Greier Maria do Carmo, Runge Annette, Dudas Jozsef, Hartl Roland, Santer Matthias, Dejaco Daniel, Steinbichler Teresa Bernadette, Federspiel Julia, Seifarth Christof, Konschake Marko, Sprung Susanne, Sopper Sieghart, Randhawa Avneet, Mayr Melissa, Hofauer Benedikt Gabriel, Riechelmann Herbert
Department of Otorhinolaryngology, Head and Neck Surgery, Medical University of Innsbruck, Innsbruck, Austria.
Institute for Clinical and Functional Anatomy, Medical University Innsbruck (MUI), Innsbruck, Austria.
Front Oncol. 2024 Mar 7;14:1364577. doi: 10.3389/fonc.2024.1364577. eCollection 2024.
Head and neck squamous cell carcinomas (HNSCC) are highly heterogeneous tumors. In the harsh tumor microenvironment (TME), metabolic reprogramming and mitochondrial dysfunction may lead to immunosuppressive phenotypes. Aerobic glycolysis is needed for the activation of cytotoxic T-cells and the absence of glucose may hamper the full effector functions of cytotoxic T-cells. To test the effect of mitochondrial dysfunction on cytotoxic T cell function, slice cultures (SC) of HNSCC cancer were cultivated under different metabolic conditions.
Tumor samples from 21 patients with HNSCC were collected, from which, SC were established and cultivated under six different conditions. These conditions included high glucose, T cell stimulation, and temporarily induced mitochondrial dysfunction (MitoDys) using FCCP and oligomycin A with or without additional T cell stimulation, high glucose and finally, a control medium. Over three days of cultivation, sequential T cell stimulation and MitoDys treatments were performed. Supernatant was collected, and SC were fixed and embedded. Granzyme B was measured in the supernatant and in the SC via immunohistochemistry (IHC). Staining of PD1, CD8/Ki67, and cleaved-caspase-3 (CC3) were performed in SC.
Hematoxylin eosin stains showed that overall SC quality remained stable over 3 days of cultivation. T cell stimulation, both alone and combined with MitoDys, led to significantly increased granzyme levels in SC and in supernatant. Apoptosis following T cell stimulation was observed in tumor and stroma. Mitochondrial dysfunction alone increased apoptosis in tumor cell aggregates. High glucose concentration alone had no impact on T cell activity and apoptosis. Apoptosis rates were significantly lower under conditions with high glucose and MitoDys (p=0.03).
Stimulation of tumor-infiltrating lymphocytes in SC was feasible, which led to increased apoptosis in tumor cells. Induced mitochondrial dysfunction did not play a significant role in the activation and function of TILs in SC of HNSCC. Moreover, high glucose concentration did not promote cytotoxic T cell activity in HNSCC SC.
头颈部鳞状细胞癌（HNSCC）是高度异质性肿瘤。在恶劣的肿瘤微环境（TME）中，代谢重编程和线粒体功能障碍可能导致免疫抑制表型。细胞毒性T细胞的激活需要有氧糖酵解，而葡萄糖的缺乏可能会妨碍细胞毒性T细胞的完全效应功能。为了测试线粒体功能障碍对细胞毒性T细胞功能的影响，在不同代谢条件下培养了HNSCC癌的切片培养物（SC）。
收集21例HNSCC患者的肿瘤样本，从中建立SC并在六种不同条件下培养。这些条件包括高糖、T细胞刺激，以及使用FCCP和寡霉素A暂时诱导线粒体功能障碍（MitoDys），有无额外的T细胞刺激、高糖，最后是对照培养基。在三天的培养过程中，进行了连续的T细胞刺激和MitoDys处理。收集上清液，固定并包埋SC。通过免疫组织化学（IHC）在上清液和SC中测量颗粒酶B。在SC中进行PD1、CD8/Ki67和裂解的半胱天冬酶-3（CC3）染色。
苏木精伊红染色显示，在三天的培养过程中，总体SC质量保持稳定。单独的T细胞刺激以及与MitoDys联合刺激均导致SC和上清液中颗粒酶水平显著升高。在肿瘤和基质中观察到T细胞刺激后的凋亡。单独的线粒体功能障碍增加了肿瘤细胞聚集体中的凋亡。单独的高糖浓度对T细胞活性和凋亡没有影响。在高糖和MitoDys条件下，凋亡率显著降低（p = 0.03）。
刺激SC中的肿瘤浸润淋巴细胞是可行的，这导致肿瘤细胞凋亡增加。诱导的线粒体功能障碍在HNSCC的SC中TILs的激活和功能中未发挥重要作用。此外，高糖浓度并未促进HNSCC SC中细胞毒性T细胞的活性。
