Imaging of Primary and Metastatic Tumors Treated with Radiotherapy-Directed Antigen-Capturing Nanoparticles, Reducing Metastasis-Seeding and Colonization, under PDL-1 Blockade

Abstract

<h3>Purpose/Objective(s)</h3> We tested a treatment combining radiotherapy with antigen-capturing nanoparticles (AC-NPs; 112 ± 73 nm) containing digitoxin and anti-STAT-3 inhibitor (HJC0152) encapsulated in nanocapsules (545 ± 24 nm), which release their contents upon radiation exposure. We conducted two radiotherapy sessions: 1) treatment of primary tumor and metastases by immuno-radiotherapy and abscopal effect with PDL-1 blockade, respectively, and 2) reduction of new metastasis seeding and colonization by dissociation of circulating tumor cell (CTC)-clusters using digitoxin, and by impairing premetastatic niche (PMN) formation using a STAT-3 inhibitor (HJC0152), respectively. <h3>Materials/Methods</h3> In session one, nanocapsules were generated by mixing iopamiron, 400 mg of an anti-PD-L1 antibody (Ab), and 10 µg/mL HJC0152 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. In session two, 400 nM digitoxin was 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 mmol/L FeCl₂ with 1 µg/mL anti-P-selectin Ab. This yielded encapsulated PLGA AC-NPs containing digitoxin. In session one, 1 × 10¹⁰ nanocapsules were intravenously injected into BALB/c mice exhibiting a primary 4TI mammary carcinoma in the left hind leg and lung metastases. Tumor accumulation was monitored by computed tomography (CT). Subsequently, 10 or 20 Gy ⁶⁰Co γ-radiation was locally administered to primary tumors and lung metastasis. In session two, 1 × 10¹⁰ nanocapsules were injected i.v. and allowed to interact with P-selectin for 9 h; additionally, 10 or 20 Gy ⁶⁰Co γ-radiation was administered solely to the primary tumor. <h3>Results</h3> In session one, CT imaging of the accumulation of anti-VEGFR-1/2 nanocapsules around the primary and metastatic tumors improved their diagnosis. The nanocapsules released P-selectin, anti-PD-L1 Ab, and HJC0152 in response to the initial radiation dose. After session two, the nanocapsules accumulated 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 intensified DC-mediated CD8⁺ T-cell priming. The primed CD8⁺ T-cells attacked PD-L1-suppressed primary and metastatic tumors. PLGA AC-NPs also released digitoxin, which dissociated CTC-clusters and reduced new metastasis seeding; the colonization of seeded tumor cells was subsequently inhibited through impaired PMN formation by HJC0152. Additionally, HJC0152 intensified DC-mediated CD8⁺ T-cell priming. These treatments significantly increased the antitumor effect (EF 1.7) and reduced metastasis by 88.8%. <h3>Conclusion</h3> Our CT-detectable nanocapsules will lead to better diagnosis and tumor treatment.