Exploring the induction of immunogenic cell death (ICD) by high-intensity focused ultrasound (HIFU)

Immunogenic cell death (ICD) is an intriguing concept within cancer immunotherapy. Indeed, by inducing a specific means of cellular demise, a protective immune response can be triggered against the dying cells. As such, the dying cancer cells are—in a way—turned into a vaccine, attracting immune cells to the site of cell death, and activating them to trigger an anticancer immune response. Since the early discovery of ICD, more mechanistic insights into the hallmarks of ICD have been unraveled and several state-of-the-art cancer therapies have been identified as bona fide ICD inducers (1, 2).One of the cancer therapies which could be a potential candidate to induce ICD is high-intensity focused ultrasound (HIFU) (3). HIFU is currently used as a way of debulking solid tumors in a non-invasive, image-controlled and precise way without the use of ionizing radiation. Depending on the ultrasound parameters, tumor tissue can be ablated either in a mechanical (tissue disruption) or thermal way (heating and coagulative necrosis). Interestingly, there are some indications that HIFU may also affect the immune system: experiments in mice and man have shown attraction/activation of immune cells to/in the tumor site (4, 5). Of note: these immunostimulating effects were reported to be more prominent for mechanical HIFU than thermal HIFU. However, neither the mechanisms behind these observations, nor the possible link to the induction of ICD have been investigated. Therefore, the key aim of work is to investigate the potential of HIFU to induce ICD. Using a custom-designed HIFU set-up, we were able to—in a precise, automated and controlled way—expose B16F0 melanoma cells to HIFU in vitro. The HIFU-exposed tumor cells were subsequently evaluated for the occurrence of apoptosis and ICD hallmarks. More specifically, we evaluated the exposure of calreticulin (CRT) on the cell membrane and release of high mobility group box 1 (HMGB-1) and adenosine triphosphate (ATP). Our data show that application of mechanical HIFU protocols (using short, repeated ultrasound bursts) induce a significant fraction of apoptotic cells (as measured by a loss of mitochondrial membrane potential) within 4h after treatment. Importantly, already within the first hour after HIFU exposure, high levels of the ICD hallmark and Toll-like receptor 4 agonist HMGB-1 were found in the supernatant of the HIFU-treated cells, compared to untreated controls. To determine the effect of HIFU on ATP release, a quinacrine assay was performed to stain intracellular ATP-containing vesicles. Using flow cytometry, we observed that HIFU triggered exocytosis of these vesicles, with 2- to 3-fold reductions in quinacrine fluorescence at 4h and 24h after HIFU exposure, respectively. In addition, at 24h after HIFU exposure, >30% of the cells exposed CRT on the outer leaflet of the cell membrane, which was a drastic increase compared to blanks (