Metabolic network analysis of DB1 melanoma cells: how much energy is derived from aerobic glycolysis?

A network model has been developed for analysis of tumor glucose metabolism from (13)C MRS isotope exchange kinetic data. Data were obtained from DB1 melanoma cells grown on polystyrene microcarrier beads contained in a 20-mm diameter perfusion chamber in a 9.4 T Varian NMR spectrometer; the cells were perfused with 26 mM [1,6-(13)C(2)]glucose under normoxic conditions and 37°C and monitored by (13)C NMR spectroscopy for 6 h. The model consists of ∼150 differential equations in the cumomer formalism describing glucose and lactate transport, glycolysis, TCA cycle, pyruvate cycling, the pentose shunt, lactate dehydrogenase, the malate-aspartate and glycerophosphate shuttles, and various anaplerotic pathways. The rate of oxygen consumption (CMRO(2)) was measured polarographically by monitoring differences in pO(2). The model was validated by excellent agreement between model predicted and experimentally measured values of CMRO(2) and glutamate pool size. Assuming a P/O ratio of 2.5 for NADH and 1.5 for FADH2, ATP production was estimated as 46% glycolytic and 54% mitochondrial based on average values of CMRO(2) and glycolytic flux (two experiments).

Effect of lucanthone hydrochloride on the radiation response of intestine and bone marrow of the Chinese hamster.

A sublethal dose of 100 mg lucanthone hydrochioride/kg (Miracil D, Nilodin; NSC-14574) administered ip into Chinese hamsters [median lethal dose for 30-day survival (LD50/30) of 315 mg/kg] reduced the radiation tolerance of the small intestine and had little or no effect on the radiation tolerance of the bone marrow. Lucanthone hydrochloride was administered at various times before and after whole-body 60Co gamma-irradiation. The median lethal dose for 7-day survival (LD50/7), indicative of death from gastrointestinal epithelial denudation, was reduced from 1,235 rads to minimum values of 995 rads or 985 rads by lucanthone hydrochloride inoculation 10 hours before irradiation or 7.5 hours post irradiation, respectively. The LD50/30, indicative of death from bone marrow stem cell depletion, remained unaltered at approximately 990 rads over the entire treatment scheme, which indicated that the radioresponsiveness of bone marrow stem cells was unaffected by lucanthone hydrochloride. The lucanthone hydrochloride effect was reversible in that control values of LD50/7 were attained by 40 hours post inoculation. Serum concentration of lucanthone hydrochloride in the Chinese hamster, determined spectrophotometrically, reached a peak of 8 microgram/ml by 1.5 hours post inoculation and then decreased exponentially with a half-life of approximately 6 hours, so that by 30 hours post inoculation it was unmeasurable.

The effect of adriamycin and radiation on G2 progression.

The effect of the DNA-intercalating antibiotic adriamycin on the progression of Chinese hamster ovary cells into mitosis, and on the delay induced by ionizing radiation, was studied using the mitotic cell selection procedure to monitor the rate of cell division. Following the addition of adriamycin, the mitotic rate remained unaltered for a refractory period and then decreased to zero. This effect was concentration dependent with transition points between the S-G2 boundary for 0.1 mug/ml and late G2 for 250 mug/ml. Cells treated with either a 10- or 30-min pulse of 1.0 mug adriamycin per ml exhibited a refractory period identical to that observed for continuous treatment. However, after a delay of congruent to 3.5 or congruent to 5 hr, respectively, cell division resumed. The mitotic rate of cells that received 150 rads of X-ray at the oneset of an adriamycin pulse declined coincident with that of radiation only, but resumed coincident with those receiving adriamycin only. This implies that radiation-induced division delay (congruent to 3 hr) was repaired before cells recovered from adriamycin-induced division delay and that the two agents were not additive. This lack of synergism is in contrast to that observed for cell lethality.

Superoxide dismutase levels in Chinese hamster ovary cells and ovarian carcinoma cells after hyperthermia or exposure to cycloheximide.

