Department of Biomedical Engineering, University of Mississippi, University, Oxford, MS, USA.
Department of Chemical Engineering, University of Mississippi, Oxford, MS, USA.
Methods Mol Biol. 2022;2543:45-55. doi: 10.1007/978-1-0716-2553-8_5.
Apoptotic cells are cleared from the body principally through recognition and engulfment by neighboring phagocytes, a process known as efferocytosis. During efferocytosis, phagocytes are recruited to the site/activated by "find me" signals released from apoptotic cells, precisely identify apoptotic cells by the recognition of "eat me" signals on the apoptotic cell surface, and engulf the apoptotic cells to prevent secondary necrosis and inflammation. Thus, efferocytosis is critical for tissue homeostasis in normal physiology. However, efferocytosis of apoptotic tumor cells-performed by tumor-associated macrophages-suppresses immunity within the tumor microenvironment and limits the antitumor response. This phenomenon is further exacerbated in tumor residual disease because of the high apoptotic cell burden generated by cytotoxic therapies. Blocking efferocytosis could be a powerful approach to boost tumor immunogenicity, particularly as a combination approach with cytotoxic therapies that produce many apoptotic cells, but little is currently known about the immune response to efferocytosis. Moreover, there is a dearth of in vivo models available to study the immunologic and therapeutic consequences of blocking efferocytosis in tumor residual disease.Here, we describe a model that enables in vivo studies of tumor immunology in the aftermath of cytotoxic therapy with an emphasis on the impact of efferocytosis. Orthotopic HER2+ mammary tumors are established in immune-competent mice, followed by a single administration of lapatinib, a receptor tyrosine kinase inhibitor of HER2, to the mice that induces widespread, transient apoptosis in the tumor microenvironment. In the days following lapatinib treatment, agents that block efferocytosis such as BMS-777607 are administered. Tissue is collected from cohorts of mice at day 2 (after lapatinib treatment only) to assess apoptosis, day 8 (after lapatinib treatment followed by blockade of efferocytosis) to assess the immune response to apoptosis and efferocytosis, and day 28 (after 4 consecutive weeks of treatment) to assess therapeutic efficacy. This model enables mechanistic studies of tumor immunology in residual disease as well as therapeutic efficacy studies of targeted agents that disrupt efferocytosis.
凋亡细胞主要通过邻近的吞噬细胞的识别和吞噬来从体内清除，这个过程被称为吞噬作用。在吞噬作用过程中，吞噬细胞被凋亡细胞释放的“找我”信号募集到凋亡细胞所在部位/激活，通过识别凋亡细胞表面的“吃我”信号，准确识别凋亡细胞，并吞噬凋亡细胞，从而防止二次坏死和炎症。因此，吞噬作用对于正常生理中的组织稳态至关重要。然而，肿瘤相关巨噬细胞对凋亡肿瘤细胞的吞噬作用会抑制肿瘤微环境中的免疫反应，并限制抗肿瘤反应。由于细胞毒性疗法产生的高凋亡细胞负担，在肿瘤残留疾病中，这种现象进一步加剧。阻断吞噬作用可能是增强肿瘤免疫原性的一种有力方法，特别是作为与产生许多凋亡细胞但很少有免疫反应的细胞毒性疗法相结合的方法，但目前对吞噬作用的免疫反应知之甚少。此外，目前缺乏可用于研究阻断肿瘤残留疾病中吞噬作用的免疫和治疗后果的体内模型。在这里，我们描述了一种模型，该模型可用于研究细胞毒性治疗后肿瘤免疫学，重点关注吞噬作用的影响。在免疫功能正常的小鼠中建立 HER2+原位乳腺肿瘤，然后给小鼠单次给予曲妥珠单抗（HER2 的受体酪氨酸激酶抑制剂），导致肿瘤微环境中广泛、短暂的细胞凋亡。在曲妥珠单抗治疗后的几天内，给小鼠给予 BMS-777607 等阻断吞噬作用的药物。在第 2 天（仅在曲妥珠单抗治疗后）、第 8 天（在曲妥珠单抗治疗后阻断吞噬作用）和第 28 天（连续 4 周治疗后）从各组小鼠中收集组织，以评估凋亡情况、评估对凋亡和吞噬作用的免疫反应、评估治疗效果。该模型能够进行残留疾病中肿瘤免疫学的机制研究以及针对破坏吞噬作用的靶向药物的治疗效果研究。
