Cheda, A., Wrembel-Wargocka, J., Lisiak, E., Nowosielska, E. M., Marciniak, M. and Janiak, M. K. Single Low Doses of X Rays Inhibit the Development of Experimental Tumor Metastases and Trigger the Activities of NK Cells in Mice. Radiat. Res. 161, 335–340 (2004).
There is evidence indicating that low-level exposures to low- LET radiation may inhibit the development of tumors, but the mechanism of this effect is virtually unknown. In the present study, BALB/c mice were irradiated with single doses of 0.1 or 0.2 Gy X rays and injected intravenously 2 h later with syngeneic L1 sarcoma cells. Compared to the values obtained for sham-irradiated control mice, the numbers of pulmonary tumor colonies were significantly reduced in the animals exposed to either 0.1 or 0.2 Gy X rays. Concurrently, a significant stimulation of NK cell-mediated cytotoxic activity was detected in splenocyte suspensions obtained from irradiated mice compared to sham-exposed mice. Intraperitoneal injection of the NK-suppressive anti-asialo GM1antibody totally abrogated the tumor inhibitory effect of the exposures to 0.1 and 0.2 Gy X rays. These results indicate that single irradiations of mice with either 0.1 or 0.2 Gy X rays suppress the development of experimental tumor metastases primarily due to the stimulation of the cytolytic function of NK cells by radiation.
INTRODUCTION
Carcinogenesis is the most important stochastic effect of the exposure of humans to ionizing radiation. In fact, radiogenic solid tumors and leukemias have been detected in people acutely irradiated with intermediate to high2 doses after nuclear detonations in Hiroshima and Nagasaki or during radiotherapy (2–4). On the other hand, in the case of low-dose and/or low-dose-rate exposures, a number of epidemiological studies indicate that cancer incidence and cancer mortality are not elevated among the inhabitants of high compared to low background radiation areas (5, 6). Moreover, the results of animal studies have demonstrated that whole-body exposures to low doses of X or γ rays are associated with a reduced cancer rate and increased latency of spontaneous lymphomas and leukemias in the exposed animals (7–9).
While high doses of ionizing radiation such as those used in radiotherapy suppress tumor growth by direct cell killing, immune stimulation has been suggested as a possible mechanism of the anti-neoplastic effects of low-dose whole-body exposures. Evidence for this assumption is provided by the results of experimental studies indicating that low doses of low-LET radiation inhibit the development of experimental and spontaneous metastases when the exposures occur prior to the injection of tumor cells (10–13). Some reports indicate that low-dose whole-body irradiation of mice and rats with X or γ rays leads to stimulation of the proliferation and/or function of various immunological effector cells (13–15). The results of these experiments suggest that low- level radiation may be employed as a supplementary therapy for primary and/or metastatic tumors. Low doses of ionizing radiation have already been used in the treatment of non-Hodgkin's lymphoma patients (16, 17).
In mice, effective antitumor activity is provided by both circulating and organ-associated natural killer (NK) lymphocytes (18). As demonstrated in many models of experimental and spontaneous tumors, these cytolytic effectors are particularly potent killers of metastatic cells, and depletion of the function of NK cells leads to strongly increased formation of metastases (19, 20).
In light of these data, we sought to determine in the present investigation whether irradiation of mice with low doses of X rays affects the development of experimental metastases and modulates the activity of NK lymphocytes. The results demonstrate that single exposures to 0.1 or 0.2 Gy X rays significantly reduce the number of L1 sarcoma cell colonies grown in the lungs and that the effect is causatively related to the markedly enhanced NK cell-mediated cytotoxicity.
MATERIALS AND METHODS
Animals and Irradiation
For the experiments, male BALB/c mice aged 6–8 weeks were used. The animals were obtained from the Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw. Total-body irradiation (TBI) of the animals was performed using a HS320 Pantak X-ray generator operating at 230 kV, 20 mA, with 1-mm aluminum and copper filters, at a dose rate of 2.2 Gy/h, to obtain absorbed doses of 0.1 or 0.2 Gy per mouse (the absorbed doses were verified using thermoluminescent dosimeters implanted s.c. in the middle abdominal region). Control mice were sham-exposed under the same conditions. All the animal studies were carried out in accordance with the regulations and with the permission of the Local Ethical Committee for Experimentation on Animals at the National Institute of Public Health in Warsaw.
