METHODS: Blood samples were obtained from a total of 6 healthy volunteers (HC), 6 Chronic Pancreatitis (CP) and 34 PDAC patients consisting of 22 resectable (RPC), 8 locally advanced (LAPC) and 4 metastatic (MPC). Real-time Quantitative Polymerase Chain reactions (RT-qPCR), Metabolomics, Enzyme-Linked Immunosorbent Assay (ELISA), Reactive Oxygen Species (ROS), and Immunophenotyping assays were conducted. Statistical analysis was conducted in R (v 4.3.2). Additional analysis of single-cell RNA data from 20 patients (16 PDAC and 4 controls) was conducted to interrogate the distribution of T-cell and Natural Killer cell populations.
RESULTS: Granulocyte and neutrophil levels were significantly elevated while lymphocytes decreased with PDAC severity. The total percentages of CD3 T-cell subpopulations (helper and double negative T-cells) decreased when compared to HC. Although both NK (p = 0.014) and NKT (p < 0.001) cell levels increased as the disease progressed, their subsets: NK CD56dimCD16- (p = 0.024) and NKTs CD56+ (p = 0.008) cell levels reduced significantly. Of note is the negative association of NK CD56dimCD16- (p < 0.001) cell levels with survival time. The gene expression analyses showed no statistically significant correlation when comparing the PDAC groups with the controls. The inflammatory status of PDAC was assessed by ROS levels of serum which were elevated in CP (p = 0.025), (RPC (p = 0.003) and LAPC (p = 0.008)) while no significant change was observed in MPC, compared to the HC group. ROS was shown to be positively correlated with GlycA (R = 0.45, p = 0.0096). Single-cell analyses showed a significant difference in the ratio of NKT cells per total cell counts in LAPC (p < 0.001) and MPC (p < 0.001) groups compared with HC, confirming observations in our sample group.
CONCLUSIONS: The expression of these immune cell markers observed in this pilot study provides insight into their potential roles in tumour progression in the patient group and suggests their potential utility in the development of immunotherapeutic strategies.
方法:从总共6名健康志愿者(HC)获得血液样本,6慢性胰腺炎(CP)和34例PDAC患者,包括22例可切除(RPC),8例局部晚期(LAPC)和4例转移性(MPC)。实时定量聚合酶链反应(RT-qPCR),代谢组学,酶联免疫吸附测定(ELISA),活性氧(ROS),并进行免疫表型分析。在R(v4.3.2)中进行统计学分析。对来自20名患者(16名PDAC和4名对照)的单细胞RNA数据进行额外分析以询问T细胞和自然杀伤细胞群的分布。
结果:随着PDAC的严重程度,粒细胞和中性粒细胞水平显著升高,而淋巴细胞水平降低。当与HC相比时,CD3T细胞亚群(辅助和双阴性T细胞)的总百分比降低。尽管NK(p=0.014)和NKT(p<0.001)细胞水平随着疾病的进展而增加,它们的亚群:NKCD56dimCD16-(p=0.024)和NKTCD56(p=0.008)细胞水平显着降低。值得注意的是NK⑶56dimCD16-(p<0.001)细胞水平与存活时间的负相关性。当将PDAC组与对照进行比较时,基因表达分析没有显示出统计学上显著的相关性。PDAC的炎症状态通过CP升高的血清ROS水平来评估(p=0.025),(RPC(p=0.003)和LAPC(p=0.008)),而MPC中未观察到显着变化,与HC组相比。ROS与GlycA呈正相关(R=0.45,p=0.0096)。单细胞分析显示,与HC相比,LAPC(p<0.001)和MPC(p<0.001)组的NKT细胞/总细胞计数的比率存在显着差异。确认我们样本组中的观察结果。
结论:在这项初步研究中观察到的这些免疫细胞标志物的表达提供了它们在患者组肿瘤进展中的潜在作用的见解,并表明它们在开发免疫治疗策略中的潜在效用。