Superoxide dismutase (SOD) activity in Chinese hamster ovary (CHO) and ovarian carcinoma (OvCa) cells was measured after exposure to hyperthermia and correlated with the development of thermotolerance. The SOD activity of each cell type was largely copper- and zinc-containing SOD activity. Both cell types had similar but low levels of SOD activity when the cells were grown at 37 degrees C. After exposure for 2 h at 41.5 degrees C, SOD activity of OvCa cells, but not of CHO cells, was increased. After exposure to 45 degrees C for 15 min, SOD activity was also increased in the OvCa cells, but not in CHO cells. After 15 min at 45 degrees C followed by 1 h incubation at 37 degrees C, SOD activity was increased in OvCa and CHO cells; after an 8-h incubation at 37 degrees C, SOD activity doubled in each cell type. Thermotolerance is maximal after 2 to 3 h of exposure at 41.5 degrees C and after 8 to 10 h incubation at 37 degrees C following exposure to 15 min at 45 degrees C. The turnover of SOD activity in OvCa cells was estimated by the rate at which activity was lost following addition of cycloheximide (10 micrograms/ml). Twenty-four % of the activity was lost with a half-life of 10 min, and 76% was lost with a half-life of 4.5 h. Despite restriction of general protein synthesis 3 to 4 h after 45 degrees C hyperthermia, SOD activity was increased at 1 and 8 h after exposure, presumably coincidental with heat shock protein synthesis and development of thermotolerance. These data suggest that SOD activity may be important in protecting cells exposed to heat and that it may play a role in the development of thermotolerance.

Intracellular acidification of human melanoma xenografts by the respiratory inhibitor m-iodobenzylguanidine plus hyperglycemia: a 31P magnetic resonance spectroscopy study.

In vivo 31P magnetic resonance spectroscopy demonstrates that human melanoma xenografts can be significantly acidified by induction of hyperglycemia combined with administration of m-iodobenzylguanidine (MIBG), an inhibitor of mitochondrial respiration. In melanoma xenografts (< or =8 mm diameter), intracellular pH (pHi, measured by the chemical shift of the Pi resonance) and extracellular pH (pHe, measured with 3-aminopropylphosphonate) was reduced by less than 0.2 unit during i.v. infusion of glucose for 40 min. Administration of MIBG (30 mg/kg) under hyperglycemic conditions (26 mM) reduced tumor pHi and pHe by approximately 0.4 (P < 0.001) and approximately 0.6 (P < 0.001) unit, respectively; coincidentally, the nucleoside triphosphates:Pi ratio decreased approximately 60% (P < 0.004) relative to the baseline level. Minimal changes in pHi and pHe and a small decrease in nucleoside triphosphates:Pi ratio (26%, P = 0.2) were observed in liver in response to MIBG plus hyperglycemia. These results suggest that under normoglycemic and hyperglycemic conditions, small human melanoma xenografts (< or =8 mm) may exhibit a relatively high level of oxidative phosphorylation that may be blocked by MIBG. The acidification may result from increased lactate production as a direct effect of MIBG inhibition of respiration in mitochondria of tumor cells, or through indirect systemic effects, which remain to be identified. The synergetic effects of MIBG and hyperglycemia result in significant acidification of the tumor and a decrease in tumor bioenergetic status, and the effects are largely selective for tumors in comparison with normal tissues.

Absence of Crabtree effect in human melanoma cells adapted to growth at low pH: reversal by respiratory inhibitors.

