Wang Yan, Rousset Xavier, Prunier Chloé, Garcia Paul, Dosda Emilien, Leplus Estelle, Viallet Jean
R&D Department, Inovotion, 38700 La Tronche, France.
Université Grenoble Alpes, 38000 Grenoble, France.
Cancers (Basel). 2022 Jun 23;14(13):3095. doi: 10.3390/cancers14133095.
(1) Purpose: To assess the use of the chicken embryo () model as an alternative model for immuno-oncology (IO) drug development, focusing on programmed cell death protein-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) immune checkpoint inhibitors. (2) Methods: First, the presence of immune cells in the model was detected through the immunophenotyping of chicken peripheral blood mononuclear cells (PBMCs) based on fluorescence activated cell sorting (FACS) analysis and the immunohistochemistry (IHC) analysis of tumor-infiltrating lymphocytes. Second, the cross-reactivity between one anti-human PD-1 Ab, pembrolizumab (KEYTRUDA), and chicken PD-1 was verified through the labelling of chicken splenocytes with pembrolizumab by FACS analysis. Third, the blockade effect of pembrolizumab on chicken PBMCs was assessed through cytotoxicity assay based on MTT. Fourth, the CAM assay was used to estimate the anti-tumor performance of pembrolizumab through the analyses of tumor growth and chicken immune cell infiltration in tumors. Finally, the efficacy of several PD-1 or PD-L1 inhibitors (nivolumab, atezolizumab and avelumab) on tumor growth was further assessed using the CAM assay. (3) Results: The presence of CD3, CD4, CD8 T lymphocytes and monocytes was confirmed by FACS and IHC analyses. During assays, pembrolizumab cross-reacted with chicken lymphocytes and induced PD-1/PD-L1 blockade, which permitted the restoration of chicken T-cell's cytotoxicity against human lung cancer H460 tumor cells. All these results were correlated with findings based on the CAM assay: pembrolizumab inhibited H460 tumor growth and induced evident chicken immune cell infiltration (with significant chicken CD45, CD3, CD4, CD8 and CD56 markers) in tumors. Furthermore, the potency of the CAM assay was not limited to the application of pembrolizumab. Nivolumab, atezolizumab and avelumab also led to tumor growth inhibition , on different tumor models. (4) Conclusions: The chicken embryo affords a physiological, immune reactive, environment for IO research, which allows observation of how the immune system defense against tumor cells, as well as the different immune tolerance mechanisms leading to tumor immune escape. The encouraging results obtained with PD-1/PD-L1 inhibitors in this study reveal the potential use of the chicken embryo model as an alternative, fast, and reliable model in the different fields of IO drug discovery.
(1) 目的：评估鸡胚模型作为免疫肿瘤学（IO）药物研发的替代模型，重点关注程序性细胞死亡蛋白-1（PD-1）/程序性细胞死亡配体-1（PD-L1）免疫检查点抑制剂。(2) 方法：首先，通过基于荧光激活细胞分选（FACS）分析的鸡外周血单个核细胞（PBMC）免疫表型分析和肿瘤浸润淋巴细胞的免疫组织化学（IHC）分析，检测模型中免疫细胞的存在情况。其次，通过FACS分析用派姆单抗标记鸡脾细胞，验证一种抗人PD-1抗体派姆单抗（可瑞达）与鸡PD-1之间的交叉反应性。第三，通过基于MTT的细胞毒性试验评估派姆单抗对鸡PBMC的阻断作用。第四，采用鸡胚绒毛尿囊膜（CAM）试验，通过分析肿瘤生长和肿瘤中鸡免疫细胞浸润情况，评估派姆单抗的抗肿瘤性能。最后，使用CAM试验进一步评估几种PD-1或PD-L1抑制剂（纳武单抗、阿特珠单抗和阿维鲁单抗）对肿瘤生长的疗效。(3) 结果：FACS和IHC分析证实了CD3、CD4、CD8 T淋巴细胞和单核细胞的存在。在试验过程中，派姆单抗与鸡淋巴细胞发生交叉反应并诱导PD-1/PD-L1阻断，从而恢复了鸡T细胞对人肺癌H460肿瘤细胞的细胞毒性。所有这些结果都与基于CAM试验的结果相关：派姆单抗抑制H460肿瘤生长，并在肿瘤中诱导明显的鸡免疫细胞浸润（具有显著的鸡CD45、CD3、CD4、CD8和CD56标志物）。此外，CAM试验的效能并不局限于派姆单抗的应用。纳武单抗、阿特珠单抗和阿维鲁单抗在不同肿瘤模型上也导致了肿瘤生长抑制。(4) 结论：鸡胚为IO研究提供了一个生理、免疫反应性的环境，这使得可以观察免疫系统如何抵御肿瘤细胞，以及导致肿瘤免疫逃逸的不同免疫耐受机制。本研究中使用PD-1/PD-L1抑制剂获得的令人鼓舞的结果揭示了鸡胚模型在IO药物发现的不同领域作为一种替代、快速且可靠的模型的潜在用途。
