Abstract:Histotripsy is a noninvasive, non-thermal ablation technique that uses high-amplitude, focused ultrasound pulses to fractionate tissue via acoustic cavitation. The goal of this study was to show the potential of histotripsy with electronic focal steering to achieve rapid ablation of a tissue volume at a rate matching or exceeding current clinical techniques (~1–2 mL/min). Treatment parameters were established in tissue-mimicking phantoms and applied to ex vivo tissue. 6-μs pulses were delivered by a 250 kHz ar… Show more
“…This is consistent with the unique cell death mechanisms driven by electroporation-based ablation therapy that are time-dependent, with cell death detectable up to a week after treatment [26]. Unlike other forms of non-thermal tumor ablation, such as histotripsy that causes instant cell rupture [27], the cell death induced by H-FIRE continues over an extended timeframe.Fig. 1High Frequency Irreversible Electroporation (H-FIRE) Induces Inflammatory Cell Death and Effectively Attenuates the Tumor Promoting Microenvironment In Vitro .…”
Background: Despite promising treatments for breast cancer, mortality rates remain high and treatments for metastatic disease are limited. High-frequency irreversible electroporation (H-FIRE) is a novel tumor ablation technique that utilizes high-frequency bipolar electric pulses to destabilize cancer cell membranes and induce cell death. However, there is currently a paucity of data pertaining to immune system activation following H-FIRE and other electroporation based tumor ablation techniques. Methods: Here, we utilized the mouse 4T1 mammary tumor model to evaluate H-FIRE treatment parameters on cancer progression and immune system activation in vitro and in vivo. Findings: H-FIRE effectively ablates the primary tumor and induces a pro-inflammatory shift in the tumor microenvironment. We further show that local treatment with H-FIRE significantly reduces 4T1 metastases. H-FIRE kills 4T1 cells through non-thermal mechanisms associated with necrosis and pyroptosis resulting in damage associated molecular pattern signaling in vitro and in vivo. Our data indicate that the level of tumor ablation correlates with increased activation of cellular immunity. Likewise, we show that the decrease in metastatic lesions is dependent on the intact immune system and H-FIRE generates 4T1 neoantigens that engage the adaptive immune system to significantly attenuate tumor progression. Interpretation: Cell death and tumor ablation following H-FIRE treatment activates the local innate immune system, which shifts the tumor microenvironment from an anti-inflammatory state to a pro-inflammatory state. The non-thermal damage to the cancer cells and increased innate immune system stimulation improves antigen presentation, resulting in the engagement of the adaptive immune system and improved systemic anti-tumor immunity.
“…This is consistent with the unique cell death mechanisms driven by electroporation-based ablation therapy that are time-dependent, with cell death detectable up to a week after treatment [26]. Unlike other forms of non-thermal tumor ablation, such as histotripsy that causes instant cell rupture [27], the cell death induced by H-FIRE continues over an extended timeframe.Fig. 1High Frequency Irreversible Electroporation (H-FIRE) Induces Inflammatory Cell Death and Effectively Attenuates the Tumor Promoting Microenvironment In Vitro .…”
Background: Despite promising treatments for breast cancer, mortality rates remain high and treatments for metastatic disease are limited. High-frequency irreversible electroporation (H-FIRE) is a novel tumor ablation technique that utilizes high-frequency bipolar electric pulses to destabilize cancer cell membranes and induce cell death. However, there is currently a paucity of data pertaining to immune system activation following H-FIRE and other electroporation based tumor ablation techniques. Methods: Here, we utilized the mouse 4T1 mammary tumor model to evaluate H-FIRE treatment parameters on cancer progression and immune system activation in vitro and in vivo. Findings: H-FIRE effectively ablates the primary tumor and induces a pro-inflammatory shift in the tumor microenvironment. We further show that local treatment with H-FIRE significantly reduces 4T1 metastases. H-FIRE kills 4T1 cells through non-thermal mechanisms associated with necrosis and pyroptosis resulting in damage associated molecular pattern signaling in vitro and in vivo. Our data indicate that the level of tumor ablation correlates with increased activation of cellular immunity. Likewise, we show that the decrease in metastatic lesions is dependent on the intact immune system and H-FIRE generates 4T1 neoantigens that engage the adaptive immune system to significantly attenuate tumor progression. Interpretation: Cell death and tumor ablation following H-FIRE treatment activates the local innate immune system, which shifts the tumor microenvironment from an anti-inflammatory state to a pro-inflammatory state. The non-thermal damage to the cancer cells and increased innate immune system stimulation improves antigen presentation, resulting in the engagement of the adaptive immune system and improved systemic anti-tumor immunity.
