Fred W. Hetzel

Lymph flow from murine footpad tumors before and after sublethal hyperthermia

The effect of local hyperthermia (43.5 degrees C for 1 h) on lymph flow from B16-F10 tumor-bearing foot pads of C57BL/6 mice was measured by monitoring the clearance of 99mTc-labeled human serum albumin. The foot was represented by a single-compartment model enabling a quantitative computation of lymphatic flow from the tumor to regional lymph nodes. Lymphatic flow from untreated tumors was 0.0059 +/- 0.0011 ml/min cm3 compared to 0.0118 +/- 0.0027 ml/min cm3 lymphatic flow from tumors immediately following heating. Morphological alterations in tumor blood vessels result in their high vascular permeability. The increase in lymphatic clearance from tumors after sublethal hyperthermia is compatible with the increase in interstitial fluid formation in tumors based on Starlings Law.

Regional lymph node and pulmonary metastases after local hyperthermia of melanomas in C57BL/6 mice

The effects of local tumor hyperthermia on regional lymph node metastases are inconclusive. We studied the effects of hyperthermia on the incidence of popliteal, femoral, and abdominal lymph node metastases in C57BL/6 mice with primary B16 melanomas (F10 variant) growing subcutaneously in the left foot. Tumors were heated to 42.3, 43.5, and 44.2 degrees C for 90 minutes either 7 days after inoculation of 5 X 10(4) viable cells (microscopic tumor = mic) or when the tumors were approximately 3 mm in diameter (macroscopic tumor = mac). Femoral lymph node metastases occurred in 0/21 control animals and in 8/22 (36%), 11/19 (58%), and 11/17 (65%) animals whose primary tumors were heated to 42.3, 43.5, and 44.2 degrees C, respectively. For all three treatments, the increase in metastases as compared to controls was statistically significant (p less than 0.004, Fishers exact test). The incidence of abdominal lymph node metastasis was slightly higher in the treated groups than controls. Twenty of 21 (95%) control mice developed popliteal lymph node metastases and hyperthermia-induced increases could not be demonstrated. Fifteen of 21 control mice killed 3 weeks after amputation of tumor-containing leg had pulmonary metastases with an average of 6 +/- 4 (standard deviation) lesions per affected mouse. Pulmonary metastases occurred in 22/22 (100%), 17/19 (89%), and 13/17 (76%) of mice whose tumors were heated to 42.3, 43.5, and 44.2 degrees C, respectively. The numbers of metastases for affected mice were significantly increased compared to controls for tumors heated to 43.5 and 44.2 degrees C (28 +/- 43, 43 +/- 52, 119 +/- 121, p greater than 0.02, p less than 0.006, p less than 0.002, for two sample T-test). While 0/8 mic tumors were cured 5/9 mac tumors heated to 44.2 degrees C disappeared (p less than 0.03, Fishers exact test) and there was a growth delay in the remaining mice. Mic tumors, heated to 43.5 degrees C, had an accelerated onset of growth while mac tumors heated to this temperature had a slight growth delay. Growth of both mic and mac primary tumors heated to 42.3 degrees C was similar to controls. These results show that therapeutic and subtherapeutic local hyperthermia increases metastases to regional lymph nodes and to lungs even when primary tumor growth rate is partially or totally controlled.

Dose-dependent metabolic response of mammary carcinoma to photodynamic therapy

The metabolic response of mammary carcinoma in the C3H mouse to photodynamic therapy (PDT) was measured using in vivo 31P nuclear magnetic resonance (31P-NMR) spectroscopy and pH microelectrodes. Twenty-four hours after administration of Photofrin II (12.5 mg/kg), the tumor was subjected to photoactivation using an argon dye laser. Optical treatment doses were 200, 400, and 600 J/cm2 and corresponded to the following tumor control doses: TCD10/30, TCD50/30, and TCD90/30, respectively. In vivo 31P-NMR spectra and pH micro-electrode measurements were obtained prior to treatment and at 4, 24, 48, and 72 h and 1 week post-treatment. The data revealed a significant (P less than 0.0002) alkalosis as indicated by the pH measured by NMR compared to pH measured by microelectrodes at all treatment levels and time points. Spectral differences between treatment groups were apparent as early as 4 h after treatment. The ratio of beta-nucleoside triphosphate to inorganic phosphate at 4 h after treatment was significantly (P less than 0.01) smaller for 600 J/cm2 treatment than for 200 J/cm2 treatment. At curative (600 J/cm2) levels, from 48 h on, no phosphate resonances were detected in the spectra. The pH measured by NMR transiently decreased from pretreatment levels after 200 and 400 J/cm2 treatment (P less than 0.002, P less than 0.009, respectively), while no change in pH from pretreatment values was found after 600 J/cm2 treatment. The data suggest that the early metabolic response of mammary carcinoma to PDT, as indicated by 31P-NMR spectroscopy, is dose dependent, and may be a sensitive indicator of biological outcome to treatment.

Dose-Dependent Thermal Response of Tumor pH and Energy Metabolism Evaluated by in Vivo 31P NMR Spectroscopy and Microelectrodes

In vivo 31P NMR spectroscopy and pH microelectrodes were employed to measure the energy metabolism and pH of a mammary carcinoma in the flank of the C3H mouse before and serially up to a week after various hyperthermia treatments. Water bath hyperthermia was used to treat the tumor at 43.5 degrees C for 30 min (TCD0/30, NMR measurement only), 1 h (TCD10/30), and 2 h (TCD60/30), respectively. The data indicate that, except at 4 h after TCD60/30 treatment, all pH values measured by NMR (pHn) were significantly higher (P less than or equal to 0.001) compared to pH values measured by microelectrodes (pHe) at all treatment levels and times. The magnitude of the difference between pHn and pHe (delta pH) was significantly decreased from the pretreatment level only at 4 h after hyperthermia treatment (0.51 pH units for TCD60/30 and 0.21 pH units for TCD10/30). The ratio of beta-nucleoside triphosphate to inorganic phosphate (beta-NTP/Pi) and pHn were more sensitive to hyperthermia treatment than pHe. The beta-NTP/Pi ratio failed to recover to the pretreatment ratio after 1 or 2 h hyperthermia treatment, while a total recovery was observed within 72 h for 30 min hyperthermia treatment. Our data suggest that the temporal profile of beta-NTP/Pi, pHn, and delta pH may be indicative of the biological outcome of hyperthermia treatment.

The effects of vasopressin on the radiation response of cultured mammalian cells.

Since vasopressin is known to exert radioprotective effects in vivo, its effects on mammalian cells in vitro were examined. No toxicity or changes in cellular plating efficiency were observed at concentrations up to 10 units/ml. There was no direct radioprotective effect attributable to exposure of the cells to vasopressin prior to and during irradiation.