Influence of high-intensity focused ultrasound on the development of metastases.

OBJECTIVE: The hypothesis that exposure of a solid tumor to high-intensity focused ultrasound (HIFU) could lead to an increase of metastases was investigated in an animal model. METHODS: The highly metastatic AT-6 Dunning R3327 rat prostate cancer subline was implanted into the hind limb of Fisher-Copenhagen rats and was exposed to 1 pulse/mm2 tumor (acoustical power 1,000 W/cm2) delivered by an experimental machine (Ablatherm, Technomed, France), or the animals were sham treated, as soon as the tumor had reached a volume of 175-225 mm3. The tumor-bearing leg was amputated 24 h later and the number of metastases examined 12 weeks thereafter at autopsy. RESULTS: Metastases were seen in 3 (23%) of the 13 animals exposed to HIFU and in 4 (25%) of the 16 sham-treated animals; this is not significantly different. There was also no significant difference in weight of the lungs that contained metastases between sham-treated and HIFU-treated animals. CONCLUSION: Metastatic spread of animal tumors with a high metastatic potential is not enhanced by HIFU exposure.

The influence of high-energy shock waves on the development of metastases.

The hypothesis that exposure of a solid tumor to high-energy shock waves (HESW) could lead to an increase of metastases was investigated in an animal model. The highly metastatic AT-6 Dunning R3327 rat prostate cancer subline was implanted in the hind limb of a Fisher-Copenhagen rat and was exposed to 6000 shock waves delivered by an experimental lithotripter, or sham-treated, as soon as the tumor had reached a volume of 175-225 mm3. The tumor-bearing leg was amputated 24 h later and the number of metastases was examined 12 weeks thereafter at autopsy. Metastases were seen in 82% of the animals exposed to HESW and in 25% of the sham-treated animals. There was no significant difference in weight of the lungs that contained metastases, between sham and treated animals. These results were confirmed in a second experiment. We conclude that the metastatic spread of tumors with a high metastatic potential may be enhanced by shock-wave exposure.

In vitro proton magnetic resonance spectroscopy of four human prostate cancer cell lines.

There is accumulating evidence that some biochemical pathways observable by magnetic resonance spectroscopy, e.g., citrate acid and phospholipid metabolism, are altered in human prostate cancer. Four well-established human prostate cancer cell lines were therefore studied with magnetic resonance spectroscopy to compare differences in metabolic content with tumor biological behavior. Herein we demonstrate that, although each cell line has its own metabolic profile, relative creatine and citrate levels can be used to discriminate the androgen-dependent LNCaP cell line from the androgen-independent DU-145, TSU, and PC-3 cell lines.

The effects of successive high-energy shock-wave tumor administration on tumor blood flow.

The effects of repeated high-energy shock wave (HESW) tumor administration on tumor blood flow (TBF) were studied in NU-1 human kidney cancer xenografts. Deuteriated water was used as a magnetic resonance spectroscopic detectable tracer for measuring tumor blood flow. Tumors were exposed twice to 800 electromagnetically generated HESW, with a 24-h interval or sham exposed. No changes in TBF occurred after sham exposure to HESW. TBF levels 2 h after the first and second HESW application were, respectively, 46% and 37% lower than the mean preexposure TBF value and returned to normal levels within 16 h. There was statistically no difference found between the effects on tumor blood flow after the first and second HESW exposure. These observations are in agreement with earlier studies and provide a rationale to shorten the time interval between HESW monotreatments to 2 to 3 h.

Effects of high energy shock waves on tumor blood flow and metabolism: 31P/1H/2H nuclear magnetic resonance study.

The effects of high energy shock waves (HESW) on tumor cell metabolism and tumor blood flow were studied in the NU-1 kidney cancer xenograft by multinuclear 1H/2H/31P magnetic resonance spectroscopy. Tumor xenografts were exposed to 800 HESW using an experimental electromagnetic shock wave emitter based on the Siemens Lithostar Plus, which is used for clinical lithotripsy. Exposure of tumors to 800 HESW resulted in a temporary decrease of tumor blood flow (TBF) determined by the 2H NMR monitoring of the 2HO1H wash-out after intratumoral injection. By concomitant recording of 31P and 1H NMR spectra, tumor pH, high-energy phosphates and lactate levels were followed. Tumor treatment with HESW transiently resulted in acidification, ATP decrease, P(i) increase and lactate increase. In contrast, HESW administration adjacent to the tumor did not significantly influence TBF, tumor pH, high-energy phosphates or lactate levels, showing that the observed alterations are caused by an interaction of HESW and tumor tissue. The most likely explanation for these observations is that HESW administration causes local vascular malfunctioning followed by a reduction in oxygen and nutrient supply to the tumor which leads to a decreased aerobic energy metabolism. The results of this study may be used to aid the design of HESW-based therapies.

Magnetic resonance spectroscopy detects metabolic differences between seven Dunning rat prostate tumor sublines with different biological behavior.

