Radiation therapy has been long utilized as localized cancer treatment. Recent studies have also demonstrated that it has a distant effect by the enhanced immunity, but it rarely occurs. The purpose of this study was to investigate whether X-ray irradiation combined with anti-PD-L1 and anti-CTLA-4 antibodies (P1C4) provides a higher probability of this distant effect as well as enhanced local antitumor efficacy for osteosarcoma. LM8 mouse osteosarcoma cells were inoculated into both legs of C3H mice assigned to one of four groups, namely no treatment (No Tx), P1C4, X-ray irradiation (RAD) to the leg of one side, and combination (COMB) groups. Survival and treatment-related immune molecular changes were analyzed. Administration of P1C4 produced a tumor growth delay on day 30 in 18% of the mice. In contrast, combination therapy produced the strongest tumor growth inhibition not only at the irradiated tumor but also at unirradiated tumor in 67% of the mice. Accordingly, lung metastasis in the COMB group was strongly reduced by 98%, with a significant survival benefit. Unirradiated tumor in mice in the COMB group significantly recruited CD8 + tumor-infiltrating lymphocytes with a moderate reduction of Treg, producing a significant increase in the CD8/ Treg ratio. These results suggest that radiation enhances the efficacy of P1C4 treatment against distant metastasis as well as local control in osteosarcoma. Our data suggest that radiation therapy combined with dual checkpoint blockade may be a promising therapeutic option for osteosarcoma.

Significant numbers of malignant tumor cells that have spread to surrounding tissues and other distant organs are often too small to be picked up in a diagnostic test, and prevention of even such small metastases should improve patient outcomes. Using a mouse model, we show in this article that intravenous administration of a human CCL3 variant carrying a single amino acid substitution after mild local hyperthermia not only induces tumor growth inhibition at the treated site but also inhibits metastasis. Colon26 adenocarcinoma cells (1 × 105 cells/mouse) were grafted subcutaneously into the right hind leg of syngeneic BALB/c mice and after nine days, when tumor size reached ~11 mm in diameter, the local tumor mass was exposed to high-frequency waves, by which intratumoral temperature was maintained at 42 °C for 30 min. Mice received the CCL3 variant named eMIP (2 μg/mouse/day) intravenously for five consecutive days starting one day after heat treatment. We found that tumor growth in eMIP recipients after hyperthermia was inhibited markedly but no effect was seen in animals treated with either hyperthermia or eMIP alone. Furthermore, the number of lung metastases evaluated at 18 days after hyperthermia treatment was dramatically reduced in animals receiving the combination therapy compared with all other controls. These results encourage future clinical application of this combination therapy.

Osteosarcoama is one of the common malignancies at bone in children and adolescent. Recent study demonstrated that combination of anti-PD-L1 and anti-CTLA-4 antibodies provided grate control of metastatic osteosarcoma. However, a strong local treatment strategy including surgery or high precision radiation therapy is necessary to elucidate osteosarcoma. Radiation therapy plays an important role in local control for malignant tumors but previous studies demonstrated that radiation enhanced immune response in which not only local tumor regression at irradiated sites but also regression of metastatic tumor outside the radiation field were observed. Although this phenomenon, so called the abscopal effect, is rarely seen, recent studies demonstrated that combination of X-ray irradiation with checkpoint blockades provided higher probability of the abscopal effect for some kind of tumors. However, the effect of x-ray irradiation combined with checkpoint blockade on the abscopal effect for osteosarcoma has been totally unknown. We investigated whether local X-ray irradiation combined with the anti-PD-L1 and anti-CTLA-4 antibodies enhances local and distant antitumor efficacy for osteosarcoma. LM8 mouse osteosarcoma cells were inoculated into both legs of C3H mice. Mice were treated by 10 Gy X-ray irradiation alone to the tumor in the one side leg (RAD group) at day 12, 150 ug of anti-PD-L1 and anti-CTLA-4 antibodies (P1C4 group) at days 9, 12, and 15, or those combination (COMB group). Administration of anti-PD-L1 and anti-CTLA-4 antibodies provided tumor growth delay or complete response at day 37 for about 20% of the mice. X-ray irradiation strongly inhibited tumor growth at irradiated tumor but not in unirradiated tumor. On the other hand, the combination therapy provided the strongest tumor growth inhibition not only at irradiated tumor but also at unirradiated tumor for about 89% of the mice. Accordingly, lung metastasis in mice in COMB group was strongly reduced by 97% with the significant survival benefit compared with the mice in P1C4 group. Flow cytometric analysis revealed that mice in COMB group significantly recruited CD8 tumor infiltrating lymphocytes with moderate reduction of regularly T cells (Tregs), thereby increasing the CD8/Treg ratio. Furthermore, quantitative real time PCR showed significant induction of PD-L1 on irradiated LM8 cells in vitro. The radiation-induced upregulations of PD-L1, B7-1, and B7-2, the ligands of CTLA-4, were also confirmed by flow cytometry, indicating that the enhanced efficacy anti-PDL1 and CTLA-4 antibodies may associate with these upregulations by radiation. These results suggest that X-ray irradiation contributes to the enhancement of the efficacy for the distant metastasis as well as local control in the treatment of anti-PD-L1 and anti-CTLA-4 antibodies for osteosarcoma. Our data provide a rational to establish a new therapeutic strategy and start up a clinical trial for osteosarcoma. Citation Format: Yutaka Takahashi, Tomohiro Yasui, Keisuke Tamari, Kazumasa Minami, Masahiko Koizumi, Yuji Seo, Fumiaki Isohashi, Keisuke Ohtani, Ryosuke Kambe, Kazuhiko Ogawa. Radiation combined with checkpoint blockades enhanced antitumor efficacy for osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4585. doi:10.1158/1538-7445.AM2017-4585