DataSheet_1_Functional reprogramming of peripheral blood monocytes by soluble mediators in patients with pancreatic cancer and intraductal papillary mucinous neoplasms.pdf

BackgroundMonocytes and monocyte-derived tumor infiltrating cells have been implicated in the immunosuppression and immune evasion associated with pancreatic adenocarcinoma (PDAC). Yet, precisely how monocytes in the periphery and tumor microenvironment in patients with intraductal papillary mucinous neoplasm (IPMN), a precursor lesion to PDAC, change during disease progression has not been defined. Here we functionally profiled the peripheral immune system and characterized the tumor microenvironment of patients with both IPMN and PDAC. We also tested if sera from patients with IPMN and PDAC functionally reprogram monocytes relative to that of healthy donors.
MethodsPancreatic tissue and peripheral blood were collected at the time of resection from 16 patients with IPMN and 32 patients with PDAC. Peripheral blood and pancreatic tissue/tumor were immunophenotyped using flow cytometry. Whole blood was plated and incubated with R848 (a TLR 7/8 agonist) or LPS (a TLR4 agonist) for 6 hours and TNF expression in monocytes was measured by flow cytometry to measure monocyte activation. To test if TLR sensitivity is determined by factors in patient sera, we preconditioned healthy donor monocytes in serum from PDAC (n=23), IPMN (n=15), or age-matched healthy donors (n=10) followed by in vitro stimulation with R848 or LPS and multiplex cytokine measurements in the supernatant.
ResultsTNF expression in R848-stimulated peripheral blood monocytes was higher in patients with low grade vs high grade IPMN (65% vs 32%, p = 0.03) and stage 1 vs stage 2/3 PDAC (58% vs 42%, p = 0.03), this was not observed after LPS stimulation. TLR activation correlated with increasing grade of dysplasia from low grade IPMN to high grade IPMN. Serum from patients with IPMN and PDAC recapitulated suppression of TNF induction after R848 stimulation in naïve, healthy donor monocytes.
ConclusionPeripheral blood monocyte TNF secretion inversely correlates with the degree of dysplasia in IPMN and cancer stage in PDAC, suggesting innate immune reprogramming as IPMNs progress to invasive disease. These effects are, at least in part, mediated by soluble mediators in sera.

背景
单核细胞及单核细胞来源的肿瘤浸润细胞与胰腺导管腺癌（pancreatic adenocarcinoma, PDAC）相关的免疫抑制及免疫逃逸密切相关。然而，作为胰腺导管腺癌前驱病变的胰腺导管内乳头状黏液性肿瘤（intraductal papillary mucinous neoplasm, IPMN）患者的外周血单核细胞及肿瘤微环境，在疾病进展过程中的具体变化尚未明确。本研究对IPMN及PDAC患者的外周免疫系统进行了功能谱分析，并对其肿瘤微环境进行了表征。此外，本研究还对比了健康供体、IPMN及PDAC患者的血清，以探究其是否可在功能上重编程单核细胞。

方法
本研究纳入16例IPMN患者及32例PDAC患者，在其手术切除时收集胰腺组织及外周血样本。采用流式细胞术（flow cytometry）对外周血及胰腺组织/肿瘤组织进行免疫表型分型。将全血接种培养后，分别与TLR7/8激动剂R848或TLR4激动剂脂多糖（lipopolysaccharide, LPS）共孵育6小时，通过流式细胞术检测单核细胞中肿瘤坏死因子（Tumor Necrosis Factor, TNF）的表达水平，以评估单核细胞的活化状态。为探究TLR敏感性是否由患者血清中的因子所决定，本研究采用PDAC患者（n=23）、IPMN患者（n=15）或年龄匹配的健康供体（n=10）的血清预处理健康供体单核细胞，随后在体外用R848或LPS进行刺激，并对上清液进行多重细胞因子检测。

结果
经R848刺激后，低级别IPMN患者外周血单核细胞的TNF表达水平高于高级别IPMN患者（65% vs 32%，p = 0.03），1期PDAC患者的该水平亦高于2/3期PDAC患者（58% vs 42%，p = 0.03）；而LPS刺激后未观察到该差异。TLR活化程度与IPMN异型增生程度从低级别向高级别进展呈正相关。IPMN及PDAC患者的血清可重现初始健康供体单核细胞经R848刺激后TNF诱导被抑制的现象。

结论
外周血单核细胞的TNF分泌水平与IPMN的异型增生程度及PDAC的临床分期呈负相关，提示随着IPMN进展为侵袭性疾病，先天免疫重编程过程发生。上述效应至少部分由血清中的可溶性介质所介导。