Abstract
Background: Mouse regulatory T cells (Treg) may be deleted by intraperitoneal injection of anti-CD25 monoclonal antibody (mAb) (PC61). When Treg populations are thus suppressed, the immune system attacks tumours in autoimmune reactions. Materials and Methods: An osteosarcoma (LM8) was transplanted subcutaneously into C3H/He mice. Serial injections of PC61 were conducted from seven days before (pre-PC61 group) or from two days thereafter (post-PC61 group) and tumour growth and metastasis were examined four weeks later. A control group received PBS injections. Results: Subcutaneous tumours were reduced in size and the numbers of lung and liver metastatic colonies were significantly decreased in both pre- and post-PC61 groups compared to the control group. Conclusion: Tumour suppression was effective even when injection of PC61 was performed two days after LM8 transplantation. These results indicate that such treatments might be suitable to be applied in the clinic after surgical operations.
Osteosarcomas constitute about 20% of all primary bone malignancies (1). At present, 20-30% of affected patients die in the first five years from metastatic spread, particularly to the lungs (2). However, no new anticancer drugs targeting osteosarcoma have been introduced in recent years and the development of new therapies is a high priority.
PC61 is an anti-interleukin 2 receptor (IL2R) α (CD25) mouse monoclonal antibody (mAb). It was reported earlier that mice receiving injections of PC61 develop autoimmune disease with a decreased CD4+CD25+ cell population, suggesting this population to be comprised of regulatory T cells (Tregs) (3-5). Subsequently, when several kinds of tumours were transplanted into mice previously administered PC61, tumour growth was strongly suppressed (6, 7). However, to date there have been no reports dealing with osteosarcomas. Therefore, the present study used an osteosarcoma cell line (LM8) that metastasises to the lungs at a high rate to determine whether establishment and growth, as well as metastasis of LM8 may be suppressed by prior administration of PC61. Furthermore, the effects of PC61 injections subsequent to tumour transplantation were examined.
Materials and Methods
LM8 cell line. LM8 is an osteosarcoma cell line originally from the C3H/He mouse, which has high metastatic potential (8). It was maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc, Grand Island, NY, USA) supplemented with 10% foetal calf serum in an air incubator with 5% CO2 at 37°C. After suspending LM8 cells in phosphate-buffered saline (PBS), 100 μl of the cell suspension (1×106 cells) was injected subcutaneously under the dorsal skin of C3H male mice.
Treatment with PC61. Aliquots of 0.2 mg rat mAb (IgG1) against CD25 (PC61) were injected intraperitoneally into C3H mice at −7, −2, 2, 7 and 14 days (pre-PC61 group) or at 2, 7, 10, 14 and 19 days after LM8 transplantation (post-PC61 group). Controls received 100 μl PBS injected intraperitoneally in the same way as the pre-PC61 group.
Mice. C3H/He male mice were obtained from CLEA (Osaka, Japan) and were six weeks old when infused with LM8 cells, subcutaneously under the back skin. There were 35 mice in the control group, 10 in the pre-PC61 and 11 in the post-PC61 group. All animals were housed in a specific pathogen-free animal room and all animal studies were performed in accordance with the institutional guidelines at Aichi Cancer Center Research Institute.
Volume of tumours of C3H/He mice. LM8 cells (1×106) were injected subcutaneously into C3H mice that received injections of PC61 before and after (pre-PC61) or only after (post-PC61) LM8 transplantation. The sizes of the grafted tumours, measured every week, were smaller in the pre-PC61 and post-PC61 groups than in the control group. Data are expressed as mean±standard deviation.
Weight of tumours of C3H/He mice. The subcutaneous tumours at the time of sacrifice were lighter in the pre-PC61 and post-PC61 groups than in the control group. Data are expressed as mean±standard deviation; *p=0.034 (pre-PC61), p=0.026 (post-PC61).
Evaluation items. The major (a) and minor (b) axes of grafted tumours were measured every week and tumour volumes were calculated according to the formula: volume (mm3)=(π/6)ab2. All mice were sacrificed 28 days after inoculation with LM8 cells. Transplanted subcutaneous tumour tissues and various organs, including the lung and liver, were excised and weighed. The excised tumours and organs were fixed in formalin, embedded in paraffin, sectioned (6 μm thickness) and stained with haematoxylin and eosin for histological observation. The numbers of metastatic colonies in the lung and liver were counted under a light microscope (Nikon ECLIPSE 80i, Tokyo, Japan).
Flow cytometry. Spleen cells obtained from three post-PC61 and three PBS-injected (control) mice from the study were stained with phycoerythrin-conjugated anti-CD4 (GK1.5) and fluorescein isothiocyanate-conjugated anti-CD25 (7D4) (BD Biosciences Pharmingen, San Diego, CA, USA) and assayed using a fluorescence-activated cell sorter (FACScan; BD Biosciences, San Jose, CA, USA).
