Imaging of Primary and Metastatic Tumors Treated With Radiotherapy-Directed Antigen Capturing Nanoparticles, Dissociation of Circulating Tumor Cell (CTC)-Clusters, and Reduction of CTC Extravasation Under PDL-1 Blockade

Abstract

<h3>Purpose/Objective(s)</h3> We investigated the efficacy of a combination of radiotherapy with antigen-capturing nanoparticles (AC-NPs; 123 ± 45 nm) containing digitoxin and anti-PODXL siRNA encapsulated in nanocapsules (567 ± 38 nm) that release their contents upon radiation exposure. We performed two radiotherapy sessions. Treatment with AC-NPs achieved successful immuno-radiotherapeutic effects on the primary tumors. An abscopal effect was also observed in the metastatic tumors by dendritic cell (DC)-mediated T-cell priming with PD-L1 blockade. New metastasis reduction was achieved by the dissociation of circulating tumor cell (CTC)-clusters by digitoxin and by reducing the extravasation of the dissociated CTCs via siRNA-mediated silencing of PODXL. <h3>Materials/Methods</h3> For session 1; nanocapsules were generated by mixing iopamiron and 400 μg anti-PD-L1 antibody (Ab) with a 1.0 mL solution containing 4.0% alginate, 3.0% hyaluronate, and 1 µg/mL P-selectin. This mixture was sprayed into 0.5 mmol/l FeCl₂ supplemented with 1 µg/mL anti-VEGFR-1/2 Ab. For session two, 400 nM digitoxin and 50 nM anti-PODXL siRNA were encapsulated in poly lactic-co-glycolic acid (PLGA) AC-NPs using the nanoprecipitation method. The particles were mixed with the above cocktail and sprayed into 0.5 mM/L FeCl₂ with 1 µg/mL anti-P-selectin Ab. This yielded encapsulated PLGA AC-NPs containing digitoxin. In session 1; 1 × 10¹⁰ nanocapsules were intravenously injected in BALB/c mice exhibiting primary 4TI mammary carcinoma in the left hind leg and lung metastases. Tumor accumulation was monitored using CT imaging. Subsequently, 10/20 Gy ⁶⁰Co γ-radiation was locally administered to the primary tumors. In session 2; 1 × 10¹⁰ nanocapsules were intravenously injected and allowed to interact with P-selectin for 9 h; 10/20 Gy ⁶⁰Co γ-radiation was locally administered. <h3>Results</h3> In session 1, CT revealed the accumulation of anti-VEGFR-1/2 nanocapsules around the primary and metastatic tumors. The microcapsules released P-selectin and anti-PD-L1 Ab in response to the initial irradiation. After session 2, the nanocapsules amassed around the primary tumor via a P-selectin Ag-Ab reaction. PLGA AC-NPs captured tumor-derived protein antigens released by the second radiation dose and transported them to DCs. This intensified DC-associated cross-priming of CD8⁺ T-cells. Primed CD8⁺ T-cells attacked PD-L1 and suppressed primary and metastatic tumors. PLGA AC-NPs also released digitoxin and anti-PODXL siRNA. The released digitoxin dissociated CTC clusters into single CTCs, and extravasation was reduced by siRNA-mediated silencing of PODXL, which reduced new metastasis formation. These treatments significantly increased the antitumor effect (EF 1.5) and reduced metastasis by 87.2%. <h3>Conclusion</h3> CT-detectable microcapsules exhibited targeted AC-NP-mediated immunotherapeutic benefits. They caused abscopal effects and reduced new metastasis formation, which improved the diagnosis and treatment of the tumor.