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Biomechanics

New Tools and Applications in Biomechanics

Ultrasound Mechanotherapy to Non-invasively Sensitize Prostate Tumors to TRAIL-Mediated Apoptosis

Saturday, October 14, 2023
2:45 PM – 3:00 PM PDT
Location: 333 (Center, Level 3)
Introduction:: The King Lab has previously demonstrated how fluid shear stress (FSS) sensitizes breast, colon, and prostate cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis through Piezo1 activation of the intrinsic apoptotic pathway [1]. Piezo1 is a mechanosensitive ion channel (MSC) that is activated by external stimuli and cancer cells recognize and respond to these signals, leading to calcium influx to promote pro-apoptotic pathways [1,2]. We aimed to explore the biochemical response that occurs in prostate cancer (PC) when treated with therapeutic ultrasound (US) combined with TRAIL. TRAIL binds to death receptors 4 and 5 on cancer cells, while sparing healthy cells, however soluble TRAIL on its own lacks efficacy in humans to be used as a cancer therapy.


Materials and Methods:: Sensitization of TRAIL-mediated apoptosis via US was examined in two PC cell lines: weakly metastatic LNCaPs, and highly metastatic PC3s. The US system is shown in Figure 1A. For the results in Figures 1B-D, a 1.1 MHz transducer, with a 17% duty cycle, 0.33 Hz pulse repetition frequency, and 500 ms burst duration was employed, over a range of pressures: 262.5, 380, 472, 944, and 1993 kPa. Cells were treated with TRAIL (200 ng/mL), and exposed to a 60 s US. A Piezo1 inhibitor, GsMTx-4, and a calcium chelator, EGTA, were used to confirm MSC activation during TRAIL+US treatment. Viability and apoptosis were assessed using the Annexin-V/ propidium iodide (AV/PI) assay and mitochondrial depolarization was measured using a JC-1 assay. A pilot in vivo study used male NU/NU mice (Fig 1E-F) bearing subcutaneous PC3 tumors inoculated on each flank. Soluble TRAIL (2 mg/kg) was delivered intratumorally, followed by a single US treatment (5 min/ tumor), using the same parameters described above, at 944 kPa. Tumors were processed by the Translational Pathology Core at Vanderbilt for immunohistochemistry (IHC) analysis upon resection.


Results, Conclusions, and Discussions::
Combination treatment delineated how increasing pressure (60 s US) led to a pressure-dependent increase in TRAIL-mediated apoptosis and mitochondrial depolarization for PC3 cells (Fig 1B-C), complimented by an increase in TRAIL sensitization (Fig 1D). Treatment of PC3 cells with GsMTx-4 and EGTA reduced TRAIL sensitization upon combination treatment (results not shown), demonstrating activation of pro-apoptotic pathways, such as Piezo1. The in vivo study demonstrates for the first time, the use of US to decrease tumor burden when applied with soluble TRAIL (Fig 1E-F), evident by the decreased tumor burden. IHC analysis of the tumors revealed significantly upregulated DR5 and Piezo1 expression for the TRAIL+US-treated tumors.





TRAIL and US combination therapies can provide an alternate, non-invasive mechanobiology-based therapeutic, which employs technology already widely available. This is critical considering that the prostate is located near neurovascular bundles that risk being damaged during invasive treatments options [3]. Future work will apply these studies to additional cancer types, test alternate US parameters, and determine if additional MSCs are being activated and contributing to the TRAIL-sensitization of cancer cells via US exposure.




Acknowledgements (Optional): : This research was funded by the NSFGRFP.

References (Optional): : [1] JM Hope, Cell Death & Dis, 2019, 10, 1-15.
[2] JA Dombroski, Cells, 2021, 10, 1-14.
[3] Y Wu; Nature Biomed. Engr, 2021, 5, 1336-47.