Association of tumour-associated macrophages with cancer cell EMT, invasion, and metastasis of Kazakh oesophageal squamous cell cancer

Abstract

Background: Tumour-associated macrophages (TAMs) play an important role in the growth, progression, and metastasis of tumours. Epithelial-mesenchymal transition (EMT) is a mechanism for tumour invasion and metastasis. In this study, we aimed to determine whether TAMs can induce EMT for the invasion and metastasis of Kazakh oesophageal squamous cell cancer (ESCC).

Methods: CD163 was used as a marker for TAMs, and the density of TAMs in tumour nest and surrounding stroma was quantified using immunohistochemistry (IHC). IHC staining was used to evaluate the expression of E-cadherin (epithelial marker) and vimentin (mesenchymal marker) in Kazakh ESCC and cancer-adjacent normal tissues (CANs). Additionally, 6-well transwell plates (0.4 μm) were used to establish the co-culture system of ESCC (EC109 or EC9706) cells and macrophages. Real-time quantitative polymerase chain reaction (qPCR) and western blot experiments were used to determine whether ESCC cells undergo EMT transformation after co-culture with macrophages. Transwell assays were used to detect the migration and invasion of the ESCC cells.

Results: The distribution of CD163-positive TAMs in cancer tissues was closely related to EMT in Kazakh ESCC. The expression of vimentin in the ESCC cells was significantly upregulated, the expression of E-cadherin was significantly downregulated, and the invasion and migration of the ESCC cells were significantly enhanced after tumour-associated macrophages were added to the co-culture.

Conclusions: Tumour-associated macrophages promote EMT in ESCC, which may be one of the important factors involved in the invasion and progression of Kazakh ESCC.

Keywords: Epithelial-mesenchymal transition; Kazakh; Migration and invasion; Oesophageal squamous cell carcinoma; Tumour-associated macrophages.

Fig. 1

Distribution of CD163-positive M2 macrophages in Kazakh ESCC and CAN tissues. (a) and (b) showed the distribution of M2 macrophages in ESCC tumour stromal and islet, respectively. CD163 revealed diffuse staining of membranes and cytoplasm of M2 macrophages and showed the high density of M2 macrophages located in ESCC tissues (especially in tumour stroma). (c) and (d) showed the distribution of TAMs in CAN stroma and epithelia, respectively. A small number of CD163-positive M2 macrophages appear in CAN tissues

Fig. 2

Detection of E-cadherin and Vimentin expression in Kazakh ESCC and CANs by IHC E-cadherin and vimentin staining was localized predominantly in the cytomembrane and (or) cytoplasm. (a) Positive E-cadherin staining is shown in CAN tissues (scored as 3). (b) Negative E-cadherin staining is shown in CAN tissues (scored as 0). (c) and (d) show weak and moderate E-cadherin staining in Kazakh ESCC tissues, respectively (scored as 1 and 2, respectively). (e) Negative vimentin staining is shown in CAN tissues (scored as 0). (f) Weak vimentin staining is shown in ESCC tissues (scored as 1). (g) and (h) show moderate and strong vimentin staining in Kazakh ESCC tissues, respectively (scored as 2 and 3, respectively)

Fig. 3

TAMs induce the cancer cells to undergo EMT. (a) PMA-treated THP-1 cells were differentiated into adherent macrophages. (b) EC109 and EC9706 cells presented typical cobblestone and epithelial-like appearances, respectively; with CM treatment, they were scattered and spindle shaped, and exhibited fibroblast-like appearances. The photographs were taken at 100x magnification. (c) The relative mRNA expression levels of E-cadherin and vimentin were determined by qPCR, respectively. The housekeeping gene GAPDH was used as the control. (d) The EMT-related proteins E-cadherin and vimentin were examined by western blotting, respectively; β-actin was used as a loading control. (e) and (f) Macrophages promote the migration and invasion of ESCC cells, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001