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. 2019 Jan 7;38(1):6.
doi: 10.1186/s13046-018-1003-0.

Tumor-associated neutrophils induce EMT by IL-17a to promote migration and invasion in gastric cancer cells

Sen Li  1   2 , Xiliang Cong  2 , Hongyu Gao  2 , Xiuwen Lan  2 , Zhiguo Li  2 , Wenpeng Wang  3 , Shubin Song  2 , Yimin Wang  2 , Chunfeng Li  2 , Hongfeng Zhang  2 , Yuzhou Zhao  4 , Yingwei Xue  5
Affiliations

Tumor-associated neutrophils induce EMT by IL-17a to promote migration and invasion in gastric cancer cells

Sen Li et al. J Exp Clin Cancer Res. .

Erratum in

Abstract

Purpose: Epithelial to mesenchymal transition (EMT) can contribute to gastric cancer (GC) progression and recurrence following therapy. Tumor-associated neutrophils (TANs) are associated with poor outcomes in a variety of cancers. However, it is not clear whether TANs interact with the EMT process during GC development.

Methods: Immunohistochemistry was performed to examine the distribution and levels of CD66 + neutrophils in samples from 327 patients with GC. CD66b + TANs were isolated either directly from GC cell suspensions or were conditioned from healthy donor peripheral blood polymorphonuclear neutrophils (PMNs) stimulated with tumor tissue culture supernatants (TTCS) and placed into co-culture with MKN45 or MKN74 cells, after which migration, invasion and EMT were measured. Interleukin-17a (IL-17a) was blocked with a polyclonal antibody, and the STAT3 pathway was blocked with the specific inhibitor AG490.

Results: Neutrophils were widely distributed in gastric tissues of patients with GC and were enriched predominantly at the invasion margin. Neutrophil levels at the invasion margin were an independent predictor of poor disease-free survival (DFS) and disease-specific survival (DSS). IL-17a + neutrophils constituted a large portion of IL-17a-producing cells in GC, and IL-17a was produced at the highest levels in co-culture compared with that in TANs not undergoing co-culture. TANs enhanced the migration, invasion and EMT of GC cells through the secretion of IL-17a, which activated the Janus kinase 2/signal transducers and activators of transcription (JAK2/STAT3) pathway in GC cells, while deprivation of IL-17a using a neutralizing antibody or inhibition of the JAK2/STAT3 pathway with AG490 markedly reversed these TAN-induced phenotypes in GC cells induced by TANs.

Conclusions: Neutrophils correlate with tumor stage and predict poor prognosis in GC. TANs produce IL-17a, which promotes EMT of GC cells through JAK2/STAT3 signalling. Blockade of IL-17a signalling with a neutralizing antibody inhibits TAN-stimulated activity in GC cells. Therefore, IL-17a-targeted therapy might be used to treat patients with GC.

Keywords: Epithelial mesenchymal transition; Gastric cancer; IL-17a; Migration and invasion; Neutrophils.

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Conflict of interest statement

Ethics approval and consent to participate

The present study was authorized by the Ethics Committee of Harbin Medical University Cancer. All procedures performed in studies were in accordance with the ethical standards. Informed consent was obtained from all patients and volunteers before they were included in the study.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Representative picture of immunohistochemical staining of CD66b + neutrophils at the nontumoral, invasive margin and tumor center tissues of GC sampers. a High CD66b + neutrophils density at the nontumoral gastric tissues. b Low CD66b + neutrophils density at the nontumoral gastric tissues. c High CD66b + neutrophils density at the invasive margin of GC tissues. d Low CD66b + neutrophils density at the invasive margin of GC tissues. e High CD66b + neutrophils density at the tumor center of GC tissues. f Low CD66b + neutrophils density at the tumor center of GC tissues. Magnifications: × 200
Fig. 2
Fig. 2
Kaplan-Meier curves of DFS and DSS based on the number of neutrophils at the nontumoral, invasive margin and tumor center tissues of patients with GC. a, d Higher number of neutrophils at the nontumoral tissues were not correlated with prognosis (P = 0.981 and P = 0.896). b, e Higher number of neutrophil at the invasive margin of GC tissues were closely correlated with poor DFS and DSS (P = 0.001 and P = 0.001). c, f Higher number of neutrophil at the tumor center of GC tissues were closely correlated with poor DFS and DSS (P < 0.001 and P < 0.001)
Fig. 3
Fig. 3
IL-17a protein is primarily expressed by neutriphils in GC. a, b Association of CD66b + neutriphils and IL-17a + cells at the invasive margin of GC tissues by immunohistochemical staining. Magnifications: × 200. c Analysis of IL-17a and CD66b distribution in GC tissues by immunofluorescence microscope. One of 10 representative micrographs is shown. Magnifications: × 400. d The production of IL-17a in the cell supernatant in the gastric cell lines (GES-1, MKN45 and MK74), the production of IL-17a in the cell supernatant in the neutrophils isolated from GC tissues and nontumoral tissues, neutrophils activated by TTCS and NTCS, the production of IL-17a in the co-culture system were quantified 24 h after change the culture medium by ELISA. * P < 0.05. e the expression level of IL-17a mRNA in the GC cells (MKN45 and MK74), GC cells co-cultured neutrophils, neutrophils, and neutrophils co-cultured GC cells were quantified 24 h after change the culture medium by qRT-PCR. ** P < 0.001
Fig. 4
Fig. 4
Neutrophils enhance the migration, invasiveness and EMT of GC cells through IL-17a. (a, b) The effect of neutrophils on the migration and invasion ability of GC cells (MKN45 and MKN74) was determined 24 h when IL-17a neutralizing antibody or IgG isotype control antibody was added to Transwell co-culture chamber. Magnifications: × 100. *, P < 0.05; **, P < 0.001. c Protein expression of E-cadherin, Vimentin, and ZEB1 in GC cells (MKN45 and MKN74) co-cultured with neutrophils were analyzed by western blot when IL-17a neutralizing antibody or IgG isotype control antibody was added to the Transwell co-culture system. Densitometric analysis of E-cadherin, Vimentin, and ZEB1 expression were shown. **, P < 0.001
Fig. 5
Fig. 5
IL-17a activates the STAT3 signalling pathway and promotes EMT, migration and invasiveness of GC cells. a, b Protein expression of p-JAK2, JAK2, p-STAT3, and STAT3 in GC cells (MKN45 and MKN74) co-cultured with neutrophils were analyzed by western blot when IL-17a neutralizing antibody or AG490 was added to the Transwell co-culture system. Densitometric analysis of p-JAK2, JAK2, p-STAT3, and STAT3 expression were shown. *, P < 0.05. c, d The effect of neutrophils on the migration and invasion ability of GC cells (MKN45 and MKN74) were determined when AG490 was added to the Transwell co-cultured chamber. Magnifications: × 100. *, P < 0.05; **, P < 0.01. e Protein expression of E-cadherin, Vimentin, and ZEB1 in GC cells (MKN45 and MKN74) co-cultured with neutrophils were analyzed when AG490 was added to Transwell co-culture system. Densitometric analysis of E-cadherin, Vimentin, and ZEB1 expression were shown. *, P < 0.05
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