In this study, it was investigated whether prostate tumor biological parameters correlate with metabolic profiles. 1H and 31P magnetic resonance spectra were acquired from perchloric acid extracts of seven Dunning R-3327 prostate tumor sublines. Several metabolic ratios, for example, phosphocholine/total phosphate, choline/total creatine, and inositol/total creatine, did not correlate specifically with one biological characteristics but, based on each of these ratios, the well-differentiated, nonmetastatic, and hormone-dependent sublines could be discriminated from the poorly differentiated or anaplastic, metastatic, and hormone-independent sublines. The glycerophosphoethanolamine/total phosphate, glycerophosphocholine/total phosphate, and phosphocreatine/total phosphate ratios correlated with differentiation grade, and the differences in glycerophosphorylglycerol/total phosphate ratio between metastatic and nonmetastatic sublines was highly significant. No correlation for hormonal sensitivity with any of the metabolites measured could be found, neither by 31P nor by 1H MRS.

Characterization of human prostate cancer, benign prostatic hyperplasia and normal prostate by in vitro 1H and 31P magnetic resonance spectroscopy.

In vitro 1H and 31P magnetic resonance spectra were acquired from perchloric acid extracts of human prostate tissue obtained by transurethral resection. This included tissue of patients with benign prostatic hyperplasia and prostatic adenocarcinoma; one tissue sample was obtained from a patient without any sign of BPH or malignancy. Major resonances in the magnetic resonance spectra were assigned to prostate compounds and were quantified. The citrate/lactate, citrate/total choline, phosphocholine/total creatinine, choline/total creatine, alanine/total creatine, phosphoethanolamine/total phosphate, phosphocholine/total phosphate and glycerophosphoethanolamine/total phosphate ratios were statistically different for the prostate cancer samples as compared with the BPH specimens. These observations may contribute to the understanding of in vivo magnetic resonance spectra of the prostate and indicate that magnetic resonance spectroscopy can aid in the diagnosis of prostate malignancy.

Biological effects of high energy shock waves in mouse skeletal muscle: correlation between 31P magnetic resonance spectroscopic and microscopic alterations.

To investigate the in vivo effects of electromagnetically generated high energy shock waves (HESW) on skeletal muscle, we used in vivo 31P nuclear magnetic resonance (NMR) spectroscopy (MRS) measurements and correlated the results with microscopical studies. Mouse skeletal muscle (calf muscle) was exposed to 200 or 800 HESW (Pmax: 37.5 MPa, Pmin: 5.2 MPa, tr: 30-120 ns, tw: 340 ns, frequency: 1.25 Hz). In the 31P MRS spectra, transient alterations were observed. A prominent increase of inorganic phosphate (Pi) peaks was found, as well as the appearance of Pi with different chemical shifts, reflecting the presence of different pH values (5.4-7.1) in cellular or tissue compartments. Within 20-96 h after exposure, pH values and Pi levels returned to normal. The changes were more pronounced in the animals treated with 800 HESW as compared to 200 HESW. Light and electron microscopy demonstrated focal degenerations of muscle fibers. This process consisted of disorganization of myofilaments and structural changes in sarcoplasmic organelles and was progressive in time. The (ultra)structural changes were not present in all myofibers (i.e., between affected degenerating fibers unaffected intact fibers were seen). Several ultrastructural abnormalities were also found in capillaries even up to severe dilatation and disruption, as well as in the peripheral nerves. The degeneration of the preexisting myofibers was predominantly confined to type 1 fibers and was followed by a regeneration of the muscle tissue by proliferation of myoblasts. A notable amount of myotubes still showed vacuolization. We conclude that in vivo HESW exposure of skeletal muscle tissue results in a degeneration of myofibers. The cellular effects are present in foci and associated with changes in the 31P NMR spectra. The NMR spectroscopy technique provides us with a noninvasive method to evaluate in a longitudinal way the biological effects of HESW.

Cytotoxic effects of high energy shock waves in different in vitro models: influence of the experimental set-up.

High energy shock waves (electromagnetically generated, Siemens Lithostar) were studied for their effects in vitro on different (tumor) cell types. Cells were exposed to the shock waves as a single cell suspension or as a cell pellet on the bottom of a test tube. In both cases, a dose dependent direct cytotoxicity, established by trypan blue dye exclusion, was observed after treatment with 1000 or 2000 shock waves. Also, the antiproliferative capacity as determined by clonogenic potential (double layer soft agar) and growth rate (plastic) were affected in this way. However, comparing the results after treatment in suspension or pellet, a discrepancy was evident. The cell lines showed a different susceptibility in pellet vs. suspension. Also the differential sensitivity of the cell types varied in these two treatment models. Furthermore the outcome depended on the cell concentration; direct cytotoxicity in a cell suspension was more pronounced at higher cell concentrations, while in a pellet this was increased by decreasing the number of cells. Finally, no shock wave induced cytotoxicity could be seen after fixation of cells in gelatine or by placing the pellet on a bottom layer of gelatine. Pressure measurements revealed no adequate explanation for this phenomenon. These results indicate that in vitro effects depend on the way cells are exposed to the shock waves and can be greatly influenced by changing the conditions of the microenvironment. Therefore, precise descriptions of the experimental set-up and careful interpretations of their outcome are obligatory.