Tumor Cells and Cell Lines
L1 sarcoma cells were obtained from the Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw. These cells developed spontaneously in the lung of a BALB/c mouse and have been maintained ever since by serial s.c. passages in that strain (21). YAC-1 cells, a murine lymphoma cell line, were obtained from the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw. The cells were cultured in RPMI-1640 medium (Sigma, Poznan, Poland) supplemented with 10% FBS (GIBCO BRL, Karlsruhe, Germany), 100 U/ml penicillin, 100 μg/ml streptomycin (Polfa, Warsaw), and 2 mMl-glutamine and stabilized with Na2CO3(Sigma).
Immunogenicity Assay
BALB/c mice were injected i.p. with 106L1 sarcoma cells, 106splenocytes obtained from allogeneic C57BL/6 mice, or culture medium. Four and 7 days later, the animals were killed humanely and cells from the mesenteric lymph nodes were collected and pooled. After washing, the cells were resuspended in the wells of 96-well culture plates (Corning, Warsaw) at 3 × 105cells per well and incubated for 24 h at 37°C in a humidified atmosphere of 95% air and 5% CO2with 14.8 kBq of [3H]thymidine (Polatom, Otwock-Swierk, Poland). After that, the cells were washed and their radioactivity was measured in the Tri-Carb 2100TR Counter (Canberra-Packard, Warsaw). For each experimental group, three mice were used and five samples were taken for in vitroexperiments.
Antibodies
To suppress the NK cell-mediated cytotoxicity in vivo,the rabbit anti- asialo GM1antibody (WAKO Chemicals, Neuss, Germany) was used to block the activity of the NK cells (22, 23). For this purpose, 1 day before the irradiation and injection of the L1 cells, mice were treated i.p. with the anti-asialo GM1antibody (20 μl Ab in 0.5 ml PBS). The mice were assayed 2 or 14 days later for the activity of NK splenocytes or the number of the pulmonary tumor colonies, respectively. Normal rabbit serum (NRS; DAKO, Gdynia, Poland) was used as the control antibody. For each experimental group, four (NK cell-mediated cytotoxicity assay) or 12 (tumor lung colonies assay) mice were used.
FITC-labeled anti-mouse Pan-NK Cells antibody (DX5) (Becton-Dickinson, Warsaw) was used to identify NK cells in the spleen cell populations obtained from irradiated and sham-irradiated mice, as described below. Each experimental group consisted of six mice.
Lung Tumor Colony Assay
An assay of L1 sarcoma cells was used as a mouse model of experimental tumor metastases. To obtain the cells for the assay, 14 days after s.c. transplantation of 106L1 cells, the tumors were removed, minced and incubated for 30 min at room temperature with 0.25% trypsin-EDTA (GIBCO BRL) and standard DNase I enzyme solution (Sigma). After that, the cells were washed and resuspended in culture medium at a concentration of 106cells/ml. The lung colony assay was performed as described previously (24). Briefly, 2 h after irradiation, mice were injected i.v. with 0.2 ml of the L1 cell suspension per mouse. Fourteen days later, the animals were killed humanely, their lungs were injected with India ink, and the total numbers of superficial macroscopic colonies per lung were counted using a magnifying glass. Each experimental group consisted of 12 mice.
Preparation of the NK Cell Suspension
NK cells were purified as described previously (25). Briefly, single cell suspensions in culture medium were prepared from the spleens of irradiated and sham-irradiated mice and incubated on glass petri dishes for 40 min at 37°C in a humidified atmosphere of 95% air and 5% CO2; in each case the cells were collected from at least three mice and pooled. The nonadherent cells were then collected, washed and incubated for 30 s at room temperature in an ammonium chloride solution to lyse the erythrocytes. After they were washed and resuspended in culture medium, the cells were passed through a nylon wool column, and the cells that did not adhere to the wool were used for the NK cell-mediated cytotoxicity assay and flow cytometry.
