Silica-induced chronic inflammation promotes lung carcinogenesis in the context of an immunosuppressive microenvironment

Abstract

The association between inflammation and lung tumor development has been clearly demonstrated. However, little is known concerning the molecular events preceding the development of lung cancer. In this study, we characterize a chemically induced lung cancer mouse model in which lung cancer developed in the presence of silicotic chronic inflammation. Silica-induced lung inflammation increased the incidence and multiplicity of lung cancer in mice treated with N-nitrosodimethylamine, a carcinogen found in tobacco smoke. Histologic and molecular analysis revealed that concomitant chronic inflammation contributed to lung tumorigenesis through induction of preneoplastic changes in lung epithelial cells. In addition, silica-mediated inflammation generated an immunosuppressive microenvironment in which we observed increased expression of programmed cell death protein 1 (PD-1), transforming growth factor-β1, monocyte chemotactic protein 1 (MCP-1), lymphocyte-activation gene 3 (LAG3), and forkhead box P3 (FOXP3), as well as the presence of regulatory T cells. Finally, the K-RAS mutational profile of the tumors changed from Q61R to G12D mutations in the inflammatory milieu. In summary, we describe some of the early molecular changes associated to lung carcinogenesis in a chronic inflammatory microenvironment and provide novel information concerning the mechanisms underlying the formation and the fate of preneoplastic lesions in the silicotic lung.

Figure 1

Effects of oropharyngeal administration of silica on mouse lungs. The left panel shows the histologic analysis of lung silica-induced chronic inflammation in sections stained with hematoxylin and eosin. Silica alone-treated mice were sacrificed at months 2 (n = 4), 4 (n = 4), 6.5 (n = 4), 9 (n = 4), and 12 (n = 4) after treatment. Lungs were harvested and all the lung lobes were analyzed (one field per lobe). (A–E) Silicotic granulomas at different experimental points following silica treatment. Insets show the details of the granulomatous proliferations. (F–O) Alveolar (F–J) and bronchiolar (K–O) hyperplasia in areas close to the granulomas at 2, 4, 6.5, 9, and 12 months after silica treatment. Alveolar proteinosis (J), hyperplastic goblet cells (N), and perivascular, peribronchiolar inflammatory reaction (asterisk, O) and lung fibrotic lesions (data not shown) are common features in this model. Images were obtained at the same magnification. The right panel includes representative examples of p-H2AX staining in lungs from NDMA-only- and NDMA-silica-treated mice. Samples were considered positive when six or more cells presented positive immunoreactivity in at least two of three fields analyzed (x40).

Figure 2

Incidence and multiplicity of adenomas and lung tumors in mice treated with NDMA-only or NDMA-silica. (A) Evaluation of adenomatous structures at different experimental time points. (B) Quantitation of adenocarcinomas at month 12 after treatment. NDMA-only-treated mice were analyzed at months 4 (n = 4), 6.5 (n = 4), 9 (n = 4), and 12 (n = 19) after treatment. NDMA-silica-treated mice were analyzed at months 4 (n = 8), 6.5 (n = 8), 9 (n = 8), and 12 (n = 21) after treatment. Incidence rate was calculated as the percentage of mice with at least one lung lesion. Multiplicity is represented as the average of the number of lesions found in mouse lungs ± SEM. P values were calculated using the Kruskal-Wallis test in adenomas, whereas the test chosen for the analysis of adenocarcinomas was the Mann-Whitney U test.

Figure 3

Histopathologic features of lung lesions from NDMA and NDMA-silica-treated mice. All lesions classified as adenomas and adenocarcinomas in Figure 2 were analyzed. (A–H) Hematoxylin and eosin staining of bronchiolar (A) and alveolar (B) epithelium, a solid adenoma (C), and a papillary adenocarcinoma (D) from NDMA-treated mice and bronchiolar hyperplasia (E), alveolar epithelium (F), a solid adenoma (G), and papillary adenocarcinoma (H) from NDMA-silica-treated mice. (I–P) Immunohistochemical analysis of pro-SPC and CC10 in tumors and adenomas from NDMA-silica- and NDMA-treated mice. Slides were counterstained with Harris' hematoxylin. All adenomas (I, M) and adenocarcinomas (J, N) exhibited positive staining for pro-SPC in both NDMA-silica- and NDMA-treated mice. Adenomas (K, O) and adenocarcinomas (L, P) were negative for CC10 staining in both experimental groups, whereas normal bronchiolar epithelial cells close to the tumors (*) exhibited CC10 positive staining. Scale bar, 100 µm.

Figure 4

K-RAS mutational analysis of adenocarcinomas from the NDMA-silica- and NDMA-only-treated mice. (A) Total number of adenocarcinomas with either K-RAS codon 12 or 61 mutations in NDMA-silica- and NDMA-treated mice. (B) Representative electropherograms showing G-to-A transition in K-RAS codon 12 and A-to-G transition in codon 61. Differences between groups were analyzed using the Fisher exact test.

Figure 5

Differentially expressed genes after a comparison of gene expression profiles of adenomas from NDMA and NDMA-silica-treated mice. (A) Matrix expression of the representative signature for the set of genes differentially expressed (B > 0; |log FC| > 1.5). Each line represents the gene expression profile of a mixture of RNA from at least two different adenomas (total n = 14). Red and green represent upregulated and downregulated genes, respectively, when compared to the corresponding normal lung tissue expression profile. (B) Retnla negative staining in an NDMA adenoma (left panel) and Retnla positive staining in an NDMA-silica adenoma located close to silica-induced lung inflammation (right panel). Retnla staining was performed in adenomas from 10 mice treated with NDMA and 16 mice treated with NDMA-silica. Differences between groups were analyzed using the Fisher exact test.

Figure 6

Expression of different immunomodulators in adenomas from NDMA-silica- and NDMA-treated mice. (A) Signature of immunosuppressive genes expressed in adenomas from NDMA-silica-treated mice compared to NDMA-treated mice. Each line represents the gene expression profile of a mixture of RNA from at least two different adenomas (total n = 14). Upregulated and downregulated genes are shown in red and green, respectively. (B) Percentage of lesions with FOXP3-positive cells within adenomas and adenoma peripheral areas (“capsule”) from NDMA-silica-treated (n = 15) and NDMA-treated (n = 15) mice. (C) Percentage of FOXP3-positive lymphocytes to total lymphocytes counted on lymphoid clusters located in non-injured lung parenchyma areas from NDMA-silica- and NDMA-treated mice. Values are represented as the means ± SEM. (D) FOXP3 immunohistochemical staining of lymphoid clusters from NDMA- and NDMA-silica-treated mice. The P value was calculated using the Mann-Whitney U test.