Development of irreversible electroporation-based rational combinations to potentiate the activity of cancer immunotherapy against pancreatic ductal adenocarcinoma

项目总结 摘要胰腺导管腺癌是人类最致命的癌症之一，总生存期为5年。 (OS)转移性疾病的发生率为7%，局部晚期疾病的发生率低于20%。受益于当前 包括放化疗和手术在内的治疗通常是温和和短暂的。世界银行面临的重大挑战 领域是如何将免疫寒冷的PDAC转变为对免疫检查点阻断有反应的热肿瘤 (ICB)治疗。我们最近发现了不可逆电穿孔(Ire)，这是目前的一种肿瘤消融技术。 在临床上使用，显著致敏PDAC对抗PD-1 ICB，导致长期存活约40%的小鼠在 一种积极的原位PDAC模型。显著的抗PDAC活性归因于有效的诱导 免疫原性细胞死亡和间质扰动有利于肿瘤CTL的侵袭。作为努力的一部分， 确定进一步提高IRE+抗PD-1联合治疗PDAC疗效的方法，我们发现 飞行时间质谱仪(CyTOF)免疫图谱和免疫抑制机制的研究 PDAC的单细胞RNAseq，显示明显的CXCR2表达的髓系抑制 细胞(MDSCs)。此外，我们还发现ire抑制糖酵解和氧化磷酸化(OxPhos)。 同时上调谷氨酰胺酶和谷氨酸，提示谷氨酰胺分解是一种代偿机制 满足IRE处理细胞的能量和生物合成需求。这些数据与已知的关键数据一起 MDSCs和谷氨酰胺代谢增强在免疫抑制中的作用，其他人的发现是 抗糖尿病药物二甲双胍和苯福明从根本上改变肿瘤代谢程序以致敏 肿瘤到ICB治疗，以及我们的初步发现，IRE和Re-Phen，一种新开发的类似物 苯福明下调OxPhos途径，同时对谷氨酸的产生产生相反的影响， 使我们假设通过耗尽MDSCs或抑制来减弱免疫抑制TME Re-Phen对谷氨酰胺的分解可增强IRE+ICB，进一步延长总生存期，提高 持久的反应。为了验证我们的假设，我们将追求以下具体目标：1)确定 免疫抑制因子与IRE+ICB的长期和短期反应相关。我们将使用 细胞免疫图谱、scRNAseq和细胞因子阵列分析以充分表征IRE在 抗PD-1抗体在免疫抑制的TME上的存在和缺失。2)确定以下内容的程度 针对MDSCs的治疗可增强IRE+ICB。3)确定网络中断的程度 治疗药物Re-Phen的代谢程序增强IRE+ICB。这个项目的发现是 有望揭示MDSCs在免疫中先前未定义的角色和未受调控的代谢编程 IRE+ICB联合治疗中的抑制作用。该项目的成功将产生非同寻常的影响 因为它将为PDAC提供一种潜在有效的治疗方法。

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers, with a 5-year overall survival (OS) rate of 7% for metastatic disease and less than 20% for locally advanced disease. Benefit from current therapies including chemoradiation and surgery is often modest and transient. The significant challenge in the field is how to turn immunologically cold PDAC into hot tumors that respond to immune checkpoint blockade (ICB) therapy. We recently showed that irreversible electroporation (IRE), a tumor ablative technique currently used in the clinics, significantly sensitized PDAC to anti-PD-1 ICB, leading to long-term survival in ~40% mice in an aggressive orthotopic PDAC model. The remarkable anti-PDAC activity was attributed to efficient induction of immunogenic cell death and stromal perturbation in favor of tumor infiltration of CTLs. As part of an effort to define approaches to further enhance the efficacy of IRE + anti-PD-1 combination against PDAC, we uncovered novel immune suppressive mechanism through time-of-flight mass cytometry (CyTOF) immune profiling and single cell RNAseq of PDACs, which showed significant infiltration of CXCR2-expressing myeloid suppressive cells (MDSCs). Furthermore, we found that IRE collapsed glycolysis and oxidative phosphorylation (OxPhos) while upregulated glutaminase and glutamate, suggesting glutaminolysis as a compensatory mechanism to satisfy energy and biosynthesis needs of IRE-treated cells. These data, taken together with the known critical role of MDSCs and heightened glutamine metabolism in immune suppression, the findings by others that the anti-diabetic drugs metformin and phenformin fundamentally change the tumor metabolic program to sensitize tumors to ICB therapy, and our preliminary findings that both IRE and Re-Phen, a newly developed analogue of phenformin, downregulated the OxPhos pathway while displaying an opposite effect on glutamate production, lead us to hypothesize that attenuation of the immunosuppressive TME by depletion of MDSCs or suppression of glutaminolysis by Re-Phen potentiates IRE + ICB to further prolong overall survival and increase the rate of durable response. To test our hypothesis, we will pursue the following specific aims: 1) To identify immunosuppressive factors associated with long-term versus short-term response to IRE + ICB. We will use CyTOF immune profiling, scRNAseq, and cytokine array analyses to fully characterize the impact of IRE in the presence and absence of anti-PD-1 on the immunosuppressive TME. 2) To determine the extent to which therapies directed at MDSCs potentiate IRE + ICB. 3) To determine the extent to which disruption of the metabolic program by theranostic agent Re-Phen potentiates IRE + ICB. The findings from this project are expected to reveal previously undefined roles of MDSCs and deregulated metabolic programming in immune suppression in the context of combined IRE + ICB therapy. Success of this project will have exceptional impact because it will offer a potentially effective therapy for PDAC.