Because many tumors are acidic and hypoxic relative to normal tissues, glycolysis and oxygen consumption were investigated in early-passage human melanoma cells adapted to growth at pH 6.7. In the absence of glucose, the basal rate of oxygen consumption in low pH-adapted cells was 75% of that in cells grown at pH 7.3. The rate of lactic acid production in low pH-adapted cells was increased 4-fold by exposure to 16.7 mM glucose compared with a 10-fold increase in cells grown at pH 7.3. Furthermore, in low pH-adapted cells the rate of oxygen consumption was stimulated by the addition of glucose in contrast to the inhibition of oxygen consumption by elevated glucose in cells grown at pH 7.3 (i.e., the Crabtree effect). Both low pH-adapted cells and cells grown at pH 7.3 exposed to glucose plus 0.35 mM meta-iodo-benzylguanidine (MIBG), an inhibitor of mitochondrial respiration, had oxygen consumption reduced by approximately 60% and lactic acid production increased by approximately 65% relative to glucose alone. Although adaptation to growth at low pH was associated with a loss of the Crabtree effect and a higher ratio of oxygen consumption to lactic acid production, the rate of glycolysis was the same in both growth conditions in the presence of 0.1 mM dinitrophenol, an uncoupler of ATP synthesis. This indicates that the glycolytic capacity of low pH-adapted cells remains unchanged. Therefore, tumor acute acidification and oxygenation can be achieved by exposure to hyperglycemia combined with MIBG to improve therapeutic response.

Thermoradiotherapy in the management of superficial malignant tumors.

In recent years there have been numerous randomized and nonrandomized studies conducted to assess the efficacy of hyperthermia combined with either radiation therapy or chemotherapy, especially in the treatment of superficially seated malignant tumors. The major impact of hyperthermia is currently on locoregional control of tumor. Heat may be directly cytotoxic to tumor cells or inhibit repair of both sublethal and potentially lethal damage after radiation. These effects are augmented by the physiological conditions in tumors which lead to states of acidosis and hypoxia. Blood flow is often impaired in tumor relative to normal tissue, and hyperthermia may lead to a further decrease in blood flow and augment heat sensitivity. Three major areas of clinical investigation have borne the greatest fruit for hyperthermia as adjunctive therapy to radiation therapy. These include recurrent and primary breast lesions, melanoma, and head and neck neoplasms. The thermal enhancement ratio was increased in all cases and is estimated to be 1.4 for neck nodes, 1.5 for breast, and 2 for malignant melanoma. In general, the most important prognostic factors for complete response are radiation dose, tumor size, and minimal thermal parameters (minimum thermal dose, mean minimum temperature or temperature exceeded by 90% of thermal sensors). The number of heat fractions administered per week appears to have no bearing on the overall response, which may be indicative of the effects of thermotolerance. The total number of heat fractions delivered also appears to be irrelevant provided adequate heat is delivered in one or two sessions. The major prognostic factors for the duration of local control are tumor histology, concurrent radiation therapy dose, tumor depth, and mean minimum temperature.

The thermal response and development of thermotolerance of the bone marrow stromal progenitor CFU-F.

The effect of hyperthermia on the murine bone marrow stromal progenitor (fibroblast colony-forming unit, CFU-F) was evaluated and its ability to develop thermotolerance demonstrated. CFU-F were obtained from nucleated marrow of Balb/c mice and heated in vitro in alpha minimum essential medium plus 15% fetal bovine serum. Thermotolerance development was tested two ways. 1) The development of thermotolerance during prolonged hyperthermia was observed with a "step-up" heating protocol (i.e., cells were incubated at 41 degrees C and at regular intervals challenged with 15 min at 44 degrees C). 2) The development of thermotolerance at 37 degrees C after a short exposure to a high temperature (greater than or equal to 43 degrees C) was observed with a split treatment protocol that consisted of two 15-min treatments of 44 degrees C separated with time at 37 degrees C. The inverse of the slopes of the hyperthermia dose-response relationships (Do +/- SE) for CFU-F were 118 +/- 14, 53 +/- 7, 23 +/- 0.6, 11 +/- 0.3, 7 +/- 0.3, and 5 +/- 0.5 min for exposures of 41.5 degrees, 42 degrees, 42.5 degrees, 43 degrees, 43.5 degrees, and 44 degrees C, respectively. The plot of the slopes of the heat "dose-response" relationships versus the inverse of the absolute temperature (Arrhenius plot) yields a change in slope at approximately 43 degrees C, and the inactivation enthalpies (slopes above and below the inflection point at 43 degrees C) were 606 +/- 100 kJ/mol (145 +/- 24 kcal/mol) and 1372 +/- 29 kJ/mol (328 +/- 7 kcal/mol) above and below 43 degrees C, respectively. The maximum thermotolerance ratio (TTR, surviving fraction after maximum thermotolerance development to surviving fraction of normotolerant CFU-F) at 37 degrees C after an acute thermal exposure to 15 min at 44 degrees C occurred after 12 h, with a half time of 60 min and a TTR of 41. The maximum TTR during prolonged hyperthermia at 41 degrees C was 2.4 by approximately 50 min. These results show that CFU-F are as sensitive as committed hematopoietic precursors (e.g., granulocyte-macrophage colony-forming units, CFU-GM) to hyperthermia over a wide range of thermal exposures and are capable of thermotolerance development during prolonged hyperthermic exposures and at 37 degrees C after short exposures. We conclude that at least one of the stromal elements of normal marrow may be compromised during whole-body or regional clinical hyperthermia protocols.

