Prognostic significance of interleukin-8 and CD163-positive cell-infiltration in tumor tissues in patients with oral squamous cell carcinoma

Abstract

Purpose: We investigated whether serum interleukin (IL)-8 reflects the tumor microenvironment and has prognostic value in patients with oral squamous cell carcinoma (OSCC).

Experimental design: Fifty OSCC patients who received radical resection of their tumor(s) were enrolled. Preoperative sera were measured for IL-8 by ELISA. Expression of IL-8 and the infiltration of immune cells in tumor tissues were analyzed by an immunohistochemical staining of surgical specimens.

Results: We found that disease-free survival (DFS) was significantly longer in the Stage I/II OSCC patients with low serum IL-8 levels compared to those with high levels (p = 0.001). The tumor expression of IL-8, i.e., IL-8(T) and the density of CD163-positive cells in the tumor invasive front, i.e., CD163(IF) were correlated with the serum IL-8 level (p = 0.033 and p = 0.038, respectively), and they were associated with poor clinical outcome (p = 0.007 and p = 0.002, respectively, in DFS) in all patients. A multivariate analysis revealed that N status, IL-8(T) and CD163(IF) significantly affected the DFS of the patients. Further analysis suggested that combination of N status with serum IL-8, IL-8(T) or CD163(IF) may be a new criterion for discriminating between OSCC patients at high and low risk for tumor relapse. Interestingly, the in vitro experiments demonstrated that IL-8 enhanced generation of CD163-positive M2 macrophages from peripheral blood monocytes, and that the cells produced IL-10.

Conclusions: These findings indicate that IL-8 may be involved in poor clinical outcomes via generation of CD163-positive M2 macrophages, and that these factors in addition to N status may have prognostic value in patients with resectable OSCSS.

Figure 1. The relationship between serum IL-8 levels and the clinical outcome in OSCC patients who underwent radical resection of their tumor(s).

The differences in the serum IL-8≥7.5 pg/ml vs. IL-8<7.0 pg/ml in all patients, in the Stage I/II patients and in the Stage III/IV patients were calculated by log-rank test. *P<0.05. NA: not analyzed because the number of OS events was only one.

Figure 2. Relationship between IL-8 expression, CD163-positive cell-infiltration in tumor tissues and clinical outcome of OSCC patients.

(A) Immunohistochemical staining for IL-8 and CD163. Most of the IL-8-positive cells were tumor cells, not stromal cells in tumor tissues. CD163-positive cells had infiltrated into the tumor invasive front but not the intra-tumor region. (B) Differences in IL-8(T)(+) vs. IL-8(T)(−) and CD163(IF)High vs. CD163(IF)Low in OS and DFS in OSCC patients, calculated by log-rank test. *P<0.05.

Figure 3. The combination of independent factors for predicting the DFS of OSCC patients.

Differences in the DFS of OSCC patients with (A) N0 and IL-8(T)(−) vs. N(+) or IL-8(T)(+), (B) N0 and CD163(IF)Low vs. N(+) or CD163(IF)High, (C) IL-8(T)(−) and CD163(IF)Low vs. IL-8(T)(+) or CD163(IF)High, (D) N0 and IL-8(T)(−) and CD163(IF)Low vs. N(+) or IL-8(T)(+) or CD163(IF)High, (E) N0 and IL-8<7 pg/ml vs. N(+) or IL-8≥7 pg/ml were calculated by log-rank test. *P<0.05.

Figure 4. Generation of CD163-positive M2 macrophages by IL-8.

Healthy donor-derived monocytes were treated with M-CSF (25 ng/ml) for 5 days and then IL-4 (20 ng/ml) and IL-13 (20 ng/ml) for 2 days, or with M-CSF (25 ng/ml) for 5 days and then IL-8 (10 ng/ml) for 2 days. The cell-surface expression of CD163 (A) and CD206 (B) of the cells was evaluated by using a flow cytometric analysis and IL-10 in the culture supernatants was measured by ELISA (R&D Systems) (C). Bars denote SD of 5 samples.