212Bi-Macroaggregated Albumin Inhibited Mouse Melanoma Growth by Regulating Cell Cycle Checkpoint Markers Without Promoting Living Cell Repopulation

Abstract

Radiotherapy using an a-particle emitting radionuclide has emerged as a promising candidate for cancer treatment; however, the efficacy of 212Bi for mouse melanoma treatment has not yet been studied. Here, we evaluated the efficacy of 212Bi-labeled macroaggregated albumin (MAA) in delivering radiation to mouse melanoma cells in vitro and in vivo. Methods: The efficacy of 212Bi efficacy in killing melanoma cells was assessed by in vitro clonogenic and cell survival assays. Immunoblot assays were used to investigate downstream pathways, radioresistance, and epithelial-to-mesenchymal markers. We assessed melanoma cells’ repopulation using a conditioned medium (CM; 50%) from 212Bi-MAA–irradiated B16F10 cells. 212Bi-MAA was intratumorally injected in B16F10 melanoma–bearing C57BL/6 mice to study the efficacy, stability, and internal organ toxicity of 212Bi-MAA. Results: 212Bi-MAA effectively killed and inhibited the clonogenic capacity of B16F10 cells. Furthermore, 212Bi-MAA induced the expression of DNA damage (gH2AX) and cell death (cleaved caspase-3) markers, which was at maximum at a dose of 3.7 MBq. Cell cycle checkpoint markers (ATR, Chk1, and Wee1) were also elevated after 212Bi treatment; however, these were reduced at 3.7 MBq compared with 0.93- and 1.85-MBq doses. Minimal to no upregulation of radioresistance (Trex1 and STAT1), cancer stemness (Nanog), and epithelial–mesenchymal transition (E-cadherin, N-cadherin, and Vimentin) markers was found after 212Bi-MAA treatment. CM from 212Bi-MAA–irradiated B16F10 cells did not alter the cell proliferation, colony-forming, and migration capacity of living B16F10 cells. CM did not change epithelial–mesenchymal transition and cell proliferation marker expression. Studies in mice showed that 212Bi-MAA was retained in B16F10 tumors and effectively reduced tumor growth in vivo without causing toxicity. Conclusion: These findings suggested that 212Bi-MAA was an effective therapy for mouse melanoma and did not induce factors that aid melanoma repopulation.