Inflammatory events during murine squamous cell carcinoma development

Abstract

Background: Squamous cell carcinoma (SCC) is one of the most common human cancers worldwide. In SCC, tumour development is accompanied by an immune response that leads to massive tumour infiltration by inflammatory cells, and consequently, local and systemic production of cytokines, chemokines and other mediators. Studies in both humans and animal models indicate that imbalances in these inflammatory mediators are associated with cancer development.

Methods: We used a multistage model of SCC to examine the involvement of elastase (ELA), myeloperoxidase (MPO), nitric oxide (NO), cytokines (IL-6, IL-10, IL-13, IL-17, TGF-β and TNF-α), and neutrophils and macrophages in tumour development. ELA and MPO activity and NO, IL-10, IL -17, TNF-α and TGF-β levels were increased in the precancerous microenvironment.

Results: ELA and MPO activity and NO, IL-10, IL -17, TNF-α and TGF-β levels were increased in the precancerous microenvironment. Significantly higher levels of IL-6 and lower levels of IL-10 were detected at 4 weeks following 7,12-Dimethylbenz(a)anthracene (DMBA) treatment. Similar levels of IL-13 were detected in the precancerous microenvironment compared with control tissue. We identified significant increases in the number of GR-1+ neutrophils and F4/80+/GR-1- infiltrating cells in tissues at 4 and 8 weeks following treatment and a higher percentage of tumour-associated macrophages (TAM) expressing both GR-1 and F4/80, an activated phenotype, at 16 weeks. We found a significant correlation between levels of IL-10, IL-17, ELA, and activated TAMs and the lesions. Additionally, neutrophil infiltrate was positively correlated with MPO and NO levels in the lesions.

Conclusion: Our results indicate an imbalance of inflammatory mediators in precancerous SCC caused by neutrophils and macrophages and culminating in pro-tumour local tissue alterations.

Figure 1

Squamous cell carcinoma induced by DMBA/TPA in mice. SCC mice were treated according to a chemical carcinogenic protocol using DMBA and TPA for 16 weeks. Papilloma incidence (A) and tumor volume (B) were determined in SCC mice. Each value represents mean ± SEM of 9 different mice. *P<0.05, **P<0.01 and *** P< 0.001. Panels C, E and G are representative photomicrographs of dorsal tissue from SCC mice. Haematoxylin and eosin staining of skin tissue sections from BALB/c mice 4(D), 8(F) and 16 (H) weeks after chemical carcinogenesis. Data are from one experiment that is representative of three independent experiments (n = 9 mice per group). Arrows indicate inflammatory cells.

Figure 2

MPO, NO and ELA levels in the tumour microenvironment. MPO (A), NO (B) and ELA (C) levels were analyzed in the tumor and control untreated tissue as described in the methods and materials section. Results are expressed as the mean ± SEM from each individual mouse analyzed. *P<0.05, **P<0.01 and *** P< 0.001.

Figure 3

Cytokine levels in the tumour microenvironment. IL-6 (A), IL-10 (B), IL-13 (C), IL-17 (D), TNF-α (E) and TGF-β (F) levels were analyzed by ELISA. Results are expressed as the mean ± SEM from each individual mouse analyzed (n=9 mice per group). *P<0.05, **P<0.01 and *** P < 0.001.

Figure 4

Inflammatory infiltrates in mouse squamous cell carcinoma. The total number of leukocytes (A) and the number of cells expressing GR1 and F4/80 (B) were determined during the 4^th (4W), 8^th (8W) and 16^th (16W) weeks after DMBA protocol. Results are expressed as the mean ± SEM from each individual mouse analyzed (n=9 mice per group). *P< 0.05 and *** P< 0.001. Representative photomicrograph of GR1⁺ (green), F4/80⁺ (red) and IL-17⁺ cells (red) infiltrating tumour lesions. Representative tumour is shown. Blue, DAPI.