Tumor-associated macrophages correlate with the clinicopathological features and poor outcomes via inducing epithelial to mesenchymal transition in oral squamous cell carcinoma

Abstract

Background: Both tumor-associated macrophages (TAMs) and the epithelial to mesenchymal transition (EMT) of cancer cells play key roles in promoting tumor progression. However, whether TAMs could induce EMT in the progression of oral squamous cell carcinoma (OSCC) remains undefined.

Results: Here we detected the expression of macrophages markers CD68 and CD163, epithelial marker E-cadherin and mesenchymal marker vimentin in 127 OSCC patients by using semi-quantitative immunohistochemistry. CD68 and CD163 expression was not confined to the infiltrating TAMs, but also detected in cancer cells. The high number of CD68-positive macrophages was correlated with poor overall survival. Meanwhile, the expression of CD163 both in macrophages and in cancer cells was associated with poor overall survival and had a significant prognostic impact in OSCC. Importantly, the expression of CD163 in cancer cells had a significant relationship with E-cadherin and vimentin. Furthermore, the incubation of TAMs conditioned medium resulted in a fibroblast-like appearance of cancer cells (HN4, HN6 and SCC9) together with the decreased/increased expression of E-cadherin/ vimentin, which were correlated with the enhanced ability of migration and invasion.

Conclusions: Our results indicate that TAMs could promote the EMT of cancer cells, thereby leading to the progression of oral cancer.

Fig. 1

CD68-positive macrophages and CD163-positive macrophages were examined by immunohistochemistry. CD68 staining a1, a2, a3 and negative staining of CD163 was found in peritumoral stroma (b1, b2, b3). CD68-positive macrophages (c1, c2, c3) and CD163-positive macrophages (d1, d2, d3) were found both in tumor nest and stroma. (a1-d1, 100×; a2-d2, 200×; a3-d3, 400× )

Fig. 2

CD68 and CD163 expression was examined in tumor cells by immunohistochemistry. High expression of CD68 a1, a2, a3 and low expression of CD163 (b1, b2, b3) was found in tumor cells. Low expression of CD68 (c1, c2, c3) and high expression of CD163 (d1, d2, d3) was found in tumor cells. (a1-d1, 100×; a2-d2, 200×; a3-d3, 400×)

Fig. 3

CD68-positive macrophages, CD163-positive macrophages, E-cadherin and vimentin was examined by immunohistochemistry. In oral squamous cell carcinoma patients, the absence or reduced expression of CD68-positive macrophages a1, a2, a3 and increased expression of CD163-positive macrophages (b1, b2, b3) was observed in tumor tissue, where have no expression of E-cadherin (c1, c2, c3), but strong cytoplasmic staining of vimentin (d1, d2, d3). (a1-d1, 100×; a2-d2, 200×; a3-d3, 400×)

Fig. 4

Expression of CD68, CD163, E-cadherin and vimentin in tumor cells. Increased expression of CD68 a1, a2, a3 and CD163 (b1, b2, b3) was observed in tumor cells, whereas there was absence or reduced expression of E-cadherin (c1, c2, c3), but strong cytoplasmic expression of vimentin (d1, d2, d3) at the invasive front of oral squamous cell carcinoma (a1-d1, 100×; a2-d2, 200×; a3-d3, 400×)

Fig. 5

Survival cures of 127 oral squamous cell carcinoma patients. a Overall survival (OS) curves of 127 oral squamous cell carcinoma patients. In OSCC patients, the expression of CD68-positive macrophages and CD163-positive macrophages in tumor nest, CD163 in tumor cells, vimentin both in tumor and at the tumor invasion front was associated with poor OS (b, d, g, j, k). The expression of CD68-positive macrophages and CD163-positive macrophages in tumor stroma, CD68 in tumor cells, E-cadherin both in tumor and at the tumor invasion front was not associated with poor OS (c, e, f, h, i). L, low (≤ 6/ high-power field); H, high (≥ 8/ high-power field); N, negative; L, low expression; H, high expression. CD68+ 1: CD68-positive macrophages counts in tumor nest; CD68+ 2: CD68-positive macrophages counts in tumor stroma; CD163+ 1: CD163-positive macrophages counts in tumor nest; CD163+ 2: CD163-positive macrophages counts in tumor stroma; CD68: CD68 expression in tumors; CD163: CD163 expression in tumors; E-cad 1: E-cadherin expression in tumor; E-cad 2: E-cadherin expression at the invasive front of tumor; vim 1: vimentin expression in tumor; vim 2: vimentin expression at the invasive front of tumor

Fig. 6

The establishment and characterization of tumor-associated macrophages. a The PMA-treated THP-1 cells were differentiated into adherent macrophages. b The Double immunofluorescence staining of CD68 (red) and CD163 (green) in the TAMs. The nuclei were stained with DAPI. The photographs were taken at 200x magnification. c The inflammatory cytokines and chemokines were detected in conditioned medium by using mass spectral analysis

Fig. 7

TAMs induce the cancer cells undergo epithelial to mesenchymal transition (EMT). a The HN4, HN6 and SCC9 cells presented typical cobblestone and epithelial-like appearances. The HN4-M, HN6-M and SCC9-M cells were scattered and spindle shaped, exhibited fibroblast-like appearances. The photographs were taken at 40x magnification. b The relative mRNA expression levels of E-cadherin, and vimentin was performed by real-time RT-PCR. The housekeeping gene GAPDH was used as the control. The data were reported as mean ± SD. c The EMT-related proteins E-cadherin and vimentin were examined by the Western blot, β-actin was used as a loading control. The data were reported as mean ± SD. * P < 0.05, ** P < 0.01

Fig. 8

The Double immunofluorescence staining of E-cadherin (red) and vimentin (green) in the cells. The nuclei were stained with DAPI. The HN4-M, HN6-M and SCC9-M cells were scattered, exhibited fibroblast-like appearances, showed increased expression of vimentin and decreased expression of E-cadherin (400x magnification)

Fig. 9

TAMs enhance the cancer cells migration and invasion in vitro. a The HN4-M, HN6-M and SCC9-M cells could grow to confluency easier than the control cells after 24 h. b The cells invading the filters coated with the Matrigel. The photographs were taken at 100x magnification. (c) The quantification of the cell invasion in five randomly chosen fields. The data were reported as mean ± SD. *** P < 0.001