NK Cell-Mediated Cytotoxicity Assay
The cytotoxic activity of NK cells was measured using the standard in vitro51Cr release assay (26). In brief, 106YAC-1 target cells suspended in 0.1 ml culture medium were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2for 1 h with 5.55 MBq of Na251CrO4(Polatom, Otwock-Swierk, Poland). After the incubation, the cells were washed with PBS and 100-μl aliquots containing 104cells were placed in wells of microtiter plates (Corning). The NK-enriched cell populations were then added at effector:target cell ratios of 100:1, 50:1 and 25:1; five samples were taken for each in vitroexperimental group. After the 4-h incubation at 37°C in a humidified atmosphere of 95% air and 5% CO2, aliquots of the cell-free supernatants were harvested and the radioactivity of the 51Cr released from the target cells was measured in a γ-ray counter (Auto-Gamma Cobra II; Canberra-Packard, Warsaw). The rate of cytotoxic activity was calculated using the following formula: 100% × [(experimental release − spontaneous release)/(maximum release − spontaneous release)]. The release of 51Cr from the target cells cultured in the medium alone was taken as the spontaneous release, while 51Cr release from the target cells lysed with 1% Triton X-100 (Sigma) was regarded as the maximum release.
Flow Cytometry
One hundred microliters of the NK-enriched cell suspension (107cells/ ml) was stained with the DX5 antibody (10 μl) by incubation for 25 min at room temperature in the dark. After that, the cells were washed with cold PBS, and five samples from each experimental group were analyzed in a flow cytometer (FACSCalibur; Becton-Dickinson) equipped with CellQuest software (Becton-Dickinson) to estimate the percentage of the DX5-positive lymphocytes in the spleen cell suspensions under study.
RESULTS
Immunogenicity of L1 Sarcoma Cells
The immunogenicity of L1 sarcoma cells and allogenic (C57BL/6) cells is summarized in Table 1. Based on the standard published by Ryzewska et al.(27), cells are regarded as immunogenic when the index of stimulation, i.e. the ratio of the [3H]dThd incorporated into the mesenteric lymph nodes obtained from mice injected with the examined cells to that incorporated into the nodes dissected from mice given only culture medium, exceeds 3.0. Thus the results indicate that L1 sarcoma cells are clearly nonimmunogenic for BALB/c mice.
Effect of TBI with X Rays on the Development of Experimental Metastases
Figure 1 shows the results of quantification of the pulmonary tumor colonies (expressed as percentages of the control values obtained in the sham-exposed animals) that developed in mice pre-exposed to 0.1 or 0.2 Gy TBI and injected i.v. 2 h later with syngeneic L1 sarcoma cells. In all four independent experiments, irradiation with 0.1 or 0.2 Gy X rays consistently led to a comparable significant inhibition of the development of experimental lung metastases.
Effects of TBI with X Rays on NK cells
Since NK effectors were obtained from the spleens in the present experiments, we wanted to see whether a single low-level whole-body exposure of mice affected the NK cell content of this organ. As revealed by the cytofluorimetric analysis, TBI of mice with 0.1 and 0.2 Gy X rays did not lead to any marked changes in the numbers of DX5- positive (NK) cells in the spleen (Table 2).
As indicated in Fig. 2, a single whole-body exposure of mice to either 0.1 or 0.2 Gy X rays significantly stimulated the cytotoxic activity of NK splenocytes between 24 to 72 h postirradiation, with the most pronounced effect being detectable 48 h after the exposure (Fig. 2). Hence all the following estimations of the numbers and activities of NK cells were performed 48 h after TBI.
Figure 3 shows the results of the assessments of the in vitrocytolytic activity of NK lymphocytes obtained from the spleens of mice exposed to 0.1 and 0.2 Gy X rays and either injected i.v. or not with L1 sarcoma cells compared to the activity of NK splenocytes obtained from the sham- irradiated control mice that either were not injected or were injected with the tumor cells. As indicated, irradiation with either of the two low doses of X rays resulted in a significant boosting of the cytotoxic function of the NK-type splenocytes. Interestingly, injection of the syngeneic L1 sarcoma cells into the sham-exposed mice also appeared to be a significant stimulator of the NK cell-mediated cytolytic activity.
As shown in Fig. 4, when mice were injected with the specific suppressor of the NK lymphocytes in vivo(i.e. the anti-asialo GM1antibody), the activity of these cells as tested 2 days later was totally abrogated. This inhibition could not be reversed by TBI with 0.1 or 0.2 Gy X rays.
The data presented in Fig. 5 indicate that preinjection of the anti-asialo GM1antibody into sham-irradiated mice resulted in a significant increase in the numbers of tumor colonies developing in the lungs 14 days after i.v. injection of the L1 cells. When mice were pretreated with anti-asialo GM1antibody and exposed 1 day later to TBI with 0.1 or 0.2 Gy X rays, the inhibitory effect of such irradiations on the development of experimental metastases was totally suppressed, as indicated by the comparable numbers of pulmonary tumor colonies detected in the exposed and sham- exposed animals injected with L1 sarcoma cells (Fig. 5).
