Introduction: : The King Lab has previously demonstrated that noninvasive focused ultrasound (FUS) enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in prostate cancer (PC) by mechanically activating Piezo1, leading to mitochondrial depolarization and significant tumor regression both in vitro and in vivo [1]. In this study, we aimed to investigate the TRAIL sensitivity of prostate cancer cells treated with therapeutic FUS under different oxygen microenvironments. Soluble TRAIL binds to death receptors 4 and 5 specifically on cancer cells; however, its stand-alone efficacy is limited and requires complementary approaches to enhance its effectiveness. Also, this combinatorial approach using FUS and TRAIL is broadly applicable across a wide range of cancer types, offering a versatile strategy for improving therapeutic responses in various tumor microenvironments
Materials and
Methods: : Sensitization of TRAIL-mediated apoptosis via FUS was examined in PC3 cells under different oxygen microenvironments. The FUS parameters used were: 1.1 MHz frequency, 250 mVpp amplitude, 3-second burst period, and 500 kCyc cycle count. As shown in Fig. 1a, the experimental conditions included: (1) cells expanded, treated, and maintained under normoxia (NN); (2) cells expanded under hypoxia, treated, and then returned to normoxia (HN); and (3) cells expanded, treated, and maintained entirely under hypoxia (HH). The oxygen level was maintained at 1% in the hypoxic incubator and 20% in the normoxic incubator. PC3 cells were treated with TRAIL (200 ng/mL) immediately before FUS treatment. Viability and apoptosis were assessed 24 hours after treatment using the Annexin V/Propidium Iodide (AV/PI) assay.
Results, Conclusions, and Discussions:: The combination of FUS and TRAIL resulted in increased apoptosis in PC3 cells (Fig. 1b–c), indicating enhanced TRAIL sensitization. Notably, TRAIL or FUS treatment alone, when followed by reoxygenation, led to increased apoptosis in cells initially expanded under hypoxia and then returned to normoxia. This condition mimics the physiological transition of circulating tumor cells (CTCs), which experience hypoxia within the primary tumor and reoxygenation upon entering the bloodstream. However, the highest level of apoptosis was observed in cells expanded and treated under hypoxia and maintained in hypoxia post-treatment, reflecting the persistent hypoxic environment within solid tumors and suggesting that a fully hypoxic microenvironment further amplifies the efficacy of FUS + TRAIL therapy. These results demonstrate that combining TRAIL and FUS under controlled oxygen microenvironments offers a promising, non-invasive, mechanobiology-based therapeutic strategy. The maximum pressure applied in these experiments was 1389 kPa, corresponding to a mechanical index (MI) of 1.3, which remains below the FDA safety threshold of 1.9 for avoiding cavitation.
Future work will apply these studies to additional cancer types, test alternate FUS parameters, and investigate the underlying mechanisms of cell death in hypoxic conditions. This will involve evaluating mitochondrial function, assessing caspase activation pathways, and exploring calcium influx and the role of mechanosensitive ion channels such as Piezo1. We are currently extending these studies to examine other cancer types, including glioblastoma, colorectal cancer, and additional prostate cancer models. These investigations aim to determine whether apoptosis is mediated through extrinsic or intrinsic pathways, or if alternative forms of cell death are engaged under different oxygen microenvironments.
Acknowledgements and/or References (Optional):: Acknowledgements: This research was funded by the Cancer Prevention and Research Institute of Texas (CPRIT) to M.R.K., Grant No. RR230029.
References: [1] Fabiano, A. R., Newman, M. W., Dombroski, J. A., Rowland, S. J., Knoblauch, S. V., Kusunose, J., ... & King, M. R. (2025). Applying Ultrasound to Mechanically and Noninvasively Sensitize Prostate Tumors to TRAIL‐Mediated Apoptosis. Advanced Science, 2412995.
