Construction of nasopharyngeal carcinoma microsphere model based on 3d bioprinting and its response to immunotherapy drugs
基于3D生物打印的鼻咽癌微球体模型构建及其对免疫治疗 药物响应情况研究

摘要

目的 基于3D生物打印构建鼻咽癌微球体模型并分析该模型对免疫治疗药物的响应情况，以期为临床制定个性化治疗计划和方案提供指导。方法 通过3D生物打印技术构建含有免疫细胞与肿瘤细胞的复杂微球体模型，将未经处理的微球体模型归入至对照组，将添加PD-1抗体的微球体模型归入至PD-1抗体组，将添加CTLA-4抗体的微球体模型归入至CTLA-4模型，将同时添加PD-1抗体与CTLA-4抗体的微球体模型归入至联合组。通过体内实验验证基于3D生物打印的鼻咽癌微球体模型对免疫治疗药物的响应，将未经处理的人源化小鼠归入至对照组，将接受PD-1抗体的小鼠归入至PD-1抗体组，将接受CTLA-4抗体的小鼠归入至CTLA-4抗体组，将同时接受PD-1抗体与CTLA-4抗体的小鼠归入至联合组。分析各组体外实验结果及体内实验结果。结果 与PD-1抗体组、CTLA-4抗体组及联合组相比，对照组鼻咽癌细胞及PMBCs细胞G0/G1期占比、细胞凋亡率更第，PD-L1、CD8+键蛋白表达水平及TNF-α、IL-2、IFN-γ及肿瘤坏死因子α（TNF-α）、白细胞介素-2（IL-2）、干扰素γ（IFN-γ）水平更高（P<0.05），与联合组相比，PD-1抗体组、CTLA-4抗体组鼻咽癌细胞及PMBCs细胞G0/G1期占比、细胞凋亡率更低，PD-L1、CD8+及TNF-α、IL-2、IFN-γ表达水平更高（P<0.05）。PD-1抗体组、CTLA-4抗体组不同时间肿瘤体积均小于对照组，联合组不同时间肿瘤体积均小于对照组及PD-1抗体组、CTLA-4抗体组（P<0.05）。对照组CD45、CD8+表达水平高于PD-1抗体组、CTLA-4抗体组及合组（P<0.05），联合组CD45、CD8+表达水平低于PD-1抗体组、CTLA-4抗体组，CD4+表达水平高于PD-1抗体组、CTLA-4抗体组（P<0.05）。结论 通过3D生物打印构建鼻咽癌微球体模型可对免疫治疗药物响应情况进行准确评估，能够为临床合理选择治疗药物提供指导。

Abstract

Objective To construct a nasopharyngeal carcinoma microsphere model based on 3D bioprinting and analyze its response to immunotherapy drugs, aiming to provide guidance for the development of personalized treatment plans and protocols in clinical practice.
Methods Complex microsphere models containing immune cells and tumor cells were constructed using 3D bioprinting technology. Untreated microsphere models were assigned to the control group, microsphere models with added PD-1 antibody were assigned to the PD-1 antibody group, microsphere models with added CTLA-4 antibody were assigned to the CTLA-4 model group, and microsphere models with added PD-1 and CTLA-4 antibodies were assigned to the combined group. In vivo experiments were conducted to verify the response of a 3D bioprinted nasopharyngeal carcinoma microsphere model to immunotherapy drugs. Untreated humanized mice were assigned to the control group, mice receiving PD-1 antibody were assigned to the PD-1 antibody group, mice receiving CTLA-4 antibody were assigned to the CTLA-4 antibody group, and mice receiving both PD-1 and CTLA-4 antibodies were assigned to the combination group. The in vitro and in vivo experimental results for each group were analyzed.
Results Compared with the PD-1 antibody group, CTLA-4 antibody group, and combination group, the control group had a lower proportion of nasopharyngeal carcinoma cells and PMBCs in the G0/G1 phase, a lower apoptosis rate, and higher levels of PD-L1, CD8+ bond protein expression, TNF-α, IL-2, IFN-γ, tumor necrosis factor α (TNF-α), interleukin-2 (IL-2), and interferon-γ (IFN-γ) (P<0.05). Compared with the combination group, the PD-1 antibody group and CTLA-4 antibody group had a lower proportion of nasopharyngeal carcinoma cells and PMBCs in the G0/G1 phase, a lower apoptosis rate, and higher levels of PD-L1, CD8+, TNF-α, IL-2, and IFN-γ expression (P<0.05). The tumor volume in the PD-1 antibody group and CTLA-4 antibody group was smaller than that in the control group at different time points, while the tumor volume in the combination group was smaller than that in the control group, the PD-1 antibody group, and the CTLA-4 antibody group at different time points (P<0.05). The expression levels of CD45 and CD8+ in the control group were higher than those in the PD-1 antibody group, CTLA-4 antibody group, and the combined group (P<0.05). In the combined group, the expression levels of CD45 and CD8+ were lower than those in the PD-1 antibody group and CTLA-4 antibody group, while the expression level of CD4+ was higher than that in the PD-1 antibody group and CTLA-4 antibody group (P<0.05).
Conclusion   Constructing a nasopharyngeal carcinoma microsphere model using 3D bioprinting can accurately assess the response to immunotherapy drugs, providing guidance for the rational selection of treatment drugs in clinical practice.