“…Our choice of time points was based on the ultrasound parameters we chose to employ and our experience with their biological effects and immunohistochemistry studies; namely, we found cell death/destruction was significantly more rapid with mechanical ablation than with thermal ablation. Previously reported results with histotripsy 27 have also suggested cell death immediately following treatment. Here, matching immunohistochemistry (IHC) datasets at 72 h and 7 days and flow cytometry at 72 h were acquired for both treatments.…”
High intensity focused ultrasound (HIFU) rapidly and non-invasively destroys tumor tissue. Here, we sought to assess the immunomodulatory effects of MR-guided HIFU and its combination with the innate immune agonist CpG and checkpoint inhibitor anti-PD-1. Mice with multi-focal breast cancer underwent ablation with a parameter set designed to achieve mechanical disruption with minimal thermal dose or a protocol in which tumor temperature reached 65 °C. Mice received either HIFU alone or were primed with the toll-like receptor 9 agonist CpG and the checkpoint modulator anti-PD-1. Both mechanical HIFU and thermal ablation induced a potent inflammatory response with increased expression of Nlrp3, Jun, Mefv, Il6 and Il1β and alterations in macrophage polarization compared to control. Furthermore, HIFU upregulated multiple innate immune receptors and immune pathways, including Nod1, Nlrp3, Aim2, Ctsb, Tlr1/2/4/7/8/9, Oas2, and RhoA. The inflammatory response was largely sterile and consistent with wound-healing. Priming with CpG attenuated Il6 and Nlrp3 expression, further upregulated expression of Nod2, Oas2, RhoA, Pycard, Tlr1/2 and Il12, and enhanced T-cell number and activation while polarizing macrophages to an anti-tumor phenotype. The tumor-specific antigen, cytokines and cell debris liberated by HIFU enhance response to innate immune agonists.
“…The RBC tissue-mimicking phantoms were prepared with 1% agarose powder (DSA20070, DOT Scientific, Burton, MI, USA) and 5% v/v RBCs mixed in normal saline. This RBC tissue-mimicking phantom had a three-layer structure with one very thin (~500 μm) RBC- agarose-saline hydrogel layer suspended between two thick (7 mm) transparent gel layers as previously described [26]. The RBC-agarose mixture changed from translucent red to transparent and colorless as a result of RBC lysis, providing real-time visual feedback for histotripsy lesion development [15].…”
Residual bubbles produced after collapse of a cavitation cloud provide cavitation nuclei for subsequent cavitation events, causing cavitation to occur repeatedly at the same discrete set of sites. This effect, referred to as cavitation memory, limits the efficiency of histotripsy soft tissue fractionation. Besides passively mitigating cavitation memory by using a low pulse repetition frequency (~1 Hz), an active strategy was developed by our group. In this strategy, low-amplitude ultrasound sequences were used to stimulate coalescence of residual bubbles. The goal of this work is to remove cavitation memory and achieve rapid, homogeneous lesion formation using a single phased array transducer. A 1-MHz integrated histotripsy and bubble coalescing (BC) transducer system with a specialized electronic driving system was built in house. High-amplitude ( MPa) histotripsy pulses and subsequent low-amplitude (~1-2 MPa) BC sequences were applied to a red blood cell tissue-mimicking phantom at a single focal site. Significant reduction of the cavitation memory effect and increase in the fractionation rate were observed by introducing BC sequence. Effects of BC pulsing parameters were further studied. The optimal BC parameters were then utilized to homogenize a mm region at high rate.
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