Early metabolic response to high energy shock waves in a human tumor kidney xenograft monitored by 31P magnetic resonance spectroscopy.

The effects of electromagnetically generated high-energy shock waves (HESW, Siemens Lithostar) on the phosphate metabolite levels of the NU-1 human kidney cancer xenograft implanted under the skin of the hind limb of nude mice were monitored by 31P magnetic resonance spectroscopy (MRS). Administration of 200 and 800 HESW (18.1 kV, P(+) = 37.5 MPa, P(-) = 5.2 MPa, tr = 30-120 nsec, tw = 340 nsec, freq. = 1.25 Hz), focused on the tumor centre, resulted in an immediate tumor decline; 2 h after exposure to the HESW, the high-energy phosphate resonances had decreased drastically. This decline in energy rich molecules was accompanied by a concomitant increase in the inorganic phosphate resonance and a decrease in pH of the tumor. During the following period, a dose-time dependent recovery of the original high-energy phosphate resonance intensities was observed. These changes are qualitatively similar to those produced by ischemic inhibition of energy metabolism and are correlated with early histological features like vascular disruption, stasis within capillaries, and focal thrombosis. These results demonstrate that experimental HESW treatment of the NU-1 kidney tumor is effective in provoking a temporary reduction of both high-energy phosphate metabolism and tissue pH of the tumor. The data presented here strongly suggest that these effects are predominantly indirect by affecting tumor vascularity. Overall, this study shows that MRS is a powerful technique for longitudinal investigations of HESW-induced effects and can provide information about its mode of action.

Effects of high-energy shock waves combined with biological response modifiers in different human kidney cancer xenografts.

We have studied the effects of high-energy shock waves (HESW) alone or in combination with biological response modifiers on the growth of five human kidney cancer xenografts in mice. Exposure of the tumors to three sessions of 800 shock waves every 48 hours with 18.4 kV, 37.5 MPa, on the commercially available Lithostar resulted in a temporary growth delay. The sensitivity for HESW was related to the doubling time of the tumor. Several days after stopping the HESW administration, the tumors regained their original growth potential with the same doubling time. The systemic application of Tumor Necrosis Factor-alpha (TNF-alpha, 500 ng/g body weight, 5 times/week) and/or Interferon-alpha (IFN-alpha, 5.0 ng/g body weight, 3 times/week) subcutaneously around the tumor also had a limited effect on the growth of these established tumors (60-80 mm3). The combination of HESW with TNF-alpha and IFN-alpha resulted in an almost complete cessation of tumor growth in the NU-1 human kidney xenograft and had an additive antitumor effect in the NU-3. Synergism was also seen in the NU-1 and NU-3 with the combination of HESW and TNF-alpha, while the combination with IFN-alpha had only a limited effect on tumor growth. So TNF-alpha was the active agent, that enhanced the antiproliferative effects of shock waves. In the NC-65 tumor (same doubling time as the NU-1 and NU-3, but less well vascularized), the antiproliferative effect of HESW was not potentiated by TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo effects of high energy shock waves on urological tumors: an evaluation of treatment modalities.

We have studied the effect of high energy shock waves (HESW) alone or in combination with cytotoxic drugs on the growth of urological tumors. The effects of HESW on kidney or prostatic tumors depend largely on the tumor line used. In the rapidly growing prostate tumor no antitumor effect was evident in vivo, but in vitro examination of the in vivo shocked cells showed that the clonogenic potential of HESW treated cells was inhibited. In the more slowly growing tumors, like human kidney xenografts, a temporary growth delay is observed in vivo. The effect on tumor growth depends not only on the number of HESW administered but also on the initial tumor volume. The smaller the tumor burden, the better the antitumor effect. Single shock wave administration may cause a growth delay, and repeated administration leads to a prolonged delay in growth. This effect is temporary and several days after stopping the HESW administration the tumor regains its original growth potential (same doubling time). Finally we could show that tumor growth was suppressed in different tumor models for a longer period by a combination of HESW and a single dose of adriamycin. The effect of combined treatment resulted in a persistent longer doubling time.

The in vitro effect of electromagnetically generated shock waves (Lithostar) on the Dunning R3327 PAT-2 rat prostatic cancer cell-line. A potentiating effect on the in vitro cytotoxicity of vinblastin.

High energy shock wave lithotripsy has proven to be an effective tool in the management of renal calculi. The effects of electrohydraulically generated high energy shock waves (HESW) on tumor cells were described only recently. Here we present data on the experimental design for treatment of tumor cells, using electromagnetically generated shock waves. The determination of the focal area, in which pressures are at least 50% of the maximum pressure, appeared to be essential. In vitro HESW treatment resulted in a dose dependant anti-proliferative effect on Dunning R-3327 PAT-2 rat prostate cancer subline, determined by temporal growth curve analysis after plating of treated cells in soft agar. Furthermore, it was shown that HESW treatment had a potentiating effect on Vinblastin treatment. The combination of HESW with Vinblastin appeared to have an additive in vitro anti-proliferative effect on PAT-2 prostatic cancer cells.