Use of Bismuth-212 Labeled Macro Aggregated Albumin for Alpha Particle Therapy Delivery

Introduction: Macroaggregated albumin (MAA) was evaluated as a potential vehicle for locoregional delivery to treat solid tumors with alpha particle radiation. While theranostics agents are making great strides in both pre-clinical and clinical studies, only cancers with high expression levels of the targeted receptors are candidates for these therapies, thus limiting certain cancer applications. Intraarterial deliver of embolic agents, without receptor targeting, would expand radiation treatment of certain types of solid tumors. Alpha particles can overcome β- radiation resistance of tumors and offers distinct advantages. Development of an embolic delivery platform would expedite research into alpha particle therapy effects on cancer biology. MAA is FDA approved and widely used in nuclear medicine and was evaluated as a potential vehicle for alpha particles. Thus, this project investigated radiolabeling MAA with Bismuth-212 (Bi-212) to treat cancer cells in vitro and in vivo. Further, specific DNA damage response and immunogenic pathways were studied as potential targets for combination therapies.

Methods: MAA was radiolabeled with eluted Bi-212 from a Radium-224 generator (Oak Ridge). Bi212 (1.3-1.8mCi) was added to 2.5 mg of lyophilized Draximage MAA kit and purified. The purity of radiolabeling was measured by iTLC (instant thin layer chromatography). Clonogenic assays were conducted with 1000 4T1 breast cancer cells/well and treatments with radioabeled MAA (0, 5, 10, 20 uCi, n=3/group). Additional 4T1 cells were treated (0, 10, 20 uCi, n=2/group) for studying downstream signaling proteins (γH2A.X, cleaved caspase-3, TREX1, IFN-β) by western blot and RT-qPCR. Radiolabeled MAA was also injected directly into 4T1 orthotopic breast tumors for biodistribution (3 uCi, n=3/group) or histology (0 or 20 uCi, n=1/group). The clonogenic assay was analyzed by luminescence imaging and biodistribution by gamma counter analyses (PE Wizard).

Results: An average of 45% (0.8±0.3 mCi) of the added radioactivity was stably bound to MAA. After purification, the activity was stably bound to the MAA for 2 hours. iTLC showed 99% purity of the final Bi-212-MAA product during the 2 hour period followed. Cells treated with 20 uCi Bi-212-MAA showed statistically significant 99% reduction (1.3E+7 ± 6.6E+6 vs. 3.6E+9 ± 5.3E+8, p=0.002) in colony formation compared to untreated controls. Western blot analysis showed increased γH2A.X and cleaved caspase-3 expression in higher treated conditions. Increased Bi-212-MAA doses did not increase TREX1 nor IFN-β gene expression. Tumor tissues showed statistically higher uptake (159±32 %ID/g) than blood (4.7±1.9 %ID/g, p=0.009) or lung (1.7±0.5 %ID/g, p=0.008) in the biodistribution study. Further, H&E staining of lung slices showed no changes in treated mice compared to control animals.

Conclusions: MAA radiolabeling with Bi-212 resulted in stable binding of Bi-212. The Bi-212-MAA killed cells in vitro in a dose dependent manner. Bi-212-MAA induced DNA damage and caused cell death through the Caspase 3 pathway. A previously reported DNA damage response paradigm with TREX1 exonuclease and IFN-β cytokine release did not appear to be upregulated in the alpha particle treatment. Further DNA damage response and immunogenic pathways will be investigated in the future for potential synergy with alpha particle therapy.

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