PO.TB10.15 · 肿瘤生物学
胃肠道癌性腹膜转移中腹腔灌洗液细胞外囊泡的独特特征
Distinct profile of peritoneal lavage fluid extracellular vesicles in gastrointestinal carcinomatosis
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：胃肠道腹膜癌病（GI PC）带来重大的诊断和治疗挑战。常用的诊断工具如循环肿瘤DNA在PC中的敏感性较差。近期证据表明，腹腔小细胞外囊泡（EV）在腹膜转移的发生和进展中发挥关键作用。我们试图将腹腔灌洗液（PL）EV特征（数量和基因表达）与非癌症PL进行比较，以确定PL EV检测作为PC液体活检的潜力。
方法：从GI PC患者和非癌症患者收集PL样本。非癌症队列的纳入标准为BMI 18-30、无癌症/自身免疫病史，以及具有非炎症性手术指征（如疝修补）。通过沉淀分离EVs，并通过纳米颗粒追踪分析（NTA）、冷冻电子显微镜（cryo-EM）和Western blot进行表征。通过NTA定量粒径分布（0.5-199.5nm、200.5-500.5nm）和浓度。使用NanoString PanCancer Panel（770个基因）分析EV RNA。差异表达采用log₂倍数变化≥1.5或≤-1.5且p≤0.05。
结果：共收集66份PL样本（35份癌症和31份非癌症）。在31份非癌症PL中，仅19份样本有EV沉淀，表明其余样本中EVs过少以至无法形成EV沉淀。然而，所有癌症PL均有EV沉淀。对有足够EV的样本进行NTA显示，癌症样本的EV总量（以×10⁹颗粒/mL计）约为非癌症队列的两倍（10.9 × 10⁹ vs. 4.5 × 10⁹；p=0.0005）。即使在全身治疗后，癌症和非癌症PL EV数量之间的差异仍保持（9.7 × 10⁹ vs. 4.5 × 10⁹；p=0.0002）。Cryo-EM显示各组之间EV存在明显的结构差异，癌症来源的EVs表现出多层、多室和货物密集的结构，而非癌症样本主要含有简单的单层囊泡。基因表达谱分析显示，与非癌症队列相比，癌症队列中涉及上皮-间质转化、转移、细胞周期调控、血管生成和细胞外基质组织的基因（STAB1、PRF1、CCDC80、SPARCL1、VHL、RHOA）显著过表达。
结论：我们的研究是GI PC与非癌症PL比较的最大规模研究之一。在GI PC中，EV的数量以及生物物理和分子特征与非癌症组明显不同。PL EV可发展为GI PC的液体活检，以填补关键的临床需求。
查看英文原文 English abstract
Introduction: Gastrointestinal peritoneal carcinomatosis (GI PC) poses significant diagnostic and therapeutic challenges. The commonly used diagnostic tools such as circulating tumor DNA have poor sensitivity in PC. Recent evidence suggests that peritoneal cavity small extracellular vesicles (EV) play a critical role in peritoneal metastasis development and progression. We sought to compare the peritoneal lavage fluid (PL) EV profile (quantity and gene expression) to non-cancer PL to identify the potential of PL EV testing as a liquid biopsy in PC.
Methods: PL samples were collected from GI PC patients and non-cancer patients. The inclusion criteria for the non-cancer cohort were BMI 18-30, no cancer/autoimmune history, and had a non-inflammatory indication for surgery (ex. hernia repair). EVs were isolated by precipitation and characterized via nanoparticle tracking analysis (NTA), cryo-electron microscopy (cryo-EM), and western blot. Size distribution (0.5-199.5nm, 200.5-500.5nm) and concentration were quantified by NTA. EV RNA was profiled using NanoString PanCancer Panel (770 genes). Differential expression used log₂ fold change ≥1.5 or ≤-1.5 and p≤0.05.
Results: A total of 66 PL samples were collected (35 cancer and 31 non-cancer). Of the 31 non-cancer PL, only 19 samples had EV pellets, indicating that in the rest of the samples there were too few EVs to result in an EV pellet. However, all cancer PL had an EV pellet. NTA of the samples that had adequate EV showed that the total quantity of EV (as ×10⁵ particles/mL) was approximately two-fold higher in cancer samples compared to the non-cancer cohort (10.9 × 10⁵ vs. 4.5 × 10⁵; p=0.0005). The difference between the cancer and non-cancer PL EV quantity was retained even after systemic therapy (9.7 × 10⁵ vs. 4.5 × 10⁵; p=0.0002). Cryo-EM demonstrated distinct structural differences of EV between the groups, with cancer-derived EVs exhibiting multilayered, multicompartment, and cargo-dense structures, whereas non-cancer samples predominantly contained simple monolayer vesicles. Gene expression profiling demonstrated significant overexpression of genes involved in epithelial-mesenchymal transition, metastasis, cell-cycle regulation, angiogenesis, and extracellular matrix organization (STAB1, PRF1, CCDC80, SPARCL1, VHL, RHOA) in the cancer cohort compared to the non-cancer cohort.
Conclusion: Our study represents one of the largest studies of PL comparison of GI PC to non-cancer. In GI PC the amount of EV and the biophysical and molecular signatures are distinctly different from the non-cancer group. PL EV can be developed into a liquid biopsy in GI PC to fill a critical clinical need.
利益披露 Disclosure
F. Tajik, None..
V. Radhakrishnan, None..
A. N. Dang, None..
A. M. Khan, None..
M. Roman, None..
S. Daly, None..
A. Grigorian, None..
C. Barrios, None..
S. Burruss, None..
M. Senthil, None.