Department of Medicine III, School of Medicine, Technical University of Munich; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich;
Department of Medicine III, School of Medicine, Technical University of Munich; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich; Department of Internal Medicine III, University Hospital Regensburg; National Centre for Tumor Diseases WERA.
J Vis Exp. 2021 Sep 23(175). doi: 10.3791/62811.
Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.
肿瘤的免疫原性细胞死亡，由化疗或放疗引起，可以通过释放危险相关分子模式和诱导 I 型干扰素的产生，触发肿瘤特异性 T 细胞反应。免疫疗法，包括检查点抑制，主要依赖于预先存在的肿瘤特异性 T 细胞来发挥治疗作用。因此，利用免疫原性细胞死亡作为内在的抗癌疫苗的协同治疗方法可能会提高其反应性。然而，细胞在治疗诱导的应激下释放的免疫原性因子的范围仍然不完全清楚，特别是关于细胞外囊泡 (EVs)。EVs 是从几乎所有细胞中释放的纳米级膜性颗粒，被认为促进了细胞间的通讯，并且在癌症中已经被证明可以介导针对肿瘤抗原的交叉引发。为了评估各种条件下肿瘤来源的 EVs 的免疫原性效应，需要寻求适应性强、可扩展和有效的方法。因此，本文提出了一种相对简单和稳健的方法来评估 EVs 的体内免疫原性。该方案基于用 EVs 对小鼠进行体内免疫接种后，通过沉淀法从肿瘤细胞培养物中分离 EVs，然后对脾 T 细胞进行流式细胞术分析。例如，这项工作表明，奥沙利铂暴露于 B16-OVA 小鼠黑色素瘤细胞会导致释放出能够介导肿瘤反应性细胞毒性 T 细胞激活的免疫原性 EVs。因此，通过体内免疫接种和流式细胞术筛选 EVs，可以确定出现免疫原性 EVs 的条件。鉴定免疫原性 EV 释放的条件为测试 EV 对癌症的治疗效果并探索潜在的分子机制提供了必要的前提，最终揭示 EV 在癌症免疫学中的作用的新见解。