The development of thermotolerance in bone marrow CFU-S during chronic hyperthermia.

The purpose of this investigation was to study the response of the hematopoietic stem cell, spleen colony-forming unit (CFU-S), to hyperthermia. We have shown that CFU-S can acquire a transient resistance to further heating (thermotolerance). Hyperthermia was applied in vitro to nucleated bone marrow cells in McCoy's 5A medium plus 15% fetal bovine serum. Day-10 CFU-S (CFU-S10) were detected as spleen colonies after inoculation into the tail vein of irradiated (450 cGy plus 4 h plus 400 cGy) Balb/c male mice. Thermotolerance development was detected with a "step-up" heating protocol consisting of heating for various times at 42 degrees C followed immediately with a thermal challenge of 26 min at 44 degrees C. The inverse of the slopes of the heat "dose-response" curves (D degree +/- SE) of the normotolerant CFU-S heated to 42 degrees, 42.5 degrees, 43 degrees, 43.5 degrees, and 44 degrees C were 108 +/- 13, 54 +/- 8, 25 +/- 1, 17 +/- 2, and 12 +/- 5 min, respectively. A plot of the slopes of the heat "dose-response" relationships versus the inverse of the absolute temperature (Arrhenius plot) showed an inflection at approximately 43 degrees C. Analysis of the regression coefficient above and below the inflection point (Arrhenius analysis) yielded inactivation enthalpies (+/- SE) of 598 +/- 130 kJ/mol (143 +/- 31 kcal/mol) and 1205 +/- 171 kJ/mol (288 +/- 41 kcal/mol), respectively. The difference in inactivation enthalpy indicates a change in mechanism in the thermal inactivation of CFU-S above and below 43 degrees C, possibly due to thermotolerance development during exposure to temperatures less than 43 degrees C. Prolonged incubation at 42 degrees C for up to 180 min with a step-up to 44 degrees C for 26 min showed that CFU-S survival increased rapidly from 0.25 (26 min at 44 degrees C) to 0.52 within 10 min. The thermotolerance ratio (TTR, ratio of the surviving fraction of the maximum thermotolerant cells to that of the normotolerant cells) was 2.1. Both the higher inactivation enthalpy for exposures less than 43 degrees C and the rapid increase in survival during the "step-up" heating experiments at 42 degrees C demonstrate that CFU-S can develop thermotolerance during prolonged hyperthermia. These results suggest that thermotolerance can influence the thermal response of pluripotent bone marrow stem cells heated during whole-body or local-regional clinical hyperthermia protocols.

Lucanthone modification of cyclophosphamide toxicity in the Chinese hamster.

