Infiltration of neutrophils is required for acquisition of metastatic phenotype of benign murine fibrosarcoma cells: implication of inflammation-associated carcinogenesis and tumor progression

Abstract

QR-32 tumor cells, a clone derived from a murine fibrosarcoma, are poorly tumorigenic and nonmetastatic when injected into syngeneic C57BL/6 mice. However, they are converted to highly malignant ones once they have grown in vivo after being co-implanted in a subcutaneous site with a foreign body, a gelatin sponge. Early phase of inflammation induced by the gelatin sponge participates in the conversion and histological analysis shows predominant infiltration of neutrophils. The objective of this study was to determine whether the depletion of the infiltrating neutrophils has any effect on the tumor progression. Intraperitoneal administration of a monoclonal anti-granulocyte antibody, RB6-8C5 (RB6), depleted neutrophils from both the peripheral blood circulation and the local inflamed site in mice with co-implantation of QR-32 tumor cells and gelatin sponge. The RB6 administration did not inhibit either tumor development or growth of QR-32 tumor cells. In contrast, tumor cell lines established from RB6-administered mice showed a significant decrease in metastatic incidence as compared with the tumor cell lines obtained from the mice with administration of control rat IgG or saline. Metastatic ability was significantly suppressed when RB6 had been administered in the early phase (from day -2 to day 6 after implantation); however, the administration in the middle (from day 6 to day 14) or late (from day 14 to day 22) phase did not affect the metastatic ability. We confirmed the phenomena by using integrin beta(2) knockout mice that had impaired neutrophil infiltration into inflamed sites. In the knockout mice, neutrophils hardly infiltrated into the gelatin sponge and the tumors showed dramatically suppressed metastatic phenotype as compared with those in wild-type mice or nude mice. Immunohistochemical analysis demonstrated that expressions of 8-hydroxy-2'-deoxyguanosine and nitrotyrosine were parallel to those in the presence of neutrophils. These results suggested that inflammation, especially when neutrophils infiltrate into tumor tissue, is primarily important for benign tumor cells to acquire metastatic phenotype.

Figure 1.

Histopathological findings and immunohistochemistry with antibodies against granulocytes, 8-OHdG, and nitrotyrosine of the arising tumors after administration of RB6-8C5 antibody or control vehicles. Histological sections were obtained from the arising tumors after subcutaneous co-implantation of QR-32 tumor cells with gelatin sponge. Tissues were obtained from co-implantation sites on day 5 (A–F) or on day 12 (G–J). They were from C57BL/6J mice with RB6 antibody (C, F, H, J), rat IgG (B, E, G, I), or saline (A) administration, and from integrin β₂ knockout mice (C57BL/6J^Itgb2tm1Bay) (D). H&E staining (A–D) and immunohistochemistry were performed with monoclonal antibodies against granulocytes (E, F), 8-OHdG (G, H), or nitrotyrosine (I, J), respectively. High magnification (×400) of RB6 antibody-positive neutrophils is indicated (E, top right). Scale bar, 100 μm.

Figure 2.

Depletion of neutrophils by administration of RB6-8C5 monoclonal antibody from peripheral blood of the mice in which QR-32 tumor cells had been co-implanted with gelatin sponge. Ten mg/kg of RB6-8C5 antibody was injected intraperitoneally into mice in which QR-32 tumor cells had been co-implanted with gelatin sponge from day −2 to day 10. At various intervals, the numbers of peripheral blood leukocytes (A, all leukocytes; B, neutrophils; C, lymphocytes; D, monocytes) were counted, and the hemograms of more than 200 leukocytes were examined by May-Gruenwald’s and Giemsa staining. ▪, RB6-8C5-treated mice; ○, saline-treated mice; □, rat IgG-treated mice. Mean values are presented as the number of cells per ml from more than 17 mice with each treatment and the data indicates representative results of at least two separate experiments with similar results.

Figure 3.

Inhibition of acquisition of metastatic ability of QR-32 tumor cells by administration of RB6-8C5 anti-granulocyte monoclonal antibody. A: QR-32 tumor cells (1 × 10⁵) were co-implanted with gelatin sponge in mice. They were given the anti-granulocyte monoclonal antibody RB6-8C5, or control vehicles, rat IgG, or saline. B: Metastatic incidence of the arising tumors. Mice were injected intravenously with 1 × 10⁶ of each cell line. *, P < 0.001; **, P < 0.05 versus saline group. C: Mean numbers of metastatic nodules in lung per mouse. *, P < 0.001 versus saline group. The actual number of incidences is indicated above each bar (number of mice with tumor or metastasis/number of mice tested).

Figure 4.

Complement-dependent cytotoxicity induced by RB6-8C5 antibody is specific to neutrophils. Tumor cells (QR-32, QRsP-10, and QRsP-11), thymocytes, and neutrophils were separately incubated with a serially diluted RB6-8C5 antibody with rabbit complement. The cytotoxic indices are indicated as representative results of at least two separate experiments with similar results.

Figure 5.

Early-phase RB6 administration inhibits acquisition of metastatic ability of QR-32 tumor cells. QR-32 tumor cells (1 × 10⁵) were co-implanted with gelatin sponge in normal mice to which RB6-8C5 monoclonal antibody, rat IgG, or saline had been administered for different periods. A: Metastatic incidence of the arising tumors. Mice were injected intravenously with 1 × 10⁶ of each cell line. *, P < 0.001; **, P < 0.05 versus saline group. B: Mean numbers of metastatic nodules in lung per mouse. *, P < 0.001 versus saline group. The actual number of incidences is indicated above each bar (number of mice with tumor or metastasis/number of mice tested).

Figure 6.

Inhibition of acquisition of metastatic ability of QR-32 tumor cells grown in integrin β₂-knockout mice. A: QR-32 tumor cells (1 × 10⁵) were co-implanted with gelatin sponge in normal syngeneic C57BL/6J or C57BL/6J Itgβ2^tm1Bay mice or KSN nude mice. B: Metastatic incidence of the arising tumors. One × 10⁶ of each cell line was injected intravenously into normal C57BL/6J mice. *, P < 0.001; **, P < 0.05 versus saline group. C: Mean numbers of metastatic nodules in lung per mouse. The actual number of incidence is indicated above each bar (number of mice with tumor or metastasis/number of mice tested).