Multiple-Session Magnetic Fluid Hyperthermia: Augmented MNPs Penetration Led to Complete Eradication of Hetero-Multicellular Breast Tumor Spheroids via Apoptosis.

Magnetic fluid hyperthermia (MFH) is an emerging localized cancer therapy that uses magnetic nanoparticles to selectively induce 42 to 45 °C mild hyperthermia within tumor tissues under alternating magnetic field to kill cancer cells. Despite encouraging clinical outcomes in glioblastoma and prostate cancer, the broader clinical translation of MFH across various tumor types remains limited by heterogeneous nanoparticle distribution, inconsistent tumor heating, and the frequent use of poorly predictive preclinical two-dimensional (2D) cultures. These conventional cultures fail to recapitulate the structural and stromal complexity of solid tumors and often overestimate therapeutic efficacy. In an attempt to recreate the cellular heterogeneity of clinical breast tumors to some extent, we developed a hetero-multicellular spheroid of breast cancer by co-culturing breast adenocarcinoma cells MCF-7, with non-cancerous murine fibroblasts McCoy. This approach generated heterospheroids that mimic certain key features of the breast tumor microenvironment, including heterogeneous cell population, fibroblast activation, collagen-rich extracellular matrix deposition, and the spatial arrangement of proliferative, quiescent, and necrotic cells. Following histologic validation, the hyperthermic potential of poly(acrylic acid)-coated Mn0.9Zn0.1Fe2O4 nanoparticle-based magnetic fluid on the heterospheroids was assessed under a 10 kA/m magnetic field strength and 332 kHz frequency. A single-session MFH inhibited heterospheroid growth and induced apoptosis; however, it did not significantly ablate the spheroids. Subsequently, two MFH sessions led to the complete elimination of the spheroids, accompanied by pronounced apoptotic activity. These findings suggest that repeated MFH can overcome thermotolerance, thereby augmenting therapeutic potential against stromal-rich breast tumors.