Tumor-associated macrophage, angiogenesis and lymphangiogenesis markers predict prognosis of non-small cell lung cancer patients

Abstract

Background: The tumor microenvironment (TME) is a critical player in tumor progression, metastasis and therapy outcomes. Tumor-associated macrophages (TAMs) are a well-recognized core element of the TME and generally characterized as M2-like macrophages. TAMs are believed to contribute to tumor progression, but the mechanism behind this remains unclear. We aimed to investigate the clinical, angiogenic, and lymphangiogenic significance of TAMs in non-small cell lung cancer (NSCLC).

Methods: Utilizing combined immunohistochemistry and digital image analysis, we assessed CD68, CD163, VEGF-A, and VEGF-C expression in 349 patients with NSCLC. Subsequently, the potential association between M2 TAMs and angiogenic VEGF-A and/or lymphangiogenic VEGF-C was evaluated for its prognostic value. Furthermore, the effects of M2 TAMs on angiogenesis and lymphangiogenesis were explored via an in vitro co-culture system.

Results: CD68 and CD163 expression were found to directly correlate with VEGF-A and/or VEGF-C expression (all p < 0.001). Furthermore, elevated M2 ratio (CD163+/CD68+) was significantly associated with poor overall survival (p = 0.023). Dual expression of M2 ratio^high and VEGF-C^high (M2 ratio^highVEGF-C^high) was correlated with worse overall survival (p = 0.033). Multivariate analysis revealed that M2 ratio^high [HR (95% CI) = 1.53 (1.01-2.33), p = 0.046] and combined M2 ratio^highVEGF-C^high expression [HR (95% CI) = 2.01 (1.28-3.16), p = 0.003] were independent predictors of poor overall survival. Notably, we confirmed that M2 macrophages significantly enhanced the protein and mRNA expression of both VEGF-A and VEGF-C, while M1 macrophages induced only mRNA expression of VEGF-A in A549 cells.

Conclusions: This study suggests that TAMs are significantly associated with angiogenesis and lymphangiogenesis, contributing to the progression of NSCLC. Furthermore, elevated M2 ratio, similar to combined high M2 ratio and high VEGF-C expression, is a strong indicator of poor prognosis in patients with NSCLC, providing insight for future TAM-based immunotherapy strategies.

Keywords: Angiogenesis; CD163+/CD68+ ratio; Lymphangiogenesis; Non-small cell lung cancer; Prognosis; Tumor-associated macrophage; Vascular endothelial growth factor.

Fig. 1

Tumor-associated macrophage markers (CD68 & CD163), VEGF-A, and VEGF-C expression in human non-small cell lung cancer (NSCLC) tissues. Representative immunohistochemical images of CD68 (a), CD163 (b), double staining of CD68 and CD163 (c), VEGF-A (d), and VEGF-C (e), immunoglobulin G (IgG) isotype control (f). Arrowhead indicates dual stained CD163 (red) and CD68 (brown), and asterisk indicates CD68. Scale bar = 100 µm (30 µm in inset)

Fig. 2

Correlation among tumor-associated macrophage-, angiogenesis- and lymphangiogenesis-related markers in patients with NSCLC. CD68 expression positively correlated with CD163 (a), VEGF-A (b), and VEGF-C (c) expression. CD163 expression showed a significant positive correlation with VEGF-A (d) and VEGF-C (e) expression. There is a strong positive correlation between VEGF-A and VEGF-C (f)

Fig. 3

The Kaplan–Meier survival analysis of NSCLC patients. Patients expressing CD68 had better overall survival than patients who did not (OS rate, 78.2% vs. 68.6%, log rank p = 0.023) (a), while CD163 was not associated with patient survival (b). Patients with a high M2 ratio (CD163/CD68) had significantly shorter overall survival than patients with a low M2 ratio (OS rate, 68.4% vs. 78.3%, log rank p = 0.023) (c). There were no meaningful overall survival differences for VEGF-A (d) and VEGF-C (e) expression in NSCLC patients

Fig. 4

Survival analysis of NSCLC patients with M2 ratio expression according to angiogenesis (VEGF-A) or lymphangiogenesis (VEGF-C) marker expression in NSCLC patients. Dual expression of high M2 ratio and high VEGF-A (M2 ratio^high/VEGF-A^high) exhibited a tendency of worse overall survival (p = 0.056) (a). Survival times for patients with both high M2 ratio and high VEGF-C (M2 ratio^highVEGF-C^high) expression was significantly shorter (OS rate, 62.2%) than those for patients with M2 ratio^lowVEGF-C^low (OS rate, 75.6%), M2 ratio^lowVEGF-C^high (OS rate, 80.6%), and M2 ratio^highVEGF-C^low (OS rate, 73.9%) (log rank p = 0.033) (b)

Fig. 5

Effect of macrophage polarization on NSCLC cell angiogenesis and lymphangiogenesis. A549 cells were co-cultured with M0 macrophages, M1 macrophages, polarized by LPS, or M2 macrophages, polarized by IL4 + IL13. After 48 h of co-culture, total RNA and protein were extracted from the cells as indicated. Gene expression was assessed with quantitative real-time PCR (a, b), and protein expression was quantitated using specific ELISA kits (c, d). VEGF-A and VEGF-C mRNA levels increased in M2 macrophages significantly. However, M1 macrophages elevated only VEGF-A mRNA, not VEGF-C, in A549 cells. In order to assess the levels of VEGF-A and VEGF-C mRNA in polarized macrophages, the macrophages were collected after 72 h of culturing with LPS or IL4 + IL13, total RNA and proteins were extracted, and gene and protein expression were assessed with quantitative real-time PCR (e, f) and ELISA (g, h) as well. The mRNA levels of VEGF-A and VEGF-C were significantly decreased in M2 macrophages polarized by IL4 + IL13, compared to M1 macrophages polarized by LPS or M0 macrophages cultured with vehicles. Error bars represent S.D; *p < 0.05, **p < 0.01, ***p < 0.001. Asterisk represents the significance compared to A549 alone or macrophage with vehicle