The interaction of lucanthone and cyclophosphamide (CYC) was investigated in the Chinese hamster in terms of the LD50/7 and LD50/30. These values may be indicative of gastrointestinal stem cell depletion and bone marrow stem cell depletion, respectively. When a nonlethal dose of 100 mg/kg lucanthone preceded CYC injection, the LD50/7 for CYC reached its minimum value of 470 mg/kg at a treatment interval of 10 hours. Lucanthone administered simultaneously with CYC had no effect on the control LD50/7 of 750 mg/kg, and by 48 hours after lucanthone administration the LD50/7 had returned to the control value. When CYC administration preceded that of lucanthone, the LD50/7 reached a minimum of value of 610 mg/kg at an interval of 5 hours; however, for the entire sequence it was approximately 640 mg/kg over all intervals up to 48 hours. The LD50/30 for CYC was only slightly reduced by the presence of lucanthone, indicating that bone marrow sensitivity to CYC was only marginally affected by lucanthone. These data indicate that lucanthone may interact with CYC damage in much the same way as it interacts with radiation damage, viz, by reducing cellular capacity to accumulate and repair sublethal damage.

Effect of intestinal hyperthermia in the Chinese hamster.

If hyperthermia is to become a useful cancer therapeutic modality, normal tissue response must be thoroughly understood. The hyperthermia response of Chinese hamster intestine was studied by immersion of the exteriorized small intestine in heated tissue culture medium. After heating, the small intestine was reinserted, the incision closed, and animals observed until death. Animals exposed to 42.5 degrees, 43.5 degrees, or 44.5 degrees C intestinal hyperthermia exhibited LD50/7 values (including 95% intervals) of 56 min (52.9-59.3), 29 min (26.4-31.8), or 14 min (13.2-14.6), respectively. An Arrhenius plot of LD50/7 vs 1/T degree K exhibited an inactivation energy of 139 kcal/mole, which corresponds well with values generally reported for cellular inactivation. Hamster intestine conditioned with a sublethal exposure of 8 min at 44.5 degrees C developed thermotolerance to subsequent 44.5 degrees C hyperthermia. Thermotolerance induction was maximal by 24 hr; the LD50/7 for the second dose of hyperthermia increased from 6 min at 44.5 degrees C at zero time to 21 min at 44.5 degrees C after a treatment interval of 24 hr (thermotolerance ratio of 3.5). The LD50/7 subsequently decreased from 21 min to 12 min at 44.5 degrees C (the control value) by 96 hr. The hyperthermia response of this tissue was predicated by previous results from the Chinese hamster ovary (CHO) fibroblast cell line in tissue culture, and is also similar to several mouse normal tissues.

The effect of lucanthone on sublethal radiation damage, in vivo.

The capacity of the Chinese hamster jejunal crypt cell to accumulate and repair sublethal radiation damage was determined by analyzing the return of the shoulder of the radiation dose-crypt microcolony survival curve (Dr) after a priming dose of 1250 rad. The control split dose crypt cell survival curve exhibited a D0, Dr and "n" of 179 +/- 3 rad, 261 +/- 3 rad and 4.3 respectively; repair of sublethal radiation damage was completed by two hours post-irradiation. The effect of lucanthone (an antischistosomal DNA intercalating agent) on the crypt cell's capacity to accumulate and repair sublethal radiation damage was determined by injecting the drug (100 mg/kg, i.p.) at intervals before irradiation with a priming dose of 1250 rad, followed two hours later by graded doses. Injection coincident with the priming dose of radiation resulted in a 22 rad reduction of the Dr (compared to control Dr). Injection eight hours before the priming dose almost completely inhibited the accumulation and repair of sublethal radiation damage so that the resultant Dr two hours later was only 29 rad (a 232 rad reduction). At no time was the D0 of the crypt cell survival curve affected by lucanthone. These data confirm previous results from whole crypt analysis and LD50/7 analysis that non-toxic concentrations of lucanthone reversibly inhibit the accumulation and repair of sublethal radiation damage in a time-dependent manner with complete inhibition approximately eight hours post-injection. This drug is useful for the study of sublethal radiation damage in vivo and may be beneficial in radiation therapy of cancer when it is desirable to inhibit the repair of sublethal radiation damage.

Modification of human tumor pH by elevation of blood glucose.

