肿瘤免疫治疗领域的主要问题是缺乏可靠的生物标志物,这些生物标志物可以预先确定个体患者对治疗的敏感性,以及对抗性机制的认识不足。基于免疫细胞中代谢辅酶自发荧光的生物标志物可以成为早期肿瘤治疗反应的强有力的新预测因子。而光学FLIM方法可以成为预测免疫疗法有效性的工具,这样可以保留样品的空间结构,并实时获得免疫细胞代谢状态的结果。该研究的目的是对新鲜分离的淋巴结的免疫细胞中的NAD(P)H代谢辅酶进行代谢自发荧光成像研究,作为评估早期免疫疗法反应有效性的潜在标志物。
■在腹股沟淋巴结附近植入有B16F0黑素瘤的C57Bl/6FoxP3-EGFP小鼠上进行研究。小鼠注射CTLA-4抗体(BioX细胞,美国)(每只小鼠250微克,在肿瘤生长的第7、8、11和12天腹膜内)。烟酰胺腺嘌呤二核苷酸(磷酸)辅酶(NAD(P)H)通道中的FLIM图像(激发-375nm,接收-435-485nm)使用LSM880荧光共聚焦激光扫描显微镜(CarlZeiss,德国)配备了FLIMSimple-Tau模块152TCSPC(Becker&HicklGmbH,德国)。使用BDFACSAriaIII细胞分选仪(BDBiosciences,美国)。
■使用检查点抑制剂的免疫治疗导致应答小鼠新鲜分离淋巴结的T细胞发生明显的代谢重排,抑制肿瘤生长。NAD(P)H的荧光寿命成像数据表明NADHα1的游离分数增加,这是一种与糖酵解相关的形式,可满足活化T细胞和促炎细胞因子合成的高要求。相比之下,患有晚期肿瘤的无应答小鼠显示游离分数与结合α1/α2的比值较低,这可能与对治疗的抵抗机制有关。通过流式细胞术通过活化和增殖标志物表达的数据来验证对免疫疗法的反应。作者观察到与未处理的对照和非应答者相比,应答者小鼠中效应T细胞中促炎细胞因子IFN-γ的产生增加。此外,与未处理的对照相比,表面活化标志物CD25和CD69的表达增加。
■使用FLIM方法可以证明NAD(P)H辅酶的自发荧光对检查点免疫疗法的反应敏感,可以用作治疗反应有效性的可靠标记。
The main problem in the field of tumor immunotherapy is the lack of reliable biomarkers that allow pre-determining the susceptibility of individual patients to treatment, as well as insufficient knowledge about the resistance mechanisms. Biomarkers based on the autofluorescence of metabolic coenzymes in immune cells can become a powerful new predictor of early tumor response to treatment, whereas the optical FLIM method can be a tool to predict the effectiveness of immunotherapy, which allows preserving the spatial structure of the sample and obtaining results on the metabolic status of immune cells in real time. The aim of the study is to conduct a metabolic autofluorescence imaging study of the NAD(P)H metabolic coenzyme in immune cells of freshly isolated lymph nodes as a potential marker for assessing the effectiveness of an early response to immunotherapy.
UNASSIGNED: The study was carried out on C57Bl/6 FoxP3-EGFP mice with B16F0 melanoma implanted near the inguinal lymph node. The mice were injected with antibodies to CTLA-4 (Bio X Cell, USA) (250 μg per mouse, intraperitoneally on days 7, 8, 11, and 12 of the tumor growth). FLIM images in the nicotinamide adenine dinucleotide (phosphate) coenzyme (NAD(P)H) channel (excitation - 375 nm, reception - 435-485 nm) were received using an LSM 880 fluorescent confocal laser scanning microscope (Carl Zeiss, Germany) equipped with a FLIM Simple-Tau module 152 TCSPC (Becker & Hickl GmbH, Germany). Flow cytometry was conducted using a BD FACSAria III cell sorter (BD Biosciences, USA).
UNASSIGNED: Immunotherapy with checkpoint inhibitors resulted in marked metabolic rearrangements in T cells of freshly isolated lymph nodes in responder mice, with inhibition of the tumor growth. Fluorescence lifetime imaging data on NAD(P)H indicated an increase in the free fraction of NADH α1, a form associated with glycolysis to meet high demands of the activated T cells and pro-inflammatory cytokine synthesis. In contrast, non-responder mice with advanced tumors showed low values of the ratio of free fraction to bound α1/α2, which may be related to mechanisms of resistance to therapy.The response to immunotherapy was verified by data on the expression of activation and proliferation markers by means of flow cytometry. The authors observed an increase in the production of the pro-inflammatory cytokine IFN-γ in effector T cells in responder mice compared to untreated controls and non-responders. In addition, an increase in the expression of the surface activation markers CD25 and CD69 was registered compared to untreated controls.
UNASSIGNED: Use of the FLIM method allowed to demonstrate that autofluorescence of the NAD(P)H coenzyme is sensitive to the response to checkpoint immunotherapy and can be used as a reliable marker of the effectiveness of response to treatment.