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SIR 2025

Interventional Oncology

Scientific Session

Session: Interventional Oncology - Basic Science

Nano-catalyzed Cryo-immunotherapy through Necroptosis and Local Delivery of PD-L1 Blockade

Wednesday, April 2, 2025
11:24 AM – 11:33 AM CT
Location: Music City Center, 207 CD
Purpose: We aim to evaluate the therapeutic efficacy of our developed cryo-nano-nucleator (CNN) catalyzed cryoablation and potential cryo-immunotherapy applications.

Materials and Methods: CNN was prepared and loaded with anti-PD-L1 immune checkpoint inhibitors at a 10:1 ratio (CN:anti-PD-L1). Freezing temperature changes catalyzed by CNN were measured via differential scanning calorimetry. In vitro CNN-cryotreatment occurred at −80°C for 3 min per cycle for the assessment of cell viability using CCK-8 and AnnexinV/PI. Necroptosis programmed cell death was evaluated with antagonist and western blotting for RIPK3, MLKL, and p-MLKL. Immunogenic cell death was assessed by measuring DAMPs and DC maturation. In vivo studies in TrampC1-bearing mice (C57BL6, male, 6-7 weeks) compared control, cryoablation, CNN-cryoablation, and CNN-cryoablation+anti-PD-L1 (n = 6/group; CN, 0.4 mg/mice; anti-PD-L1, 0.2 mg/mice). In vivo CNN-cryoablation was performed with two freeze-thaw cycles for 3 min/cycle, monitored by thermal camera. Next-generation RNA sequencing identified differentially expressed genes (DEG). Tumor-infiltrating and systemic immune cells were analyzed via multi-parameter flow cytometry. 

Results: CNN exhibited a supra-nanostructure in TEM images and achieved a 67% loading efficiency for anti-PD-L1. CNN elevated the freezing temperature to -17oC compared to -22.9oC for the aqueous solution. It catalyzed cellular ice formation, causing CNN-treated cells to freeze at -28.4oC vs. -32.8oC for untreated cells. Consequently, CNN-cryoablation reduced cell viability to 18% compared to 62% in cryoablation alone. Rapid ice formation induced physical stress (necrosis), but also drastic water flux and ion condensation across the cell membrane induced osmotic stress (necroptosis). This was confirmed by the inhibition of cryo-cell death with RIPK1, RIPK3, and MLKL antagonists and increased MLKL phosphorylation in the western blot results. Enhanced cell-killing facilitated DAMPs release and DC maturation. DEGs also revealed necroptosis, cryo-induced damage, metabolic alterations, and anti-cancer immunity in tumor tissues. Furthermore, CNN-cryoablation combined with local anti-PD-L1 led to a significant increase in tumor-infiltrating immune cells, particularly the induction of IFNg-secreting and memory T cell, accompanied by abscopal tumor regression. 

Conclusion: CNN enhances anti-cancer immunity by promoting necrosis and necroptosis. Combining with anti-PD-L1 induced memory immune responses and abscopal effects, offering a potential for cancer vaccination.