Preclinical high intensity focused ultrasound: mechanisms and applications

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Abstract

Therapeutic ultrasound (TUS) is widely used in medicine as a tool for physiotherapy for rehabilitation medicine, as lithotripsy for bladder or kidney stone destruction, and as high intensity focused ultrasound (HIFU) for ablation of tissues and cancer. Thermal and non-thermal mechanical (acoustic radiation (ARF) or cavitation forces) and the subsequent molecular and biological effects varies with tissue types, type of beam configuration and sonication parameters. Image guided Focused Ultrasound (FUS) can be used to direct thermal and mechanical energy accurately deep within the body without causing demonstrable effects to the intervening soft-tissues or bone. The mechanotransductive effects of TUS in tissues induces the molecular changes in expression of cytokines, chemokines and trophic factors (CCTF) and cellular adhesion molecules (CAM) altering the tissue microenvironment (TME) and enhance cellular homing to targeted tissues. Approximately 12 years ago, the Frank laboratory started investigating the mechanotransductive effects of pFUS in experimental models that with the goal of enhancing cellular therapy by altering the TME. Following TUS, the TME can be altered by the mechanical effects resulting in an acute and short-lived cascade of CCTF was initially identified the chemo-attractant gradient or a molecular zip-codeconsistent with an enhanced homing permeability and retention (EHPR) of cells effect. The acute changes in CCTF and CAM were later described as an sterile inflammatory response (SIR) which is associated with release of damage associated molecular patterns (DAMPs), such as interleukin (IL)1, that induces an innate immune response within targeted tissues. The temporal changes in CCTF and CAM initiated the process of tethering, rolling and diapedesis of stem or immune cells accumulating in targeted tissue. We have also found that following direct implantation of cells coupled with TUS can increase mesenchymal stromal cell (MSC) potency resulting in prolonged survival of the xenographs through a complex process which warranted further investigations. Our initial hypothesis was that pFUS would EHPR of MSC in target tissue by altering CCTF and CAM in the TME. Ideal cell tropism strategies capitalize on molecular biological cues generated without tissue destruction. Our initial studies have demonstrated that pFUS resulted in transient interstitial edema without functional damage in both normal murine muscle and kidney. We have tested the following hypotheses on the molecular effects of pFUS on TME and impact on stem cell homing: 1) pFUS to muscle or kidney with total pressure exposure = 1 sec resulted in increased expression of CCTF and CAM involving the activation of the canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NFB) and cyclo-oxygenase-2 (COX2) signaling pathways; 2) Comparing one versus three daily courses of pFUS in skeletal muscle coupled with either IV human MSC or endothelial precursor cells (EPC) resulted in tunable cell tropism in which multiple treatments of pFUS with cell infusions significantly increased EHPR and human cells within targeted area compared to contralateral control; 3) Inhibition of CCTF and CAM response following pFUS could be specifically targeted by treating mice with ibuprofen (non-specific COX1 and 2 inhibitor) or etanercept (TNF inhibitor), anakinra (IL1a inhibitor) or prednisone (inhibit nuclear translocation of NFB) before sonication resulting in significant alterations in the TME by suppressing COX2 and NFB pathways along with significant decreases in MSC tropism to normal and dystrophic muscle or kidney; and 4) In an Cis-platin acute kidney injury (AKI) model, pFUS coupled with MSC infusion performed either before or after increases in serum blood urea nitrogen (BUN) or creatinine (Cr), resulted in greater numbers of human cells homing to treated kidneys associated with a global shift in renal TME as compared to mice with AKI alone. The combination of pFUS with MSC infusion prevented AKI when given before rise in serum BUN and Cr and rescued and improved survival in mice with AKI compared to animals receiving either of the interventions or saline alone. We investigated pulse focused ultrasound (pFUS) ability to activate mechanical radiation forces or indirectly through microbubble (MB) cavitation forces, including the transient receptor potential cation channels (TRPC) and voltage gated calcium channels (VGCC) can be opened following sonication in various tissues. We observed that mechanically-gated TRPC1 and verapamil-sensitive VGCC are both required to transduce pFUS forces into intracellular Ca2+ signaling that upregulates COX2 and thus increases CCTF in the kidney and muscle microenvironments. Moreover, pFUS generates TRPC1 currents that secondarily activate complexed VGCC through local membrane depolarization. These results establish a basis for a unifying mechanism that reconciles previously disparate observations of how ultrasound activation of different plasma membrane channels. We evaluated intracellular Ca2+ dynamics during pFUS sonication in TCMK1 renal tubular cells using live-cell Ca2+ imaging and a fluorescent NFkB reporter gene. Pharmacological or genetic manipulation of cytosolic-Ca2+-generating mechanisms revealed that pFUS (1 MHz at 3 MPa peak negative pressure) activated mechanosensitive transient receptor potential C1 (TRPC1) channels and voltage-gated Ca2+ channels to cause Ca2+-induced Ca2+ release (CICR) from ryanodine receptors. In addition, pFUS increased cytosolic Ca2+ from inositol triphosphate (IP3) receptor signaling. During sonication, IP3 was formed by extracellular-ATP-activation of purinergic P2Y receptors. Upregulated NFkB required increased cytosolic Ca2+ from both CICR and IP3-mediated release. Inhibiting cytosolic Ca2+ extrusion by the Na+/ Ca2+ exchanger or plasma-membrane Ca2+ ATPase, or store-operated Ca2+ entry through ORAI1 inhibition did not affect NFkB expression. We also observed that pFUS causes DNA damage within tumors, which is a potent activator of immunity and could contribute to changes in the TME. pFUS was shown to increase TUNEL reactivity (range = 1.6-2.7-fold) in all cell types. All lines displayed cytosolic Ca2+ transients during sonication. pFUS increased superoxide (range = 1.6-2.0-fold) and H2O2 (range = 2.3-2.8-fold). BAPTA-AM blocked increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reactivity, superoxide and H2O2 formation, while Trolox also blocked increased TUNEL reactivity increased after pFUS. mtTEMPOL allowed H2O2 formation and did not block increased TUNEL reactivity after pFUS. Mechanotransduction of pFUS directly induces DNA damage in tumor cells by cytosolic Ca2+ transients causing formation of superoxide and subsequently, H2O2. These results further suggest potential clinical utility for pFUS. We investigated the effects of pFUS on the molecular and immune cell changes in the Tumor microenvironment (TuME) in the murine B16 melanoma and 4T1 breast cancer to determine if non-ablative sonication would induce changes in CCTF that would be consistent with shifting from an immunosuppressive to anti-tumor microenvironment. Following pFUS at 1MHz at 6 megapascal (MPa) to B16 melanoma and 4T1 breast cancer flank tumors, proteomic analysis at 24 hrs of tumor lysates revealed differences in the molecular expression of CCTF and CAM at each time point and between the two tumors except for TGF in which there was significant decreases. pFUS suppressed anti-inflammatory cytokines in B16 tumors whereas in 4T1 tumors there were decreases in anti-inflammatory cytokines and increases in pro-inflammatory cytokines and cell adhesion molecules. pFUS at 6MPa increased calreticulin and alterations check-point proteins along with tumoral and splenic immune cell changes