Purpose In high intensity focused ultrasound (HIFU) therapy, an ultrasound beam is focused within the body to locally affect the targeted site without damaging intervening tissues. The most common HIFU regime is thermal ablation. Recently, there has been increasing interest in generating purely mechanical lesions in tissue (histotripsy). This paper provides an overview of several studies on the development of histotripsy methods toward clinical applications. Material and Methods Two histotripsy approaches and examples of their applications are presented. In one approach, sequences of high-amplitude, short (microsecond-long), focused ultrasound pulses periodically produce dense, energetic bubble clouds that mechanically disintegrate tissue. In an alternative approach, longer (millisecond-long) pulses with shock fronts generate boiling bubbles and the interaction of shock fronts with the resulting vapor cavity causes tissue disintegration. Results Recent pre-clinical studies on histotripsy are reviewed for treating benign prostatic hyperplasia (BPH), liver and kidney tumors, kidney stone fragmentation, enhancing antitumor immune response, and tissue decellularization for regenerative medicine applications. Potential clinical advantages of the histotripsy methods are discussed. Conclusions Histotripsy methods can be used to mechanically ablate a wide variety of tissues, whilst selectivity sparing structures such as large vessels. Both ultrasound and MR imaging can be used for targeting and monitoring the treatment in real time. Although the two approaches utilize different mechanisms for tissue disintegration, both have many of the same advantages and offer a promising alternative method of noninvasive surgery.

Raf kinase inhibitory protein (RKIP; also known as phosphatidylethanolamine-binding protein or PEBP) is a modulator of the Raf/MAPK signaling cascade and a suppressor of metastatic cancer. Here, we show that RKIP inhibits MAPK by regulating Raf-1 activation; specifically, RKIP acts subsequent to Raf-1 membrane recruitment, prevents association of Raf-1 and p21-activated kinase (PAK), and blocks phosphorylation of the Raf-1 kinase domain by PAK and Src family kinases. Mutation of the PAK and Src phosphorylation sites on Raf-1 to aspartate, a phosphate mimic, prevented RKIP association with or inhibition of Raf-1 signaling. Interestingly, although RKIP can interact with B-Raf, RKIP depletion had no effect on activation of B-Raf. Because c-Raf-1 and B-Raf are both required for maximal MAPK stimulation by epidermal growth factor in neuronal and epithelial cell lines, we determined whether RKIP significantly affects MAPK signaling. In fact, RKIP depletion increased not only the amplitude but also the sensitivity of MAPK and DNA synthesis to epidermal growth factor stimulation by up to an order of magnitude. These results indicate that selective modulation of c-Raf-1 but not B-Raf activation by RKIP can limit the dynamic range of the MAPK signaling response to growth factors and may play a critical role in growth and development.Signaling cascades that control fundamental cellular processes need to be tightly regulated. The MAPK 1 cascade is an evolutionarily conserved signaling module that is activated by a diverse set of signals and that stimulates numerous biological processes, including growth and differentiation. RKIP is a ubiquitously expressed and highly conserved protein with homologs in Arabidopsis thaliana, Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster that display remarkable degrees of interspecies sequence and structural similarity (reviewed in Ref. 6). Mammalian RKIP is distinct from other known proteins, and its function has remained largely enigmatic. A role for RKIP in signaling cascades was demonstrated when it was shown that RKIP binds to Raf-1 (7). However, RKIP also has a number of other reported functions, including inhibition of other kinases such as GRK2 (G protein-coupled receptor kinase-2) (8) and upstream kinase activators of IB kinase (9). Recently, RKIP has been identified as a metastasis suppressor gene, and this function correlates with MAPK activity (10).Elucidating the mechanism of RKIP action is important both for a complete understanding of Raf regulation and for generating potential therapeutic reagents. Previous studies have suggested that RKIP binds to and acts downstream of Raf-1 by competitive interference with MEK binding to Raf-1 (11). We previously identified a mechanism for overcoming inhibition of MAPK signaling by RKIP (12). Protein kinase C, activated by either phorbol esters or epidermal growth factor (EGF), phosphorylates RKIP at Ser 153 , and this phosphorylation causes the dissociation of RKIP from Raf-1 and the subsequent activation of ...

Boiling histotripsy (BH) is an experimental focused ultrasound technique that produces non-thermal mechanical ablation. We evaluated the feasibility, short-term histologic effects, and the resulting acute inflammatory response to BH ablation of renal cell carcinoma (RCC) in the Eker rat. Genotyped Eker rats were monitored for de novo RCCs with serial ultrasound (US) imaging. When tumors were ≥8 mm, rats underwent ultrasound-guided extracorporeal ablation of the tumor with BH, a pulsed focused US technique that produces non-thermal mechanical ablation of targeted tissues, or a sham US procedure. Treatments targeted approximately 50% of the largest RCC with a margin of normal kidney. BH treated rats were euthanized at 1 (n=4) or 48 (n=4) hours, and sham subjects (n=4) at 48 hours. Circulating plasma cytokine levels were assessed with multiplex assays prior to and at 0.25, 1, 4, 24 and 48 hours following treatment. Kidneys were collected and processed for histologic assessment, immunohistochemistry and intrarenal cytokine concentration measurements. For statistical analysis Student’s t-test was used. US-guided BH treatment was successful in all animals, producing hypoechoic regions within the targeted volume consistent with BH treatment effect. Grossly, regions of homogenized tissue were apparent with evidence of focal intra-parenchymal hemorrhage. Histologically, BH produced a sharply demarcated region of homogenized tumor and non-tumor tissue containing acellular debris. BH treatment was associated with significantly increased relative concentration of plasma TNF vs. sham treatment (p<0.05) and transient elevations in HMGB1, IL-10 and IL-6 consistent with acute inflammatory response to trauma. Intrarenal cytokine concentrations followed the same trend. At 48 hours, enhanced infiltration of CD8+ T cells was observed by immunohistochemistry in both the treated and un-treated contralateral RCC/kidneys in BH-treated animals vs. sham treatment. BH treatment was well tolerated with transient gross hematuria and a perinephric hematoma developing in one subject each. The study demonstrates the feasibility of BH ablation of de novo RCC and suggests activation of the acute inflammatory cascade following treatment that appears to stimulate CD8+ T cell infiltration of both treated and untreated tumors. Longer duration chronic studies are ongoing to characterize the longevity and robustness of this response.

We have established a minimally invasive dog model of prostate cancer. This model may be valuable for studying prostate cancer progression and distant metastasis.

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