Involvement of CX3CR1+ cells appearing in the abdominal cavity in the immunosuppressive environment immediately after gastric cancer surgery

Abstract

Background: Gastric cancer is primarily treated by surgery; however, little is known about the changes in the intraperitoneal immune environment and the prognostic impact of surgery. Surgical stress and cancer-associated inflammation cause immune cells to mobilize into the abdominal cavity via numerous cytokines. One such cytokine, CX3CR1, has various immune-related functions that remain to be fully explained. We characterized the intraperitoneal immune environment by investigating CX3CR1⁺ cells in intraperitoneal lavage fluid during gastric cancer surgery.

Methods: Lavage fluid samples were obtained from a total of 41 patients who underwent gastrectomy. The relative expression of various genes was analyzed using quantitative real-time PCR. The association of each gene expression with clinicopathological features and surgical outcomes was examined. The fraction of CX3CR1⁺ cells was analyzed by flow cytometry. Cytokine profiles in lavage fluid samples were investigated using a cytometric beads array.

Results: CX3CR1^high patients exhibited higher levels of perioperative inflammation in blood tests and more recurrences than CX3CR1^low patients. CX3CR1^high patients tended to exhibit higher pathological T and N stage than CX3CR1^low patients. CX3CR1 was primarily expressed on myeloid-derived suppressor cells and tumor-associated macrophages. In particular, polymorphonuclear myeloid-derived suppressor cells were associated with perioperative inflammation, pathological N, and recurrences. These immunosuppressive cells were associated with a trend toward unfavorable prognosis. Moreover, CX3CR1 expression was correlated with programmed death-1 expression.

Conclusions: Our results suggest that CX3CR1⁺ cells are associated with an acute inflammatory response, tumor-promotion, and recurrence. CX3CR1 expression could be taken advantage of as a beneficial therapeutic target for improving immunosuppressive state in the future. In addition, analysis of intra-abdominal CX3CR1⁺ cells could be useful for characterizing the immune environment after gastric cancer surgery.

Keywords: CX3CR1; Fractalkine receptor; Gastric cancer; Intraperitoneal lavage fluid; Myeloid-derived suppressor cell; Programmed death 1; Tumor-associated macrophage.

Fig. 1

a The change of CX3CR1 expression through surgical maneuvers. b, c Comparison of relapse-free survival and overall survival based on levels of various molecules. P<0.05, statistically significant. b Relapse-free survival. c Overall survival. d Comparison of OLR1, CD163, and PDCD1 gene expression between CX3CR1^high and CX3CR1^low patients. *P<0.05, statistically significant. e Correlogram. Circle size indicates the degree of correlation. Figures in circles indicate correlation coefficients. Darker colors of circles indicate more-significant correlations. *P<0.05, statistically significant

Fig. 2

Flow cytometry analysis (n=8). a Representative contour plots of CD3 and LOX-1 positivity among CX3CR1⁺ cells. b Representative contour plots of CD14 and CD163 positivity among CX3CR1⁺ cells. c Representative histograms of CX3CR1⁺ cells classified by CD3, LOX-1, CD14, and CD163 positivity. d, e Bar graphs showing the mean and standard error. (d) Comparison of CD3, LOX-1, and CD163 positivity among CX3CR1⁺ cells. *P<0.05, statistically significant. NS, not significant. e Representative contour plots of FoxP3, LOX-1, and CD163 positivity among CX3CR1⁺ cells. f Comparison of FoxP3, LOX-1, and CD163 positivity among CX3CR1⁺ cells. *P<0.05, statistically significant. g Comparison of CD163 expression among CD14⁺CX3CR1⁺ cells. *P<0.05, statistically significant.

Fig. 3

Cytokine profiles of Closure samples. a Heatmap. CX3CR1 group is divided into two subgroups: the high group is shown in dark blue, and the low group is shown in light blue. b Comparison of levels of various cytokines between the CX3CR1^high and CX3CR1^low groups. P<0.05, statistically significant.