Neutralization of tumor necrosis factor bioactivity ameliorates urethane-induced pulmonary oncogenesis in mice

Abstract

Tumor necrosis factor (TNF) has been implicated in inflammation-associated tumor progression. Although multiple reports identified a role for TNF signaling in established cancers, few studies have assessed the impact of TNF blockade on early tumor formation promotion. We aimed at exploring the effects of TNF neutralization in a preclinical mouse model of lung carcinogenesis. For this, Balb/c mice (n = 42) received four weekly intraperitoneal urethane injections (1 g/kg) and twice-weekly intraperitoneal soluble TNF receptor (etanercept; 10 mg/kg) administered during tumor initiation/promotion, tumor progression, or continuously (months 1, 6, and 1-8 after urethane start, respectively). Lung oncogenesis was assessed after 8 months. In separate short-term studies, Balb/c mice (n = 21) received a single control or urethane injection followed by twice-weekly intraperitoneal control or sTNFR:Fc injections. Lung inflammation was assessed after 1 week. We found that sTNFR:Fc treatment during tumor initiation/promotion resulted in a significant reduction of tumor number but not dimensions. However, sTNFR:Fc administered during tumor progression did not impact tumor multiplicity but significantly decreased tumor diameter. Continued sTNFR:Fc administration was effective in halting both respiratory tumor formation and progression in response to urethane. This favorable impact was associated with impaired cellular proliferation and new vessel formation in lung tumors. In addition, TNF neutralization altered the lung inflammatory response to urethane, evidenced by reductions in TNF and macrophage and increases in interferon γ and interleukin 10 content of the air spaces. sTNFR:Fc treatment of RAW264.7 macrophages downregulated TNF and enhanced interferon γ and interleukin 10 expression. In conclusion, TNF neutralization is effective against urethane-induced lung oncogenesis in mice and could present a lung chemoprevention strategy worth testing clinically.

Figure 1

Experimental setup of in vivo studies. (A) Long-term studies: 42 Balb/c mice received intraperitoneal urethane (four weekly doses of 1 g/kg) and concomitant twice-weekly intraperitoneal PBS (control) or sTNFR:Fc (10 mg/kg) during months 1, 6, or 0 to 8 after urethane start. 4EC, month of administration of four urethane (ethyl carbamate, EC) injections; n = sample size; each box represents 1 month; gray boxes indicate periods of sTNFR:Fc treatment. (B) Short-term studies: Balb/c mice received intraperitoneal PBS (control) or urethane (a single dose of 1 g/kg) followed by intraperitoneal PBS (control) or sTNFR:Fc (10 mg/kg) at days 2 and 5 after urethane. 1EC, day of administration of a single urethane (ethyl carbamate, EC) injection; n = sample size; each box represents 1 day; gray boxes indicate days of sTNFR:Fc treatment.

Figure 2

Effects of sTNFR:Fc-mediated TNF neutralization on urethane-induced lung carcinogenesis in Balb/c mice. (A) Representative photographs of tumor-bearing lungs (black arrowheads point to tumors). Scale bar, 1 cm. (B-E) Lung tumor evaluation results at 8 months, including tumor number (B), mean diameter (C), mean volume (D), and cumulative volume per lung (E). Dots indicate raw data points; lines, mean; bars, SEM. (F) Histologic typing of neoplastic lesions at 8 months. AC indicate adenocarcinoma; columns, mean; bars, SEM; P, overall probability. *P < .05, **P < .01, and ***P < .001 compared with control.

Figure 3

Effects of sTNFR:Fc treatment on tumor growth and neoangiogenesis. Balb/c mice were treated as outlined under Figure 1A. (A) Percentage of tumor cells showing immunoreactivity for PCNA. (B) Representative images of PCNA immunoreactivity. (C) Number of factor VIII-associated protein (F8A)-reactive cell clusters per high-power field (hpf). (D) Representative images of F8A immunoreactivity. (A, C) Dots indicate raw data points; lines, mean; bars, SEM; P, overall probability. *P < .05, **P < .01, and ***P < .001 compared with control. (B) Å = 400. Scale bar, 200 µm. Brown indicates PCNA immunoreactivity; blue, hematoxylin nuclear counterstaining. (D) Å = 600. Scale bar, 150 µm. Arrows point to brown F8A-immunoreactive cell clusters; blue indicates hematoxylin nuclear counterstaining.

Figure 4

Effects of TNF signaling blockade on air space inflammatory parameters in the tumor-bearing lungs of urethane-treated mice. Balb/c mice were treated as outlined under Figure 1A. (A) Bronchoalveolar lavage (BAL) absolute cell numbers. MΦ indicates macrophages; PMN, neutrophil polymorphonuclear leukocytes; LΦ, lymphocytes. (B) BAL levels of TNF, IFN-γ, and IL-10. Dots indicate raw data points; lines, mean; bars, SEM; P, overall probability. *P < .05, **P < .01, and ***P < .001 compared with control.

Figure 5

Effects of sTNFR:Fc on urethane-induced subacute lung inflammation. Balb/c mice were treated as outlined under Figure 1B. (A) Bronchoalveolar lavage (BAL) absolute cell numbers. MΦ indicates macrophages; PMN, neutrophil polymorphonuclear leukocytes; LΦ, lymphocytes. (B) BAL levels of TNF, IFN-γ, and IL-10. Dots indicate raw data points; lines, mean; bars, SEM; P, overall probability. *P < .05, **P < .01, and ***P < .001 compared with control.

Figure 6

Effects of sTNFR:Fc treatment on mouse macrophages in vitro. RAW264.7 mouse macrophages were cultured with varying concentrations of sTNFR:Fc. After 24 hours, supernatants were collected for cytokine determinations by cytometric bead array, and cells were lysed for protein assay. The experiment was done thrice (n = 3). TNF indicates tumor necrosis factor; IL, interleukin; IFN, interferon. Dots indicate mean; bars, SEM. ***P < .001 compared with no treatment (0 µg/ml sTNFR:Fc).