Glioblastoma (GBM) is the most common primary brain malignancy in the U.S. with a 5-year overall survival < 5% despite an aggressive standard of care. Laser interstitial thermal therapy (LITT) is a surgical approach to treating GBM that has gained traction, providing a safe option for reducing intracranial tumor burden. LITT is believed to potentially modulate GBM immune responses; however, the biochemical mechanisms underlying the modulation of immune checkpoints in GBM cells have been poorly characterized. The present study aimed to preliminarily evaluate the effects of thermal therapy and radiation on PD-L1 modulation in vitro, as a function of IDH mutational status. U87 cells and their IDH-mutant counterpart (U87R132H), which was generated using a crispr-cas9 knock-in approach, were utilized for this preliminary evaluation. Cell heating was achieved by harvesting with trypsin centrifugation where the cell pellets were treated on a heat block for the associated time and temperature. Following thermal therapy, cells were resuspended and irradiated using a 37-Cesium irradiator at 0.6 Gy min−1. Immediately following treatment, cells were either plated as single cells to allow colonies to form, and stained with Coomassie blue to be counted approximately 10–14 days later or harvested for Western blot analysis. Cell lysates were analyzed for PD-L1 expression with respect to various iron metabolic parameters (mortalin (HSPA9), transferrin receptor, and ferritin heavy chain) using a Western blotting approach. In both U87 and U87R132Hcell lines, thermal therapy showed a temperature-dependent cell-killing effect, but U87R132Hcells appeared more sensitive to thermal treatment when treated at 43 °C for 10 min. Moreover, thermal therapy had minimal effects on cell responses to 2 Gy irradiation. Treatment with thermal therapy downregulated PD-L1 expression in U87R132Hcells, which was associated with increased expression of the mitochondrial iron metabolic enzyme, HSPA9. Thermal therapy reversed the radiation-induced overexpression of PD-L1, transferrin receptor, and ferritin heavy chain in U87R132Hcells. No effects were observed in wild-type U87 cells. Moreover, Ga(NO3)3depleted mitochondrial iron content which, in turn, significantly enhanced the sensitivity of U87R132Hcells to thermal therapy and 2 Gy irradiation and caused a significant increase in PD-L1 expression. These results suggest that thermal therapy alone can modulate the immune checkpoint PD-L1. This effect was more pronounced when thermal therapy was combined with radiation. Mechanistically, mitochondrial iron trafficking through HSPA9 may coordinate the regulation of PD-L1 in the context of thermal therapy and ionizing radiation, which can be targeted with gallium-based therapy. These novel, preliminary findings warrant further mechanistic investigations in pre-clinical models of LITT.
胶质母细胞瘤（GBM）是美国最常见的原发性脑恶性肿瘤，尽管采用积极的标准化治疗，其5年总生存率仍低于5%。激光间质热疗（LITT）作为一种治疗GBM的手术方法已逐渐受到关注，为减轻颅内肿瘤负荷提供了安全选择。LITT被认为可能调节GBM的免疫应答，然而，其对GBM细胞免疫检查点调节的生化机制尚不明确。本研究旨在初步评估热疗和放射治疗对PD-L1调节的影响，并探讨其与IDH突变状态的关系。本研究采用U87细胞及其通过CRISPR-Cas9基因敲入技术构建的IDH突变型细胞（U87R132H）进行初步评估。细胞加热通过胰蛋白酶消化离心收集细胞沉淀，并在加热块上进行相应时间和温度的处理实现。热疗后，细胞重悬并于37-铯照射器中以0.6 Gy·min⁻¹的剂量进行辐照。处理后，细胞立即进行单细胞铺板以形成克隆，约10-14天后用考马斯亮蓝染色计数，或收集用于Western blot分析。通过Western blot方法分析细胞裂解液中PD-L1表达与多种铁代谢参数（如mortalin（HSPA9）、转铁蛋白受体和铁蛋白重链）的关系。在U87和U87R132H细胞系中，热疗均显示出温度依赖性的细胞杀伤效应，但U87R132H细胞在43°C处理10分钟时对热疗更为敏感。此外，热疗对细胞对2 Gy辐照的应答影响甚微。热疗处理下调了U87R132H细胞中PD-L1的表达，这与线粒体铁代谢酶HSPA9的表达增加相关。热疗逆转了辐照诱导的U87R132H细胞中PD-L1、转铁蛋白受体和铁蛋白重链的过表达，而在野生型U87细胞中未观察到类似效应。此外，Ga(NO₃)₃耗竭了线粒体铁含量，从而显著增强了U87R132H细胞对热疗和2 Gy辐照的敏感性，并引起PD-L1表达显著增加。这些结果表明，单独热疗即可调节免疫检查点PD-L1，且热疗联合放射治疗时该效应更为显著。机制上，通过HSPA9介导的线粒体铁转运可能协调热疗和电离辐射背景下PD-L1的调控，而镓靶向疗法可针对此过程发挥作用。这些新颖的初步发现值得在LITT的临床前模型中进行更深入的机制研究。
Mitochondrial Iron Metabolism as a Potential Key Mediator of PD-L1 Thermal Regulation
肿瘤(癌症)患者之家