Optimized cryoablation and combinatorial chemotherapy overcome sublethal survival in pancreatic cancer cells

Optimized cryoablation protocol minimizes the sublethal zone in PDAC models. • High cooling rates, extended holding, and dual cycles elevate lethal thresholds. • Lethal temperatures are shifted upward to −20 °C (PANC-1) and −15 °C (BxPC-3). • First study combining cryoablation with Nab-paclitaxel/Gemcitabine regimen. Incomplete tumor ablation within the peripheral sublethal zone remains a critical limitation of cryoablation for pancreatic ductal adenocarcinoma (PDAC). This study aimed to enhance ablative efficacy by optimizing freezing parameters and integrating combination chemotherapy. A GelMA-based 3D tissue model was initially employed to characterize thermal gradients and define lethal thresholds. Subsequently, a programmable freezer simulated sublethal conditions (-10 to -25 °C) to evaluate specific freezing protocols on PANC-1 and BxPC-3 cells. Results demonstrated that an optimized protocol comprising rapid cooling (50 °C min -1 ), extended hold, and dual cycles significantly elevated the effective lethal isotherm. Mechanistic investigation revealed a temperature-dependent shift in cell death modalities: apoptosis predominated at -13 °C, whereas necrosis became dominant at ≤-15 °C. This transition was underpinned by intensified intracellular reactive oxygen species accumulation and mitochondrial depolarization as freezing temperatures decreased. Furthermore, combining cryoablation with gemcitabine-based regimens markedly suppressed post-cryoablation viability. Notably, cryoablation induced a significant chemo sensitizing effect. In PANC-1 cells, low-dose chemotherapy (1/10th of the high dose) achieved efficacy comparable to high-dose regimens at temperatures ≤-13 °C. Conversely, BxPC-3 cells retained dose-sensitivity down to -13 °C. In conclusion, optimizing cryoablation parameters expands the effective ablative zone and synergizes with low-dose chemotherapy via enhanced oxidative stress and mitochondrial dysfunction, offering a promising strategy for PDAC treatment.