High infiltration of CD68-tumor associated macrophages, predict poor prognosis in Kazakh esophageal cancer patients

Abstract

Tumor-associated macrophages (TAMs), the most important immune cells in tumor microenvironment, were reported to play a key role in cancer progression, but the correlation of TAMs and Kazakh esophageal squamous cell carcinoma (ESCC) was still not clear, so we sought to identify the function of TAMs in Kazakh ESCC clinicopathological and prognostic evaluation. CD68 as the TAMs marker, and immunohistochemistry (IHC) was used to quantify the TAMs infiltrated in tumor nest and stroma, the IHC staining was also used to evaluate the expression of MMP-9 in Kazakh ESCCs. The density of CD68-TAMs in ESCCs tumor nest and stromal, were significantly higher than those of CANs (P<0.05). The increasing number of CD68-positive TAMs in tumor nest and stromal were positively associated with tumors lymph node metastasis and clinical stage (P<0.05). The expression of MMP-9 in Kazakh ESCCs was higher than that of CAN tissues (P<0.05). Increased MMP-9 expression in ESCCs was significantly associated with lymph node metastasis and tumor clinical stage (P<0.05). Importantly, the number of CD68-positive TAMs in ESCCs was significantly correlated with the expression of MMP-9 (P<0.05). Furthermore, the survival analyses demonstrated that high-density of CD68-TAMs in tumor nest was positively related to the shorter overall survival time of patients (P<0.05). Increasing numbers of CD68-TAMs promote higher expression of MMP-9 and may play an important role in the occurrence and progression of Kazakh ESCCs, and which could be used as important prognostic markers for Kazakh ESCCs.

Keywords: CD68; Kazakh; MMP-9; Tumor associated macrophages; esophageal squamous cell carcinoma.

Figure 1

The distribution of CD68-positive TAMs in Kazakh ESCC tumor nest tissue, CAN epithelial, and CAN stroma. Immunohistochemical staining of CD68, which was used to mark TAMs and to evaluate the density of TAMs in ESCC and CAN tissues. A and B. Show the distribution of TAMs in CAN epithelial and stroma. A Small number of CD68-positive TAMs appear in CAN tissues. C and D. Show the distribution of TAMs in ESCC tumor nest and stromal tissues, demonstrating that CD68 reveals diffuse staining of membranes and cytoplasm of TAMs, and showing the high density of TAMs located in ESCC tissues (especially in tumor stroma).

Figure 2

Comparison with the density of CD68 positive TAM in ESCC and CAN. A. The density of CD68 positive TAM was significantly higher in ESCC nest than that in CAN epithelium (P<0.001). B. The density of CD68 positive TAM was significantly higher in ESCC stroma than that in CAN stroma (P<0.001).

Figure 3

Immunohistochemical staining of MMP-9 in Kazakh ESCC and CAN tissues. MMP-9 staining is primarily observed in tumor stroma (cell membranes and cytoplasm); some ESCC cells also show staining. A. Negative MMP-9 staining is shown in CAN tissues (scored as 0). B. Weak MMP-9 staining is shown in Kazakh ESCC tissues (scored as 1). C and D. Show moderate and strong MMP-9 staining in Kazakh ESCC tissues, respectively (scored as 2 and 3, respectively).

Figure 4

Kaplan-Meier survival curves showing 100 ESCC patients with expression of CD68. A. Patients with high expression of CD68 in nest experienced a significantly shorter postoperative survival time than those with low CD68 levels (P = 0.017). B. Patients with CD68 over expression had a higher risk of death than those with lower CD68 levels (P = 0.017). C. Patients with high expression of CD68 in stroma has negative relationship than those with low CD68 levels (P = 0.078). D. Patients with CD68 over expression had no risk of death than those with lower CD68 levels (P = 0.078).