Nine patients with metastatic or recurrent superficial tumors of varying size and histology were administered 100 g oral glucose to investigate whether hyperglycemia can selectively lower tumor pH. pH was measured by a 21 ga modified glass needle electrode inserted through an 18 ga open-ended Angiocath. Serum glucose was monitored every 7.5 min by finger stick and a blood glucose analyzer. Tumor pH was measured over 50-80 min concomitantly with determination of blood glucose. In five nondiabetic patients (eight measurement points) tumor pH decreased 0.05-0.5 units from a pre-glucose range of 6.8-7.4 (7.14 +/- 0.08) to 6.4-7.3 (6.90 +/- 0.10) as blood glucose increased from a baseline of 80-120 mg/dl to 165-215 mg/dl. There was considerable heterogeneity from patient to patient regardless whether blood glucose increased to a peak at 40-60 min post-ingestion and then decreased, or whether it remained elevated up to the end of the 80 min observation period. In general, tumor pH decreased as blood glucose increased and then continued to fall throughout the period of observation. In one patient, tumor pH did not change although blood glucose increased to 175 mg/dl. Normal tissue pH was 7.36 +/- 0.02 when determined on four occasions in three patients (subcutaneous and intramuscular sites), and was unaffected by glucose administration. As a further control for tumor tissue and pH probe stability, pH probes in two patients were left in place for 30 min before glucose ingestion. The tumor pH was stable for the entire interval. Interestingly, three patients had an abnormal glucose response: two of those patients (one patient on two separate occasions) had an increase in blood glucose to 230-260 mg/dl in 40-60 min and pH actually increased 0.1-0.3 units. The third patient had a transient increase in blood glucose to 290 mg/dl along with a corresponding increase and subsequent decrease in tumor pH. In summary, whether glucose was given pre- or post-hyperthermia, independently of position in the tumor, and independently of whether pH increased or decreased, the slope of the curve of pH = f(time) was similar in a given patient tumor on all measurement occasions. These preliminary results suggest that hyperglycemia may be useful in non-diabetic patients, and perhaps in diabetic patients given insulin, to selectively reduce tumor pH and sensitize tumors to hyperthermia.

Response of human tumor blood flow to local hyperthermia.

The effect of heat on blood flow in human tumors was studied as a function of time during 1 hour of local hyperthermia induced by 915 MHz microwaves. Blood flow was determined from the rate of thermal clearance by use of the bio-heat transfer equation. The rate of thermal clearance was measured at intervals of approximately 10 minutes throughout the treatment session by turning off the microwave power for 50 seconds. Tumor blood flow increased by amounts varying from 15 to 250% during the first 20-50 minutes of heating at 41-45 degrees C, after which it remained relatively constant during the remainder of the treatment session. The sharp reduction in blood flow or vascular stasis reported in most transplantable rodent tumors after comparable heating was not observed in human tumors. The maximum blood flow observed in heated human tumors ranged from 10-40 ml/min/100 gm. The systematic error due to thermal conduction was estimated to be equivalent to a blood flow of less than 3 ml/min/100 gm.

Hyperthermia and radiation in advanced malignant melanoma.

