Radiotherapy and OncoImmunology Laboratory, Department of Radiation Oncology, Radboudumc, Geert Grooteplein Zuid 32, 6525GA, Nijmegen, The Netherlands.
Department of Medical Imaging, Radboudumc, Geert Grooteplein 10, Nijmegen, 6525GA, The Netherlands.
Mol Imaging Biol. 2024 Oct;26(5):835-846. doi: 10.1007/s11307-024-01934-w. Epub 2024 Jul 15.
Combined radiotherapy and immune checkpoint inhibition is a potential treatment option for head and neck squamous cell carcinoma (HNSCC). Immunocompetent mouse models can help to successfully develop radio- immunotherapy combinations and to increase our understanding of the effects of radiotherapy on the tumor microenvironment for future clinical translation. Therefore, the aim of this study was to develop a homogeneous, reproducible HNSCC model originating from the Mouse Oral Cancer 1 (MOC1) HNSCC cell line, and to explore the radiotherapy-induced changes in its tumor microenvironment, using flow cytometry and PD-L1 microSPECT/CT imaging.
In vivo growing tumors originating from the parental MOC1 line were used to generate single cell derived clones. These clones were screened in vitro for their ability to induce programmed cell death ligand 1 (PD-L1) and major histocompatibility complex class I (MHC-I) following IFNγ exposure. Clones with different IFNγ sensitivity were inoculated in C57BL/6 mice and assessed for tumor outgrowth. The composition of the tumor microenvironment of a stably growing (non)irradiated MOC1-derived clone was assessed by immunohistochemistry, flow cytometry and PD-L1 microSPECT/CT.
Low in vitro inducibility of MHC-I and PD-L1 by IFNγ was associated with increased tumor outgrowth of MOC1 clones in vivo. Flow cytometry analysis of cells derived from a stable in vivo growing MOC1 clone MOC1.3D5 showed expression of MHC-I and PD-L1 on several cell populations within the tumor. Upon irradiation, MHC-I and PD-L1 increased on leukocytes (CD45.2) and cancer associated fibroblasts (CD45.2/EpCAM/CD90.1). Furthermore, PD-L1 microSPECT/CT showed increased tumor uptake of radiolabeled PD-L1 antibodies with a heterogeneous spatial distribution of the radio signal, which co-localized with PD-L1 and CD45.2 areas.
PD-L1 and MHC-I inducibility by IFNγ in vitro is associated with tumor outgrowth of MOC1 clones in vivo. In tumors originating from a stably growing MOC1-derived clone, expression of these immune-related markers was induced by irradiation shown by flow cytometry on several cell populations within the tumor microenvironment such as immune cells and cancer associated fibroblasts. PD-L1 microSPECT/CT showed increased tumor uptake following radiotherapy, and autoradiography showed correlation of uptake with areas that are heavily infiltrated by immune cells. Knowledge of radiotherapy-induced effects on the tumor microenvironment in this model can help optimize timing and dosage for radio- immunotherapy combination strategies in future research.
放射治疗联合免疫检查点抑制是头颈部鳞状细胞癌（HNSCC）的一种潜在治疗选择。免疫功能正常的小鼠模型有助于成功开发放射免疫治疗联合疗法，并增加我们对放疗对肿瘤微环境影响的理解，以促进未来的临床转化。因此，本研究旨在开发一种源自 Mouse Oral Cancer 1（MOC1）HNSCC 细胞系的同质、可重复的 HNSCC 模型，并通过流式细胞术和 PD-L1 微 SPECT/CT 成像来探索放疗引起的肿瘤微环境变化。
在体生长的源自亲本 MOC1 系的肿瘤被用于生成单细胞衍生克隆。这些克隆在体外筛选诱导程序性死亡配体 1（PD-L1）和主要组织相容性复合体 I（MHC-I）的能力，以 IFNγ 暴露。具有不同 IFNγ 敏感性的克隆接种于 C57BL/6 小鼠中，并评估肿瘤生长情况。通过免疫组织化学、流式细胞术和 PD-L1 微 SPECT/CT 评估稳定生长（未）照射的 MOC1 衍生克隆的肿瘤微环境组成。
IFNγ 诱导 MHC-I 和 PD-L1 的低诱导能力与 MOC1 克隆在体内的肿瘤生长增加有关。来自稳定体内生长的 MOC1 克隆 MOC1.3D5 的细胞衍生的流式细胞术分析显示，肿瘤内的几个细胞群体表达 MHC-I 和 PD-L1。照射后，白细胞（CD45.2）和癌相关成纤维细胞（CD45.2/EpCAM/CD90.1）上的 MHC-I 和 PD-L1 增加。此外，PD-L1 微 SPECT/CT 显示放射性标记的 PD-L1 抗体的肿瘤摄取增加，放射信号具有异质的空间分布，与 PD-L1 和 CD45.2 区域共定位。
IFNγ 体外诱导的 PD-L1 和 MHC-I 与 MOC1 克隆在体内的肿瘤生长有关。在源自稳定生长的 MOC1 衍生克隆的肿瘤中，通过流式细胞术在肿瘤微环境中的几个细胞群体（如免疫细胞和癌相关成纤维细胞）中观察到这些免疫相关标记物的诱导。PD-L1 微 SPECT/CT 显示放疗后肿瘤摄取增加，放射自显影显示摄取与免疫细胞浸润严重的区域相关。该模型中放射治疗对肿瘤微环境的影响的知识有助于优化未来放射免疫治疗联合策略的时间和剂量。
