Biology contribution
Ionizing radiation inhibition of distant untreated tumors (abscopal effect) is immune mediated

Presented in part at the 44th Annual Meeting of the American Society for Therapeutic Radiology and Oncology, New Orleans, LA, October 6–10, 2002.
https://doi.org/10.1016/j.ijrobp.2003.09.012Get rights and content

Abstract

Purpose

Ionizing radiation can reduce tumor growth outside the field of radiation, known as the abscopal effect. Although it has been reported in multiple malignancies, the abscopal effect remains a rare and poorly understood event. Ionizing radiation generates inflammatory signals and, in principle, could provide both tumor-specific antigens from dying cells and maturation stimuli that are necessary for dendritic cells' activation of tumor-specific T cells. We therefore tested the hypothesis that the abscopal effect elicited by radiation is immune mediated. This was directly tested by enhancing the number of available dendritic cells using the growth factor Flt3-Ligand (Flt3-L).

Methods and materials

Mice bearing a syngeneic mammary carcinoma, 67NR, in both flanks were treated with Flt3-L daily for 10 days after local radiation therapy (RT) to only 1 of the 2 tumors at a single dose of 2 or 6 Gy. The second nonirradiated tumor was used as indicator of the abscopal effect. Data were analyzed using repeated measures regression.

Results

RT alone led to growth delay exclusively of the irradiated 67NR tumor, as expected. Surprisingly, growth of the nonirradiated tumor was also impaired by the combination of RT and Flt3-L. As control, Flt3-L had no effect without RT. Importantly, the abscopal effect was shown to be tumor specific, because growth of a nonirradiated A20 lymphoma in the same mice containing a treated 67NR tumor was not affected. Moreover, no growth delay of nonirradiated 67NR tumors was observed when T cell deficient (nude) mice were treated with RT plus Flt3-L.

Conclusions

These results demonstrate that the abscopal effect is in part immune mediated and that T cells are required to mediate distant tumor inhibition induced by radiation.

Introduction

Despite improvements in detection and treatment, breast cancer remains a deadly disease for many women. The tendency of breast cancer cells to spread systemically early on in its course requires an effective systemic treatment. The main limitation of currently available treatments is the failure to eradicate systemic disease, even when the tumor is clinically at an early stage. New treatment strategies are constantly explored to successfully address this problem. In this respect, antitumor immunity may have a unique role, complementary to the use of other currently available modalities of treatment. Cytolytic T cells (CTL) can eliminate tumor cells with exquisite specificity and efficacy independently from both their proliferative status and intrinsic chemosensitivity (reviewed in Ref. 1). Recent advances in understanding how the immune system functions allow for the rational design of new strategies to induce effective antitumor immunity by targeting antigens to dendritic cells (DC) (2).

Poor immunogenicity of cancer cells often prevents the development of an effective antitumor immune response. Most cancer cells do not express costimulatory molecules and, therefore, cannot trigger the activation of naive T cells 3, 4. Tumor-specific antigens need to be “transferred” from cancer cells to DC that are able to present them in the context of major histocompatibility complex (MHC) class I molecules and activate CD8+ CTL, a phenomenon termed “cross-priming” 5, 6. Recently, it has been demonstrated that DC can efficiently acquire antigens from dying cells and stimulate antigen-specific CD4+ and CD8+ T cells (reviewed in Ref. 7). In addition, inflammatory signals are also required to induce DC maturation, which results in the upregulation of CD40, MHC, and costimulatory molecules 8, 9.

Ionizing radiation therapy (RT) has the potential to enhance tumor immunogenicity by promoting cross-priming and eliciting antitumor T-cell responses. RT can induce tumor cell death and generate inflammatory signals 10, 11, 12, 13, 14. The inflammatory mediators and/or direct effects of RT on tumor-associated vessels also enhance dramatically the permeability of solid tumors to both DC and effectors T cells 15, 16. Overall, these properties may be responsible for the indirect anticancer effects of RT on cancers outside of the radiation field that have been reported in many malignancies 17, 18, 19, 20, 21, 22, 23, a phenomenon originally described as abscopal effect by R. J. Mole in 1953 (24). The definition of abscopal effect comes from the Latin ab (position away from) and scopus (mark or target). The abscopal mechanism of action remains unexplained, although a variety of underlying biologic events can be hypothesized, including a possible role for the immune system 25, 26. The abscopal effect, however, is not often observed, possibly because many tumor-bearing hosts develop impaired DC function (reviewed in Reference 27).

