Interaction of ultrasonic hyperthermia with two alkylating agents in a murine bladder tumor.

Fischer rats bearing s.c. implants of TCT-4909 bladder tumor were treated either with ultrasonic hyperthermia (US) (44.2 degrees, 20 min) or either of two alkylating agents, thiotepa or Cytoxan (CTX) or in combinations of chemotherapeutic agent and heat at specific time intervals. Applying US 20 hr before either agent (thiotepa, 2 mg/kg i.p.; or CTX, 50 mg/kg i.p.) had a less than additive effect upon tumor growth. CTX, administered 20 hr before US, resulted in a significantly increased tumor volume-doubling time compared to CTX only. This was not true for thiotepa. With both agents, a synergistic effect was obtained when US and the agent were applied within 1 hr of each other, but the maximum was observed when the US had been applied 30 min before injection of agent. The intratumor temperature had decayed to normal at the time of injection. Radiolabeled alkylating agents injected at different times after US showed decreased uptake of label up to 20 hr after heating. Tritiated thymidine uptake was also reduced over the same period. Nuclear morphometry indicated increased nuclear condensation in parallel with the reduced uptakes described above. The data suggest that the synergism was not due to increased uptake of agent into heated tissue nor to the direct activation of alkylating activity by heat. It was demonstrated that heat had a rapid and marked inhibitory action upon DNA synthesis. This could have augmented the delayed but prolonged DNA inhibition caused by the alkylating agents to produce a synergistic effect. The apparent prolongation of the growth-inhibitory effect of CTX or thiotepa by heat may be due to the thermal inhibition of the enzymes responsible for the recovery of DNA after alkylation. The precise mechanism for the synergy may vary with the agent, the dose, the equilibrium temperature and its dwell time, and the interval between modalities. The influence of each of these parameters will require further investigation.

Interaction of ultrasound with neoplastic tissue: IV. alteration of biologic activity in cells derived from rat Wilms' tumor sonicated in vivo.

Paired Wistar-Furth male rats were implanted subcutaneously with the Furth-Columbia rat Wilms' tumor. Seven days after implantation the tumors were either subjected to therapeutic ultrasound irradiation or were left as untreated controls. On the ninth day after implantation the animals were sacrificed, and suspensions of cells were made from the excised tumors by trypsinization. The cells were counted using trypan blue dye as an indicator of viability. New rats were then implanted with cells either from sonicated tumors or cells from controls. In half the recipient animals the suspensions were matched by total cell count. In the other half of the rats cell counts were matches for viability by the dye exclusion test. Tumors developed in a total of 8 of 11 rats receiving control cells and they died. A palpable tumor did not develop in any rat receiving cells fromsonicated tumors during the ninety-day study period. Surviving rats were challenged with Wilms' tumor by trocar implant. Tumor developed in all recipients, and they died.

Interaction of ultrasound with neoplastic tissue. Local effect on subcutaneously implanted Furth-Columbia rat Wilms' tumor.

Ultrasonic irradiation was employed by direct administration to the skin overlying subcutaneously implanted Furth-Columbia rat Wilms' tumors. Treated tumors were flattened and excavated and demonstrated no stigmata of hemorrhage or infection. There was a marked decrease in growth rate of the tumors with an increase in survival time of the host. Histologic assessment with the light microscope exhibited a sharp line of demarcation between the necrosed sonicated portion of tumor and the viable intact nonsonicated area of tumor. A blackened area of skin, which was not histologically similar to a burn, was interposed betwwen the site of application of the ultrasound and the necrosed tumor.

Interaction of ultrasound with neoplastic tissue. II. Systemic effects after local sonic irradiation.

Local sonic irradiation was applied to subcutaneously implanted Furth-Columbia rat Wilms' tumor. The weight and the rate of tritiated thymidine uptake were measured in host organs distal to the application field. Kidney and spleen weights were inhibited by the Wilms' tumor, and sonication of the tumor removed all or part of this inhibition. Liver weight was increased after sonication of tumor-bearing rats but not in nontumor-bearing rats. This may have been a response to tumor-specific substances released into the circulation by sonic destruction of tumor tissue. The adrenals enlarged as a response to the stresses of both tumor-bearing and of sonication. Animals were implanted on both sides with the Wilms' tumor and on without any break in the growth curve while the sonicated right tumor was inhibited. These data suggest that the therapeutic effect of ultrasound is due solely to local factors and that systemic sequelae of some irradiation are unrelated to tumor inhibition.

Ultrasonic hyperthermia system for tumor irradiation.

A laboratory ultrasonic exposure system has been developed to investigate practical energy regimes and equipment that will provide a modality for producing irreversible ultrastructural and biochemical changes to animal tumors in vivo. Electric energy is made available over a wide range of power levels and frequencies. This energy is converted to sonic pressure energy by piezoelectric element transducers. The transducer is an applicator that transmits a collimated or focused energy field to irradiate the tumor target area. That energy absorbed by the tumor gives rise to a spatial temperature distribution. Using their primary laboratory delivery system, the authors treated subcutaneous tumors in rats at controlled levels of energy and temperature. The results indicate that there are thresholds of applied energy and base temperatures above which substantial tumor damage can be expected. Histological examination supported by biochemical analysis reveals irreversible ultrastructural and chemical changes.

Interaction of ultrasound with neoplastic tissue. III. Electron microscopic demonstration of ulstrasonic destruction of Wilms tumor and its cellular membranes.

Exposure of 300 adult, male Wistar-Furth rats bearing subcutaneous implants of Wilms tumor to a vertically oriented planar ultrasound beam consistently resulted in a marked decrease in the growth rates of the tumors with an increase in the survival times of the treated animals. Grossly, the local effects consisted of a flattening of the tumors with clean excavation of their bases. Histologically, a line of demarcation was demonstrated between the sonicated (or destroyed) and non-sonicated portions of the same rat Wilms tumors. The sonicated portions exhibited a complete loss of the normal spatial relationship between the tumor epithelium and its surrounding mesenchyme. Ghost residuals of portions of individual cytoplasmic cell borders and nuclei with condensed chromatin patterns still could be discerned. Electron microscopy demonstrated a marked destruction of the nuclear and cytoplasmic cellular membranes with a migration of destroyed condensed chromatin material into the surrounding cytoplasm.

The direct effect of ultrasound upon Wilms' tumor in the rat.

The possible activation of immunologic defenses against tumor by local ultrasonic irradiation was investigated. Sonication of one tumor of a dual implant destroyed that tumor only with no contralateral effect. Injection of tumor cells previously sonicated in vivo did not induced immunity to subsequent challenge with live cells. Immunofluorescent staining of host kidneys for antibody complexes was negative. It was concluded that sonication was only effective when directly applied to the tumor.