Dendritic cell (DC)-based immunotherapy has been a promising strategy for colon cancer therapy, but the efficacy of dendritic cell vaccines is in part limited by immunogenicity of loaded antigens. In this study, we aimed to identify a putative tumor antigen that can generate or enhance anti-tumor immune responses against colon cancer. CD44⁺ colon cancer stem cells (CCSCs) were isolated from mouse colorectal carcinoma CT-26 cell cultures and induced to form defective ribosomal products-containing autophagosome-rich blebs (DRibbles) by treatment with rapamycin, bortezomib, and ammonium chloride. DRibbles were characterized by western blot and transmission electron microscopy. DCs generated from the mice bone marrow monocytes were cocultured with DRibbles, then surface markers of DCs were analyzed by flow cytometry. Meanwhile, the efficacy of DRibble-DCs was examined in vivo. Our results showed that CCSC-derived DRibbles upregulated CD80, CD86, major histocompatibility complex (MHC)-I, and MHC-II on DCs and induced proliferation of mouse splenic lymphocytes and CD8⁺ T cells. In a model of colorectal carcinoma using BALB/c mice with robust tumor growth and mortality, DC vaccine pulsed with CCSC-derived DRibbles suppressed tumor growth and extended survival. A lactate dehydrogenase test indicated a strong cytolytic activity of cytotoxic T-cells derived from mice vaccinated with CCSC-derived DRibbles against CT-26 cells. Furthermore, flow cytometry analyses showed that the percentages of IFN-γ-producing CD8⁺ T-cells were increased in SD-DC group compare with the other groups. These findings provide a rationale for novel immunotherapeutic anti-tumor approaches based on DRibbles derived from colon cancer stem cells.

中文翻译：

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癌症干细胞衍生DRibble疫苗对结直肠癌的治疗性抗肿瘤作用

基于树突状细胞 (DC) 的免疫疗法一直是结肠癌治疗的一种有前途的策略，但树突状细胞疫苗的功效部分受限于负载抗原的免疫原性。在这项研究中，我们旨在确定一种推定的肿瘤抗原，该抗原可以产生或增强针对结肠癌的抗肿瘤免疫反应。CD44 ⁺结肠癌干细胞 (CCSC) 从小鼠结肠直肠癌 CT-26 细胞培养物中分离出来，并通过雷帕霉素、硼替佐米和氯化铵处理诱导形成含有缺陷核糖体产物的富含自噬体的气泡 (DRibbles)。Dribbles 通过蛋白质印迹和透射电子显微镜进行表征。将小鼠骨髓单核细胞产生的 DCs 与 Dribbles 共培养，然后通过流式细胞术分析 DCs 的表面标志物。同时，在体内检测了 Dribble-DCs 的功效。我们的结果表明，CCSC 衍生的 DRibbles 上调 DCs 上的 CD80、CD86、主要组织相容性复合物 (MHC)-I 和 MHC-II，并诱导小鼠脾淋巴细胞和 CD8 ⁺增殖T细胞。在使用具有强劲肿瘤生长和死亡率的 BALB/c 小鼠的结肠直肠癌模型中，用 CCSC 衍生的 Dribbles 脉冲的 DC 疫苗抑制了肿瘤生长并延长了存活期。乳酸脱氢酶测试表明，来自接种了 CCSC 衍生的 DRibbles 的小鼠的细胞毒性 T 细胞对 CT-26 细胞具有很强的细胞溶解活性。此外，流式细胞术分析显示，与其他组相比，SD-DC 组产生 IFN-γ 的 CD8 ⁺ T 细胞的百分比增加。这些发现为基于结肠癌干细胞衍生的 Dribbles 的新型免疫治疗抗肿瘤方法提供了理论基础。