DISCUSSION
The results of the present study demonstrate that a single whole-body irradiation of mice with either 0.1 or 0.2 Gy X rays prior to intravenous injection of syngeneic sarcoma cells leads to suppression of the development of tumor colonies in the lungs. This observation corroborates the findings of Hosoi and Sakamoto, who detected marked reductions in the numbers of both artificial and spontaneous pulmonary metastases in mice as a result of TBI with 0.15, 0.2 or 0.5 Gy X rays (11); in those studies, the inhibitory effect of the irradiations was expressed when the tumor cells were inoculated a few hours before or after the exposure. Likewise, significant suppression of the development of pulmonary tumor nodules was reported by Ju et al.(12) and Cai (13), who irradiated mice with single doses of X rays ranging from 0.05 to 0.15 Gy 24 h before i.v. injection of B16 melanoma or Lewis lung cancer cells. A decreased incidence of lung and lymph node metastases accompanied by the enhanced infiltration of the metastatic foci by lymphocytes was also reported by Hashimoto et al.(14), who exposed rats to 0.2 Gy γ rays 14 days after s.c. implantation of hepatoma cells; in those studies, the in vitroexposure of the neoplastic cells to γ rays did not affect their growth in vivo,nor did local irradiation of the subcutaneous tumors reduce the number of spontaneous metastases. Finally, it was reported that exposure of mice to X-ray doses less than 0.2 Gy suppressed the local growth of tumors even when the neoplastic cells were inoculated after irradiation (10, 13). Collectively, these results suggest that the inhibitory effect of low doses of low-LET radiation on the development of tumors results from stimulation of anti-cancer immune mechanisms rather than from the direct impairment of the viability and/or function of the neoplastic cells (28).
Since the L1 cells used in the present investigation to induce sarcoma colonies in the lungs are not immunogenic for BALB/c mice, the specific activity of cytotoxic T lymphocytes is not likely to be involved in the observed inhibition of the growth of these colonies by 0.1 and 0.2 Gy X rays. Hence, in view of the well-established role of NK cells in nonspecific anti-metastatic surveillance (18–20, 29– 31), an important observation of this study is the significant enhancement of the cytotoxic function of NK-type lymphocytes from mice exposed to TBI with either 0.1 or 0.2 Gy X rays. Triggering the activity of NK cells after TBI with 0.075 to 0.5 Gy X or γ rays was reported previously by Liu et al.(32), Ju et al.(12), and Kojima et al.(15). To our knowledge, however, the present investigation is the first to demonstrate the association of the markedly stimulated cytolytic function of NK cells with the suppressed development of experimental tumor metastases, the two effects resulting from a single low-level exposure to X rays. In this study, the cytotoxic activity of NK cells was determined by measuring the capacity of the NK-enriched populations of splenocytes from the BALB/c mice to lyse in vitroYAC-1 lymphoma cells (a classical target for murine NK lymphocytes in the in vitrocytotoxicity assay) and not lyse the L1 sarcoma cells, which were used for the induction of the pulmonary tumor colonies. However, there are several reasons to think that in our experimental model the L1 cells were recognized and eliminated by NK lymphocytes in vivo.First, injection of L1 cells into nonirradiated mice led to a significant stimulation of the activity of NK splenocytes obtained from the recipient animals. Second, as indicated previously by others, development of neoplastic colonies in the lungs after i.v. injection of syngeneic tumor cells is closely related to the cytotoxic activity of circulating NK cells, which coincides with a similar activity in the spleen (18); in fact, in the present study, injection of the NK-suppressor anti-asialo GM1antibody in to nonirradiated mice led to a significant elevation of the number of pulmonary tumors developing in these animals after the injection of L1 cells. Third, the stimulation of the NK cell-mediated cytotoxicity by TBI with 0.1 and 0.2 Gy X rays that was detectable in the present study from 24 to 72 h after exposure coincides with the time when the i.v. injected tumor cells have already extravasated from the pulmonary capillaries and migrated into the stroma to form metastatic foci (33). Finally and most importantly, the inhibitory effect of the two low-dose exposures on the development of the colonies in the lungs was totally abrogated by pretreatment of the mice with the anti-asialo GM1antibody, a potent blocker of NK cell-mediated activity in vivo.Combined, these observations provide strong evidence for the involvement of the stimulated NK lymphocytes in suppression of the lung tumor colonies by single exposures of mice to 0.1 or 0.2 Gy X rays.
