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

项目摘要 胰腺导管腺癌（PDAC）是人类最致命的癌症之一，其5年总生存率为 (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 包括放化疗和手术的治疗通常是温和和短暂的。2010年的重大挑战 如何将免疫学上的冷PDAC转化为对免疫检查点阻断有反应的热肿瘤 (ICB)疗法我们最近表明，不可逆电穿孔（IRE），目前的肿瘤消融技术， 在临床中使用，PDAC对抗PD-1 ICB显着致敏，导致约40%的小鼠长期存活， 一个积极的原位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CTL的肿瘤浸润。作为努力的一部分， 定义进一步增强IRE +抗PD-1联合治疗PDAC疗效的方法，我们发现 通过飞行时间质谱细胞术（CyTOF）免疫分析的新的免疫抑制机制， PDAC的单细胞RNAseq，其显示表达CXCR 2的髓样抑制细胞的显著浸润， 细胞（MDSC）。此外，我们发现IRE破坏糖酵解和氧化磷酸化（OxPhos） 而上调谷氨酰胺酶和谷氨酸，表明谷氨酰胺分解是一种代偿机制， 满足IRE处理的细胞的能量和生物合成需要。这些数据与已知的关键数据一起， MDSC和谷氨酰胺代谢在免疫抑制中的作用， 抗糖尿病药物二甲双胍和苯丙氨酸从根本上改变肿瘤代谢程序， 我们的初步发现，IRE和Re-Phen，一种新开发的ICB类似物， 苯乙双胍下调OxPhos途径，同时对谷氨酸产生显示相反的作用， 导致我们假设通过MDSC耗竭或抑制免疫抑制性TME的减弱 通过Re-Phen增强IRE + ICB以进一步延长总生存期并增加 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）识别 与IRE + ICB的长期和短期反应相关的免疫抑制因素。我们将使用 CyTOF免疫谱分析、scRNAseq和细胞因子阵列分析，以充分表征IRE在免疫缺陷病毒中的影响。 免疫抑制性TME上抗PD-1的存在和不存在。2)为了确定在多大程度上 针对MDSC的治疗增强IRE + ICB。3)为了确定在多大程度上破坏了 通过治疗诊断剂Re-Phen的代谢程序增强IRE + ICB。该项目的结果是 预计将揭示以前未定义的MDSC的作用和免疫调节中代谢编程的失调。 在IRE + ICB联合治疗的背景下抑制。该项目的成功将产生特殊的影响 因为它将为PDAC提供一种潜在的有效疗法。

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.