Sections of metastatic colonies of LM8 in lung (A, top row) and liver (B, bottom row) stained with haematoxylin and eosin. Left column: sections from PBS-treated control mice; right column: sections from post-PC61 mice.
Statistical analysis. Statistical differences between the control and PC61-injected groups (pre-PC61 and post-PC61 groups) were analysed with the Student's t-test. A p-value <0.05 was considered statistically significant. All analyses were conducted using SPSS version 12.0 software (SPSS Inc., Chicago, IL, USA).
Results
Effects on subcutaneous tumour size. The sizes of subcutaneous tumours were apparently smaller in both the pre- and post-PC61 groups than in the PBS group (Figure 1) and the tumours harvested on day 28 after LM8 transplantation were significantly lighter in both the pre- and post-PC61 group than in the control group (pre-PC61: p=0.034, post-PC61: p=0.026 compared to control) (Figure 2). Histological changes such as tumour necrosis and fibrosis were not found in the tumours in any of the groups.
Effects on distant metastases. Metastasis of LM8 to lungs was evident in 0/10 of the pre-PC61 group, 1/11 of the post-PC61 group and 25/35 of the PBS group. Typical metastatic colonies in the lung and liver are shown in Figure 3. Their number was significantly smaller in the PC61-injected groups than in the control group (lung: pre-PC61 p=0.009, post-PC61 p=0.007; liver: pre-PC61 p=0.034, post-PC61 p=0.034) (Figures 4 and 5). However, the weights of the lungs and livers did not differ significantly among the groups.
The number of lung metastatic colonies was smaller in the PC61-injected groups than in the control group. Data are expressed as mean±standard deviation; **p=0.009 (pre-PC61), p=0.007 (post-PC61).
Effects on CD4+CD25+ T-cell populations. As shown in Figure 6, flow cytometry revealed the percentage of CD4+CD25+ T-cells in a mouse of the PBS (control) group to be 3.68%, while that in a mouse of the post-PC61 group was 0.24%. These results were representative of the other animals in the respective groups.
Discussion
In the present study, the sizes of subcutaneously transplanted tumours and the numbers of lung and liver metastatic colonies were significantly smaller in the PC61-injected groups (both pre- and post-PC61) than in the control group (Figure 1). The tumour regressive effects appeared to be similar in both the pre- and post-PC61 groups. Flow cytometry analysis further revealed CD4+CD25+ peripheral T-cells (spleen) to be reduced in PC61-injected mice (Figure 6).
Previously, CD25+ lymphocyte populations were shown to disappear when PC61 was administered to mice, and as a result organ-localised autoimmune disease developed spontaneously (3). In that study, injections of PC61 were started at a juvenile age, and were continued in the long term. Moreover, the mice (C57BL/6 × A/JF1) used were of a strain that readily develops autoimmune disease on depletion of the CD25+ population (3). In the present study, the injections of PC61 were performed on adults of the C3H/He strain which features a fully-established immune system. Although the CD4+CD25+ cell population assessed by FACS was remarkably decreased, no inflammatory reactions were found in the various organs examined, such as the thyroid gland and the stomach. Moreover, negative results for auto-antibodies to individual organs in the blood of PC61-treated mice were obtained by the indirect immunofluorescence technique. These results, thus, suggested that an autoimmune disease does not develop even when the CD4+CD25+ population is deleted in adult C3H/He mice lacking susceptibility (9).
The number of liver metastatic colonies were smaller in the PC61-injected groups than in the control group (pre-PC61 group: *p=0.034, post-PC61 group: *p=0.034). Data are expressed as mean±standard deviation; *p=0.034 for pre- and post-PC61.
Proportions of CD4+ and CD25+ cells in splenocytes from a PBS control mouse (left) and a post-PC61 injected mouse (right).
The results of the present study are in line with a report that progress of several types of tumours is remarkably suppressed transplanted to mice administered PC61 (4). It is thought that the immune system is capable of recognising antigens of most cells constituting the body and that it provides Tregs to regulate immunoreactions. From this perspective, a Treg population would also be expected for individual tumour antigens. When Treg populations are removed by administering a mAb against CD25, then immune reactions against tumour antigens begin. Since tumours have an active cellular metabolism, the quantity of antigens causing immunoreactions might be expected to increase, so that they are more likely to be targeted than normal organs or tissues of the body.
In the present study, Treg deletion was also associated with antitumour effects against an osteosarcoma line. In clear contrast to an earlier report of no antitumour effects of administration of PC61 two days after tumour transplantation (10), both the experimental regimens tested in the present study (pre- and post-PC61) proved effective. This is the first description of inhibition of tumour metastasis in mice deleted of Tregs. These results point to efficacy even with antibody treatment after surgical removal of tumours in humans, without any risk of autoimmunity.
Acknowledgements
We thank Sakiko Nishikawa for technical assistance. This study was supported by a Grant-in-Aid for Scientific Research (C) by the Japan Society for the Promotion of Science (19591744).
- Received October 22, 2010.
- Revision received November 12, 2010.
- Accepted November 15, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
