苷酸激活，尽管它们在癌症免疫治疗中的价值仍然存在争议。在正常大鼠脑内注射含cpg的寡核苷酸（ODN）后，我们观察到局部炎症反应，CD8+ T细胞浸润，MHC 2和ED1表达上调，证明了所使用的CpG-ODN的免疫原性。在免疫刺激序列（m-CpG）中发生突变的对照ODN中没有观察到这种情况。为了研究其在同基因肿瘤模型中的作用，我们将大鼠9L胶质肉瘤细胞植入Fisher 344大鼠纹状体。3天后，将免疫刺激性CpG-ODN、对照m-CpG-ODN或生理盐水立体定向注射到肿瘤中（第3天组）。另一组动物（第0天组），在植入前将CpG-ODN与9L细胞混合，第3天无需进一步处理。3周后处死，取脑、脾。出乎意料的是，用CpG-ODN治疗的一些动物（第0天和第3天组）的肿瘤比用生理盐水或m-CpG-ODN治疗的对照动物的肿瘤大。cpg - odn治疗动物的肿瘤大小比两组对照组变化更大。这与CpG治疗后在大多数肿瘤中观察到的炎症反应和坏死有关。然而，这并没有阻止cpg治疗动物体内实体瘤肿块的过度生长，与对照组动物相似。在对照组和cpg处理的动物的实体瘤肿块中观察到类似分枝小胶质细胞的致密浸润。在坏死区域（吞噬），ED1表达和巨噬细胞样形态提示小胶质细胞活化。在包括对照组动物在内的所有肿瘤中均检测到以CD8+ T细胞为主的密集淋巴细胞浸润和较少的NK细胞。作为T细胞或NK细胞活化标志物的穿孔蛋白的表达仅在所有治疗组的分离细胞上检测到。所有治疗组的肿瘤均显示CD25表达，表明T细胞可能维持对自身抗原的外周耐受性。体外再刺激淋巴细胞的细胞毒性T细胞试验（（51）铬释放试验）以及新鲜脾细胞产生的干扰素γ （Elispot试验）显示对9L细胞有特异性反应，但对另一种同基因细胞系（MADB 106腺癌）没有特异性反应。令人惊讶的是，与对照组相比，cpg - odn处理动物淋巴细胞的裂解率较低。在两种免疫试验中，个体动物的肿瘤大小与反应无关。综上所述，我们的数据支持CpG-ODN在正常大脑中的免疫刺激能力。然而，瘤内应用在大鼠胶质瘤模型中被证明是无效的。在神经胶质瘤患者中，CpG-ODN治疗可能不会产生有益效果。
The immunosuppressive environment of malignant gliomas is likely to suppress the anti-tumor activity of infiltrating microglial cells and lymphocytes. Macrophages and microglial cells may be activated by oligonucleotides containing unmethylated CpG-motifs, although their value in cancer immunotherapy has remained controversial. Following injection of CpG-containing oligonucleotides (ODN) into normal rat brain, we observed a local inflammatory response with CD8+ T cell infiltration, upregulation of MHC 2, and ED1 expression proving the immunogenic capacity of the CpG-ODN used. This was not observed with a control ODN mutated in the immunostimulatory sequence (m-CpG). To study their effect in a syngeneic tumor model, we implanted rat 9L gliosarcoma cells into the striatum of Fisher 344 rats. After 3 days, immunostimulatory CpG-ODN, control m-CpG-ODN, or saline was injected stereotactically into the tumors (day 3 group). In another group of animals (day 0 group), CpG-ODN were mixed with 9L cells prior to implantation without further treatment on day 3. After 3 weeks, the animals were killed and the brains and spleens were removed. Rather unexpectedly, the tumors in several of the animals treated with CpG-ODN (both day 0 and day 3 group) were larger than in saline or m-CpG-ODN treated control animals. The tumor size in CpG-ODN-treated animals was more variable than in both control groups. This was associated with inflammatory responses and necrosis which was observed in most tumors following CpG treatment. This, however, did not prevent excessive growth of solid tumor masses in the CpG-treated animals similar to the control-treated animals. Dense infiltration with microglial cells resembling ramified microglia was observed within the solid tumor masses of control- and CpG-treated animals. In necrotic areas (phagocytic), activation of microglial cells was suggested by ED1 expression and a more macrophage-like morphology. Dense lymphocytic infiltrates consisting predominantly of CD8+ T cells and fewer NK cells were detected in all tumors including the control-treated animals. Expression of perforin serving as a marker for T cell or NK cell activation was detected only on isolated cells in all treatment groups. Tumors of all treatment groups revealed CD25 expression indicating T cells presumed to maintain peripheral tolerance to self-antigens. Cytotoxic T cell assays with in vitro restimulated lymphocytes ((51)chromium release assay) as well as interferon-gamma production by fresh splenocytes (Elispot assay) revealed specific responses to 9L cells but not another syngeneic cell line (MADB 106 adenocarcinoma). Surprisingly, the lysis rates with lymphocytes from CpG-ODN-treated animals were lower compared to control-treated animals. The tumor size of individual animals did not correlate with the response in both immune assays. Taken together, our data support the immunostimulatory capacity of CpG-ODN in normal brain. However, intratumoral application proved ineffective in a rat glioma model. CpG-ODN treatment may not yield beneficial effects in glioma patients.