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It has been demonstrated that aggressive in situ tumor destruction (ablation) could lead to the release of tumor antigens, which can stimulate anti-tumor immune responses. We developed an innovative method of tumor ablation based on intratumoral alpha-irradiation, diffusing alpha-emitters radiation therapy (DaRT), which efficiently ablates local tumors and enhances anti-tumor immunity. In this study, we investigated the anti-tumor potency of a treatment strategy, which combines DaRT tumor ablation with two approaches for the enhancement of anti-tumor reactivity: (1) neutralization of immunosuppressive cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) and (2) boost the immune response by the immunoadjuvant CpG. Mice bearing DA3 mammary adenocarcinoma with metastases were treated with DaRT wires in combination with a MDSC inhibitor (sildenafil), Treg inhibitor (cyclophosphamide at low dose), and the immunostimulant, CpG. Combination of all four therapies led to a complete rejection of primary tumors (in 3 out of 20 tumor-bearing mice) and to the elimination of lung metastases. The treatment with DaRT and Treg or MDSC inhibitors (without CpG) also resulted in a significant reduction in tumor size, reduced the lung metastatic burden, and extended survival compared to the corresponding controls. We suggest that the therapy with DaRT combined with the inhibition of immunosuppressive cells and CpG reinforced both local and systemic anti-tumor immune responses and displayed a significant anti-tumor effect in tumor-bearing mice.

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CT26 model: 63-77 % of DaRT-treated mice became resistant to a re-inoculated tumor compared to 29-33 % resistant mice in the control. DA3 model: (1) The growth rate of challenge tumors was the lowest in mice which their primary tumor was treated by DaRT. (2) Most (93 %) mice in the control group developed lung metastases compared to 56 % in the DaRT group. (3) Combining DaRT with CpG resulted in a better control of the primary tumor. Our study offers a technique to eliminate local and distant malignant cells, regardless of their replication status, by stimulating specific anti-tumor immunity through the supply of tumor antigens from the destroyed tumor.

Diffusing alpha-emitters radiation therapy (DaRT) is the only known method for treating solid tumors with highly destructive alpha radiation. More importantly, as a monotherapy, DaRT has been shown to induce a systemic antitumor immune response following tumor ablation. Here, immunomodulatory strategies to boost the antitumor immune response induced by DaRT, and the response specificity, were investigated in the colon cancer CT26 mouse model. Local treatment prior to DaRT, with the TLR3 agonist poly I:C, was sufficient to inhibit tumor growth relative to poly I:C or DaRT alone. DaRT used in combination with the TLR9 agonist CpG, or with the TLR1/2 agonist XS15 retarded tumor growth and increased tumor-rejection rates, compared to DaRT alone, curing 41% and 20% of the mice, respectively. DaRT in combination with CpG, the Treg inhibitor cyclophosphamide, and the MDSC inhibitor sildenafil, cured 51% of the animals, compared to only 6% and 0% cure when immunomodulation or DaRT was used alone, respectively. Challenge and Winn assays revealed that these high cure rates involved a specific immunological memory against CT26 antigens. We suggest that DaRT acts in synergy with immunomodulation to induce a specific and systemic antitumor immune response. This strategy may serve as a safe and efficient method not only for tumor ablation, but also for in situ vaccination of cancer patients.

Diffusing alpha-emitting radiation therapy (DaRT) employs intratumoral Ra-224-coated seeds that efficiently destroy solid tumors by slowly releasing alpha-emitting atoms inside the tumor. In immunogenic tumor models, DaRT was shown to activate systemic antitumor immunity. Agonists of the membrane-bound toll-like receptors (TLRs) enhanced these effects and led to tumor rejection. Here, we examined the combination of DaRT with agents that activate a different type of pattern recognition receptors, the cytoplasmatic RIG1-like receptors (RLRs). In response to cytoplasmatic viral dsRNA, RLRs activate an antiviral immune response that includes the elevation of antigen presentation. Thus, it was postulated that in low-immunogenic tumor models, RLR activation in tumor cells prior to the induction of their death by DaRT will be superior compared to TLR activation. Intratumoral cytoplasmatic delivery of the dsRNA mimic polyIC by polyethylenimine (PEI), was used to activate RLR, while polyIC without PEI was used to activate TLR. PolyIC(PEI) prior to DaRT synergistically retarded 4T1 triple-negative breast tumors and metastasis development more efficiently than polyIC and rejected panc02 pancreatic tumors in some of the treated mice. Splenocytes from treated mice, adoptively transferred to naive mice in combination with 4T1 tumor cells, delayed tumor development compared to naïve splenocytes. Low-dose cyclophosphamide, known to reduce T regulatory cell number, enhanced the effect of DaRT and polyIC(PEI) and led to high long-term survival rates under neoadjuvant settings, which confirmed metastasis clearance. The epigenetic drug decitabine, known to activate RLR in low doses, was given intraperitoneally prior to DaRT and caused tumor growth retardation, similar to local polyIC(PEI). The systemic and/or local administration of RLR activators was also tested in the squamous cell carcinoma (SCC) tumor model SQ2, in which a delay in tumor re-challenge development was demonstrated. We conclude that RIG-I-like activation prior to intratumoral alpha radiation may serve as a potent combination technique to reduce both tumor growth and the spread of distant metastases in low-immunogenic and metastatic tumor models.

BACKGROUND:The objective of this study was to examine the combined effect of diffusing alpha‐emitter radiation therapy (DART) together with the chemotherapeutic agent cisplatin on tumor development.METHODS:BALB/c mice bearing squamous cell carcinoma tumors were treated with radium 224 (224Ra‐)–loaded stainless steel wires, releasing short‐lived, alpha‐emitting atoms from their surface. A concomitant regimen of cisplatin doses (5 mg/kg per dose) was given intravenously for the evaluation of the combined effect. Animals were monitored for tumor growth and survival.RESULTS:First, the authors observed that alpha particles and cisplatin inhibited SQ2 cell proliferation in vitro and promoted apoptosis. Treatment of tumor‐bearing mice indicated that, when a regimen of 2 separate doses of cisplatin was given concomitantly with a single intratumoral 224Ra‐loaded wire, there was moderate tumor growth inhibition relative to what was observed from each treatment alone. When tumors were treated with 2 radioactive wires positioned near the tumor base and a similar drug administration, the growth arrest effect intensified, and there also was a significant increase in survival rates. The combined treatment reduced both local tumor growth and metastatic spread to the lungs.CONCLUSIONS:Antitumor activity and overall survival of metastatic tumor‐bearing mice were improved significantly by the combined treatment. These results highlight the potential benefit of alpha radiation‐based radiotherapy in combination with chemotherapeutic drugs for anticancer treatment. Cancer 2009. © 2009 American Cancer Society.

(224)Ra-loaded wires ablate solid tumors by the release of alpha-particle emitting atoms inside the tissue, an effect that can be enhanced by combining this method with chemotherapy.