In conclusion, the results of the present study clearly show that a single whole-body exposure of mice to 0.1 or 0.2 Gy of X rays significantly inhibits the development of experimental tumor metastases and that the inhibition is primarily associated with the radiation-induced stimulation of the cytotoxic activity of NK cells. It will be interesting to determine in future studies the upper and lower dose limits of the above effects as well as a possible practical applicability of the low-level exposures to low-LET radiation.
Acknowledgments
This study was supported by grant No. 3 P05D 094 25 from the Polish Ministry of Science and Informatization.
REFERENCES
FIG. 1.
Relative numbers (percentages of the control values, indicated as solid line at 100%) of pulmonary L1 sarcoma colonies in mice exposed to 0.1 or 0.2 Gy X rays and injected i.v. with L1 sarcoma cells 2 h later. Data are mean values ± SD (bars). Results of four independent experiments (each experimental group consisted of 12 mice) are shown. An asterisk (*) indicates a statistically significant (P< 0.05) difference from the control (100%) value
FIG. 2.
Cytotoxic activity of splenic NK cells (at 50:1 E:T ratio) on three consecutive days (24 h, 48 h, 72 h) after irradiation of mice with 0.1 or 0.2 Gy X rays. C, sham-exposed (control) mice; 0.1 Gy, mice exposed to a single TBI with 0.1 Gy X rays; 0.2 Gy, mice exposed to a single TBI with 0.2 Gy X rays. Data points are means ± SD (bars) from three independent experiments; each experimental group consisted of at least three mice
FIG. 3.
Cytotoxic activity of splenic NK cells (at 50:1 E:T ratio) 48 h after irradiation of mice with 0.1 or 0.2 Gy X rays. Mean values obtained from four independent experiments ± SD (bars) are presented; each experimental group consisted of at least three mice. C, sham-exposed (control) mice; C+L1, sham-exposed mice injected with L1 sarcoma cells; 0.1 Gy, mice exposed to a single TBI with 0.1 Gy X rays; 0.2 Gy, mice exposed to a single TBI with 0.2 Gy X rays; 0.1 Gy+L1, mice exposed to a single TBI with 0.1 Gy X rays and injected with L1 sarcoma cells; 0.2 Gy+L1, mice exposed to a single TBI with 0.2 Gy X rays and injected with L1 sarcoma cells; °indicates statistically significant (P< 0.05) difference from the results obtained in group C; *indicates statistically significant (P< 0.05) difference from the results obtained in group C+L1
FIG. 4.
NK cell-mediated cytotoxic activity (at 50:1 E:T ratio) tested 48 h after i.p. injection of anti-asialo GM1antibody. PBS, sham-exposed (control) mice injected i.p. with phosphate-buffered saline; NRS, sham- exposed (control) mice injected i.p. with normal rabbit serum; Ab, sham- exposed mice injected with anti-asialo GM1antibody; 0.1 Gy+Ab, mice exposed to a single TBI with 0.1 Gy X rays and injected with anti-asialo GM1antibody; 0.2 Gy+Ab, mice exposed to a single TBI with 0.2 Gy X rays and injected with anti-asialo GM1antibody. Data are means ± SD (bars) from two independent experiments; each experimental group consisted of at least three mice. *Indicates statistically significant (P< 0.05) difference from the control (PBS or NRS) value
FIG. 5.
Numbers of tumor colonies in the lungs of mice pretreated with phosphate-buffered saline (PBS), normal rabbit serum (NRS), or anti-asialo GM1antibody (Ab) and injected with L1 sarcoma cells. Mean values obtained from two independent experiments ± SD (bars) are presented; each experimental group consisted of 12 mice. C, sham-exposed (control) mice; 0.1 Gy, mice exposed to a single TBI with 0.1 Gy X rays; 0.2 Gy, mice exposed to a single TBI with 0.2 Gy X rays. *Indicates statistically significant (P< 0.05) difference from the Ab-treated mice
TABLE 1
Immunogenicity of L1 Sarcoma Cells
TABLE 2
Percentages of DX5-Positive Cells in Spleens of the Irradiated and Sham-Exposed Micea
[1] According to the UNSCEAR 1986 Report (1), acute doses above 2 Gy, between 2 and 0.2 Gy, and below 0.2 Gy are regarded as high, intermediate and low, respectively.