Multiple mechanisms contributing to RT-induced antitumor immunity have been suggested 28, 29, 30, 31, 32. Evidence supporting the role of RT in promoting cross-priming and induction of antitumor T-cell responses was recently provided in an experimental model. If Fms-like tyrosine kinase receptor 3 ligand (Flt3-L), a growth factor that stimulates production of DC 33, 34, was administered after the ablation of a mouse lung carcinoma by local RT, the treated mice experienced a reduction in lung metastases and prolonged disease-free survival (35). However, to obtain the systemic antitumor response in this model, RT was delivered at a single high dose (60 Gy), suggesting that a massive tumor necrosis was required to promote cross-priming. Therefore, although interesting, these results seem to be of limited clinical applicability. In addition, the highly metastatic nature of the carcinoma used as a model made it difficult to determine whether the importance of RT involved only providing tumor antigens from dying cells to DC, or mainly preventing further seeding of metastases to the lungs.

Studies of concurrent chemotherapy and radiation in several models, including locally advanced breast cancer, suggest that the chemoradiation combinations favorably affect not only local control but also disease-free survival and overall survival of patients, compared to sequential chemoradiation 36, 37, 38, 39. Our group has hypothesized that immunity-related mechanisms involved in locoregional therapy might have a systemic effect on tumor growth. In the present study, we developed a model based on the nonmetastatic mouse mammary carcinoma 67NR to examine the abscopal effect. Studies were developed to determine whether local treatment with RT at low, clinically compatible doses could elicit systemic antitumor effects mediated by the immune system when combined with administration of Flt3-L to expand the DC compartment.

Section snippets

Cells and reagents

67NR is a BALB/C mouse–derived mammary carcinoma cell line (provided by Fred Miller, the Michigan Cancer Center) (40). A20 is a BALB/C mouse–derived B-cell leukemia/lymphoma (provided by G. Inghirami, New York University School of Medicine) (41). 67NR cells were grown in DMEM medium (Invitrogen Corporation, Carlsbad, CA) supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 2.5 × 10−5 M 2-mercaptoethanol, and 10% FBS (Gemini Bio-Products, Woodland, CA). These cells

The abscopal effect is induced in the 67NR mammary carcinoma model when local RT is combined with Flt3-L to expand DC

The BALB/C-derived nonmetastatic mammary carcinoma 67NR was used as a model to test whether local RT treatment can trigger systemic antitumor effects outside of the radiation field in tumor-bearing mice. These cells express MHC class I molecules but not MHC class II or the costimulatory molecules CD80 and CD86 (Fig. 1). Based on the hypothesis that the indirect antitumor effects of RT are mediated by its ability to promote cross-priming and, therefore, trigger antitumor immunity, the DC growth

Discussion

Our experiments show that, at least in the relatively radiosensitive 67NR mammary carcinoma model, the combination of local RT at a single fraction of 2 Gy with Flt3-L treatment triggered systemic antitumor effects capable of controlling tumor growth at a remote site consistent with the definition of an abscopal effect (Fig. 2). Importantly, although Flt3-L administered alone has been shown to induce tumor regression and growth control in other experimental mouse tumors (34), in our

Acknowledgements

The authors thank Dr. R. Schneider for his critical input.

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    The authors thank Elaine Thomas for Flt3-Ligand. S.D. was supported by Grant K08 CA89336 from NIH/NCI and by a grant from the Speaker's Fund for Biomedical Research: Toward the Science of Patient Care, awarded by the city of New York. S.C.F. was supported by Grants DAMD17-01-1-0345 from the Department of Defense, TURSG CCE 103174 from the American Cancer Society, and by a grant from the Breast Cancer Research Foundation.

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