PDF(Pubmed)
Abstract：
BACKGROUND: The combination of radiotherapy and immunotherapy (immunoradiotherapy) has been increasingly used for treating a wide range of cancers. However, some tumors are resistant to immunoradiotherapy. We have previously shown that MER proto-oncogene tyrosine kinase (MerTK) expressed on macrophages mediates resistance to immunoradiotherapy. We therefore sought to develop therapeutics that can mitigate the negative impact of MerTK. We designed and developed a MerTK specific antisense oligonucleotide (ASO) and characterized its effects on eliciting an anti-tumor immune response in mice.
METHODS: 344SQR cells were injected into the right legs on day 0 and the left legs on day 4 of 8-12 weeks old female 129sv/ev mice to establish primary and secondary tumors, respectively. Radiation at a dose of 12 Gy was given to the primary tumors on days 8, 9, and 10. Mice received either anti-PD-1, anti-CTLA-4 or/and MerTK ASO starting from day 1 post tumor implantation. The composition of the tumor microenvironment and the level of MerTK on macrophages in the tumor were evaluted by flow cytometry. The expression of immune-related genes was investigated with NanoString. Lastly, the impact of MerTK ASO on the structure of the eye was histologically evaluated.
RESULTS: Remarkably, the addition of MerTK ASO to XRT+anti-PD1 and XRT+anti-CTLA4 profoundly slowed the growth of both primary and secondary tumors and significantly extended survival. The ASO significantly reduced the expression of MerTK in tumor-associated macrophages (TAMs), reprograming their phenotype from M2 to M1. In addition, MerTK ASO increased the percentage of Granzyme B+ CD8+ T cells in the secondary tumors when combined with XRT+anti-CTLA4. NanoString results demonstrated that the MerTK ASO favorably modulated immune-related genes for promoting antitumor immune response in secondary tumors. Importantly, histological analysis of eye tissues demonstrated that unlike small molecules, the MerTK ASO did not produce any detectable pathology in the eyes.
CONCLUSIONS: The MerTK ASO can significantly downregulate the expression of MerTK on TAMs, thereby promoting antitumor immune response. The combination of MerTK ASO with immunoradiotherapy can safely and significantly slow tumor growth and improve survival.
METHODS: 344SQR cells were injected into the right legs on day 0 and the left legs on day 4 of 8-12 weeks old female 129sv/ev mice to establish primary and secondary tumors, respectively. Radiation at a dose of 12 Gy was given to the primary tumors on days 8, 9, and 10. Mice received either anti-PD-1, anti-CTLA-4 or/and MerTK ASO starting from day 1 post tumor implantation. The composition of the tumor microenvironment and the level of MerTK on macrophages in the tumor were evaluted by flow cytometry. The expression of immune-related genes was investigated with NanoString. Lastly, the impact of MerTK ASO on the structure of the eye was histologically evaluated.
RESULTS: Remarkably, the addition of MerTK ASO to XRT+anti-PD1 and XRT+anti-CTLA4 profoundly slowed the growth of both primary and secondary tumors and significantly extended survival. The ASO significantly reduced the expression of MerTK in tumor-associated macrophages (TAMs), reprograming their phenotype from M2 to M1. In addition, MerTK ASO increased the percentage of Granzyme B+ CD8+ T cells in the secondary tumors when combined with XRT+anti-CTLA4. NanoString results demonstrated that the MerTK ASO favorably modulated immune-related genes for promoting antitumor immune response in secondary tumors. Importantly, histological analysis of eye tissues demonstrated that unlike small molecules, the MerTK ASO did not produce any detectable pathology in the eyes.
CONCLUSIONS: The MerTK ASO can significantly downregulate the expression of MerTK on TAMs, thereby promoting antitumor immune response. The combination of MerTK ASO with immunoradiotherapy can safely and significantly slow tumor growth and improve survival.
摘要：
背景：放射疗法和免疫疗法（免疫放射疗法）的组合已越来越多地用于治疗多种癌症。然而,一些肿瘤对免疫放射疗法有抵抗力。我们先前已经表明，在巨噬细胞上表达的MER原癌基因酪氨酸激酶（MerTK）介导了对免疫治疗的抗性。因此，我们寻求开发可以减轻MerTK负面影响的疗法。我们设计并开发了MerTK特异性反义寡核苷酸（ASO），并表征了其在小鼠中引发抗肿瘤免疫反应的作用。
方法：将344SQR细胞注射到8-12周龄雌性129sv/ev小鼠第0天的右腿和第4天的左腿中，以建立原发性和继发性肿瘤，分别。在第8、9和10天对原发性肿瘤给予12Gy剂量的辐射。小鼠从肿瘤植入后第1天开始接受抗PD-1、抗CTLA-4或/和MerTKASO。通过流式细胞术评估肿瘤微环境的组成和肿瘤中巨噬细胞上的MerTK水平。用NanoString研究免疫相关基因的表达。最后,在组织学上评估了MerTKASO对眼睛结构的影响。
结果：值得注意的是，在XRT+抗PD1和XRT+抗CTLA4中加入MerTKASO显著减缓了原发性和继发性肿瘤的生长,并显著延长了生存期.ASO显著降低肿瘤相关巨噬细胞（TAMs）中MerTK的表达，将其表型从M2重新编程为M1。此外,当与XRT+抗CTLA4组合时,MerTKASO增加颗粒酶B+CD8+T细胞在继发性肿瘤中的百分比。NanoString结果表明，MerTKASO可有利地调节免疫相关基因，以促进继发性肿瘤中的抗肿瘤免疫反应。重要的是,眼组织的组织学分析表明，与小分子不同，MerTKASO在眼睛中没有产生任何可检测的病理。
结论：MerTKASO可以显著下调TAMs上MerTK的表达，从而促进抗肿瘤免疫反应。MerTKASO与免疫放射疗法的组合可以安全且显着地减缓肿瘤生长并提高生存率。
方法：将344SQR细胞注射到8-12周龄雌性129sv/ev小鼠第0天的右腿和第4天的左腿中，以建立原发性和继发性肿瘤，分别。在第8、9和10天对原发性肿瘤给予12Gy剂量的辐射。小鼠从肿瘤植入后第1天开始接受抗PD-1、抗CTLA-4或/和MerTKASO。通过流式细胞术评估肿瘤微环境的组成和肿瘤中巨噬细胞上的MerTK水平。用NanoString研究免疫相关基因的表达。最后,在组织学上评估了MerTKASO对眼睛结构的影响。
结果：值得注意的是，在XRT+抗PD1和XRT+抗CTLA4中加入MerTKASO显著减缓了原发性和继发性肿瘤的生长,并显著延长了生存期.ASO显著降低肿瘤相关巨噬细胞（TAMs）中MerTK的表达，将其表型从M2重新编程为M1。此外,当与XRT+抗CTLA4组合时,MerTKASO增加颗粒酶B+CD8+T细胞在继发性肿瘤中的百分比。NanoString结果表明，MerTKASO可有利地调节免疫相关基因，以促进继发性肿瘤中的抗肿瘤免疫反应。重要的是,眼组织的组织学分析表明，与小分子不同，MerTKASO在眼睛中没有产生任何可检测的病理。
结论：MerTKASO可以显著下调TAMs上MerTK的表达，从而促进抗肿瘤免疫反应。MerTKASO与免疫放射疗法的组合可以安全且显着地减缓肿瘤生长并提高生存率。
