低氧微环境诱导的中性粒细胞胞外诱捕网促进胃癌肿瘤生长。

Neutrophil extracellular traps induced by the hypoxic microenvironment in gastric cancer augment tumour growth.

机构信息

Department of General Surgery, The First Affiliated Hospital of Jiamusi University, Heilongjiang Province, Jiamusi, 154000, China.

Digestive Disease Center, The First Affiliated Hospital of Jiamusi University, Heilongjiang Province, Jiamusi, 154000, China.

出版信息

Cell Commun Signal. 2023 May 1;21(1):86. doi: 10.1186/s12964-023-01112-5.

DOI:10.1186/s12964-023-01112-5

PMID:37127629

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10152773/

Abstract

BACKGROUND

Inflammation-related predisposition to cancer plays an essential role in cancer progression and is associated with poor prognosis. A hypoxic microenvironment and neutrophil infiltration are commonly present in solid tumours, including gastric cancer (GC). Neutrophil extracellular traps (NETs) have also been demonstrated in the tumour immune microenvironment (TIME), but how NETs affect GC progression remains unknown. Here, we investigated the role of NET formation in the TIME and further explored the underlying mechanism of NETs in GC tumour growth.

METHODS

Hypoxia-induced factor-1α (HIF-1α), citrulline histone 3 (citH3) and CD66b expression in tumour and adjacent nontumor tissue samples was evaluated by western blotting, immunofluorescence and immunohistochemical staining. The expression of neutrophil-attracting chemokines in GC cells and their hypoxic-CM was measured by qRT‒PCR and ELISA. Neutrophil migration under hypoxic conditions was evaluated by a Transwell assay. Pathway activation in neutrophils in a hypoxic microenvironment were analysed by western blotting. NET formation was measured in vitro by immunofluorescence staining. The protumour effect of NETs on GC cells was identified by Transwell, wound healing and cell proliferation assays. In vivo, an lipopolysaccharide (LPS)-induced NET model and subcutaneous tumour model were established in BALB/c nude mice to explore the mechanism of NETs in tumour growth.

RESULTS

GC generates a hypoxic microenvironment that recruits neutrophils and induces NET formation. High mobility group box 1 (HMGB1) was translocated to the cytoplasm from the nucleus of GC cells in the hypoxic microenvironment and mediated the formation of NETs via the toll-like receptor 4 (TLR4)/p38 MAPK signalling pathway in neutrophils. HMGB1/TLR4/p38 MAPK pathway inhibition abrogated hypoxia-induced neutrophil activation and NET formation. NETs directly induced GC cell invasion and migration but not proliferation and accelerated the augmentation of GC growth by increasing angiogenesis. This rapid tumour growth was abolished by treatment with the NET inhibitor deoxyribonuclease I (DNase I) or a p38 MAPK signalling pathway inhibitor.

CONCLUSIONS

Hypoxia triggers an inflammatory response and NET formation in the GC TIME to augment tumour growth. Targeting NETs with DNase I or HMGB1/TLR4/p38 MAPK pathway inhibitors is a potential therapeutic strategy to inhibit GC progression. Video Abstract.

摘要

背景

炎症相关的致癌倾向在癌症进展中起着至关重要的作用，并与预后不良相关。缺氧微环境和中性粒细胞浸润在包括胃癌（GC）在内的实体瘤中很常见。中性粒细胞胞外诱捕网（NETs）也已在肿瘤免疫微环境（TIME）中被证实，但 NETs 如何影响 GC 的进展尚不清楚。在这里，我们研究了 NET 形成在 TIME 中的作用，并进一步探讨了 NET 在 GC 肿瘤生长中的潜在机制。

方法

通过 Western blot、免疫荧光和免疫组织化学染色评估肿瘤和相邻非肿瘤组织样本中缺氧诱导因子-1α（HIF-1α）、瓜氨酸组蛋白 3（citH3）和 CD66b 的表达。通过 qRT-PCR 和 ELISA 测量 GC 细胞及其缺氧-CM 中吸引中性粒细胞的趋化因子的表达。通过 Transwell 测定在缺氧条件下中性粒细胞的迁移。通过 Western blot 分析缺氧微环境中中性粒细胞中途径的激活。通过免疫荧光染色在体外测量 NET 的形成。通过 Transwell、划痕愈合和细胞增殖测定鉴定 NETs 对 GC 细胞的促肿瘤作用。体内，在 BALB/c 裸鼠中建立脂多糖（LPS）诱导的 NET 模型和皮下肿瘤模型，以探讨 NET 在肿瘤生长中的机制。

结果

GC 产生缺氧微环境，招募中性粒细胞并诱导 NET 形成。高迁移率族蛋白 B1（HMGB1）从缺氧微环境中 GC 细胞的核转移到细胞质，并通过中性粒细胞中的 toll 样受体 4（TLR4）/p38 MAPK 信号通路介导 NET 的形成。HMGB1/TLR4/p38 MAPK 通路抑制剂阻断缺氧诱导的中性粒细胞活化和 NET 形成。NETs 直接诱导 GC 细胞侵袭和迁移，但不诱导增殖，并通过增加血管生成加速 GC 生长的增强。NET 抑制剂脱氧核糖核酸酶 I（DNase I）或 p38 MAPK 信号通路抑制剂的治疗消除了这种快速的肿瘤生长。