Boiling Histotripsy-induced Partial Mechanical Ablation Modulates Tumour Microenvironment by Promoting Immunogenic Cell Death of Cancers

Abstract

Boiling histotripsy is a promising non-invasive High-Intensity Focused Ultrasound (HIFU) technique that employs HIFU mechanical effects to fractionate solid tumours without causing any significant thermal damage. It has been suggested that boiling histotripsy may induce a strong immune response due to the absence of denatured antigenic protein at the HIFU focus. The underlying immunological mechanisms of this technique are, however, poorly understood. In this study, we demonstrated the feasibility of using boiling histotripsy to mechanically fractionate human breast adenocarcinoma cells (MDA-MB-231) and the potential immunological effects induced by boiling histotripsy, for the first time. Our results showed that mechanical stresses produced by boiling histotripsy promote immunogenic cell death of cancer cells via TNF-induced necrosis signaling pathway. This immunogenic cell death significantly increases secretions of damage-associated molecular patterns (CRT, HSP70, HMGB-1), pro-inflammatory cytokines (IFN-γ, IL-1α, IL-1β, IL-18) and chemokines (IL-8) which are related to M1 macrophage activation. Furthermore, the levels of these signaling proteins increase with the degree of mechanical damage induced by boiling histotripsy. Together, the results presented can suggest that boiling histotripsy could be a potential therapeutic approach for not only mechanically destroying solid tumours (e.g., breast cancer) but also promoting immunogenic cell death via TNF-induced necrosis to trigger antitumour immunity.

Figure 1

(a) HIFU experimental setup used in the present in vitro study with a cluster of MDA-MB-231 breast cancer cells (●) (b) embedded in 0.6% collagen gel or (c) in liquid media.

Figure 2

Acoustic characterisation of the 2.0 MHz HIFU transducer (H148, Sonic Concepts, USA) employed. Experimentally measured two-dimensional HIFU focal pressure beam profiles along (a) the axial and (b) the lateral directions. Measurements were taken in degassed and deionised water. Comparison of one dimensional (c) axial and (d) lateral focal pressure fields measured in water by the needle hydrophone and calculated with the linearised Khokhlov-Zabolotskaya-Kuznetsov (KZK) wave equation. The lateral and axial full width half maximum (FWHM) dimensions of the transducer used were 0.89 mm and 7.25 mm, respectively.

Figure 3

HIFU pulsing protocol used in the present study. 10 ms long HIFU pulse, 1 Hz pulse repetition frequency, 1% duty cycle and the number of HIFU pulses of 5, 25, 50, 100 or 200 were employed. Simulated peak positive P₊ and negative P₋ pressures at the HIFU focus were 85 and −14 MPa in situ.

Figure 4

(a) Mechanical damage induced in the collagen gel in the absence of the 3D breast cancer cell spheroids at the HIFU focus after the boiling histotripsy exposure. Immediately after the HIFU exposure, the collagen gel was cut through the middle line (bottom view). (b) shows the cross-section of the collagen gel indicating a “tadpole” shaped mechanically fractionated lesion resulting from the boiling histotripsy exposure. The HIFU beam propagates from bottom to top. Inset: in greyscale. (c) Microscopic images of the observation of mechanically fractionated breast cancer cell spheroids in the 0.6% collagen gel. (d) Corresponding confocal images of (c) showing breast cancer cell death after the BH exposure. Live & Dead assay analysis was performed. A scale bar represents 100 μm. Five 10 ms HIFU pulses with P₊ of 85 MPa, P₋ of −14 MPa, duty cycle of 1% and PRF of 1 Hz were employed. CON = Control group (i.e., no boiling histotripsy exposure). BH = Boiling histotripsy.

Figure 5

Immunogenic cell death induced by boiling histotripsy. (a) The changes of cell viability and cytotoxicity of boiling histotripsy with HIFU pulses are shown. Results of (b) cell death pathway analysis and (c) the secretion of damage-associated molecular patterns (DAMPs). (d) Cytokine array analysis showing the secretions of pro-inflammatory cytokines and chemokines from the breast cancer cells treated with boiling histotripsy. CON = Control group (i.e., no boiling histotripsy exposure). BH = Boiling histotripsy. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001.

Figure 6

Boiling histotripsy-induced breast cancer cell death stimulates M1 macrophage polarisation. (a) The morphological changes of macrophages from M0 to M1 like cells after co-cultured with the supernatant of the boiling histotripsy-treated breast cancer cells. A scale bar represents 100 μm. (b) Increases of M1 macrophage-related markers and decreases of M2 macrophage-related markers are shown. 50 boiling histotripsy pulses were used. (c,d) Reprogramed macrophages, from M2 to M1 like, after cultured with the supernatant obtained from the boiling histotripsy-treated cancer cells. CON = Control group (i.e., no boiling histotripsy exposure). BH = Boiling histotripsy. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001.