Engineering and characterization of a novel hydrogel-based cancer spheroid system to recapitulate clinical wIRA...

Water-filtered infrared-A (wIRA) mild hyperthermia (mHT) combined with radiotherapy can effectively treat superficial tumors. While in vitro models are essential for mechanistic studies, existing approaches mostly rely on monolayer cultures and non-clinical heating. We present a multilayer agarose-based platform enabling clinically relevant wIRA-hyperthermia of 3D-tumor spheroids using a clinical device, reproducing spatial thermal gradients and thermographic feedback control without an external heat source.

We designed a multilayer stack with alternating layers of hemoglobin-agarose chips (2% Hb, 2% talcum) as infrared absorbers, spacers, and microwell chips containing tumor spheroids at 5 and 11 mm depth. Temperature profiles were characterized using thermocouples, and evaluated according to European Society for Hyperthermic Oncology (ESHO) guidelines. We monitored T47D breast cancer spheroid growth by optical scanning for 28 days, and assessed cell death by flow cytometry 48 h post-treatment.

The system achieved stable heating from room temperature within 15 ± 8 min, with mean steady-state temperatures of 41.9 ± 0.5 °C (n = 26) and 40.3 ± 0.4 °C at 5 mm and 11 mm depth, respectively. T50 (42.1 ± 0.5 and 40.5 ± 0.3 °C) and T90 values (41.3 ± 0.7 and 39.8 ± 0.7 °C) at these depths met or approached ESHO guidelines. Spheroids at higher temperatures (upper layer) were modestly larger than controls (p < 0.01) at 14 and 28 days; lower temperatures had no effect. mHT did not induce apoptosis or necrosis.

The novel model replicates in vivo wIRA temperature profiles in 3D cell culture and supports several downstream analyses. It enables investigation of cellular responses to clinically relevant mHT and provides a technical basis for future translational research.