Advanced melanoma (48 lesions in 40 patients) was treated with external microwave hyperthermia combined with radiation therapy between 1980-1988. Thirty-three lesions in 28 patients were evaluable for tumor response (mean age 64 years, 19 male, 9 female). Evaluable lesions received 13 to 66 Gy (mean 37 +/- 2 Gy) over 5 to 16 fractions (mean of 10) in 14 to 56 elapsed days (mean of 25). Tumor volume (pi/6*length*width*depth) was 62 +/- 16 cm3 (1-377 cm3). Hyperthermia was administered in 6.6 +/- 0.4 sessions (range 1-14), there were 3.2 +/- 0.4 thermal sensors per tumor (range 1-11) and 27 fields were treated twice-weekly (82%). Of the 33 evaluable lesions, 12 exhibited a complete response (36%), and 17 had a partial response (52%). Among the 12 complete responders four recurrences (33%) were observed at 8.6 +/- 1.4 months (median of 8.2 months). In superficial tumors with depth < or = 3 cm and with lateral dimensions within 2 cm of the boundaries of the microwave applicator, the complete response rate was 50% (11/22); whereas for patients with deeper tumors with depth > 3 cm, the complete response rate was 9% (1/11), p = 0.02. The minimal tumor thermal dose during the first hyperthermia treatment session correlated with response (t43min1 = 20 +/- 7 vs. 6 +/- 3 minEq43 degrees C for complete responders and noncomplete responders, respectively, p = 0.06); and 7 of 10 lesions that had t43min 1 > or = 8 minEq43 degrees C achieved a complete response whereas only 5 of 22 lesions (23%) that had t43min1 < 8 minEq43 degrees C did so (p = 0.01). However, neither the minimum tumor temperature during the first treatment, the median minimum tumor temperature over all treatment sessions nor the sum of minimum thermal dose over all treatment sessions correlated with tumor response. Twenty-three patients with 28 lesions died during follow-up (82%). The survival for complete responding patients with superficial lesions was 21.3 +/- 1.5 months compared to 4.5 +/- 0.5 months for patients with superficial lesions that did not experience a complete response (p = 0.0001). For patients with noncomplete responding lesions deeper than 3 cm survival was 4.4 +/- 0.6 months. Twenty lesions were treated without any skin reaction (42%, 20/48). Of the rest, 23 had erythema (48%, 23/48), seven had blistering (14%, 7/48) and one had ulceration of the skin.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of 42 degrees C hyperthermia on intracellular pH in ovarian carcinoma cells during acute or chronic exposure to low extracellular pH.

PURPOSE: To determine whether intracellular pH (pHi) is affected during hyperthermia in substrate-attached cells and whether acute extracellular acidification potentiates the cytotoxicity of hyperthermia via an effect on pHi. METHODS AND MATERIALS: The pHi was determined in cells attached to extracellular matrix proteins loaded with the fluorescent indicator dye BCECF at 37 degrees C and during 42 degrees C hyperthermia at an extracellular pH (pHe) of 6.7 or 7.3 in cells. Effects on pHi during hyperthermia are compared to effects on clonogenic survival after hyperthermia at pHe 7.3 and 6.7 of cells grown at pHe 7.3, or of cells grown and monitored at pHe 6.7. RESULTS: The results show that pHi values are affected by substrate attachments. Cells attached to extracellular matrix proteins had better signal stability, low dye leakage and evidence of homeostatic regulation of pHi during heating. The net decrease in pHi in cells grown and assayed at pHe = 7.3 during 42 degrees C hyperthermia was 0.28 units and the decrease in low pH adapted cells heated at pHe = 6.7 was 0.14 units. Acute acidification from pHe = 7.3 to pHe = 6.7 at 37 degrees C caused an initial reduction of 0.5-0.8 unit in pHi, but a partial recovery followed during the next 60-90 min. Concurrent 42 degrees C hyperthermia caused the same initial reduction in pHi in acutely acidified cells, but inhibited the partial recovery that occurred during the next 60-90 min at 37 degrees C. After 4 h at 37 degrees C, the net change in pHi in acutely acidified cells was 0.30 pH unit, but at 42 degrees C is 0.63 pH units. The net change in pHi correlated inversely with clonogenic survival. CONCLUSIONS: Hyperthermia causes a pHi reduction in cells which was smaller in magnitude by 50% in low pH adapted cells. Hyperthermia inhibited the partial recovery from acute acidification that was observed at 37 degrees C in substrate attached cells, in parallel with a lower subsequent clonogenic survival.

Tumor extracellular pH as a prognostic factor in thermoradiotherapy.

PURPOSE: Tumor extracellular pH measurements in 26 human tumors were evaluated for the purpose of prognostic indication of response to thermoradiotherapy. METHODS AND MATERIALS: Twenty-six patients (10 male, 16 female; mean age 62 years, range 18-89) were treated with external microwave hyperthermia (915 MHz) combined with radiation therapy. Tumor histologies included: 46% adenocarcinoma, 38% squamous cell carcinoma, 12% soft tissue sarcoma, and 4% malignant melanoma. The mean tumor depth was 1.6 +/- 0.2 cm (range 0.4-3 cm) and the mean tumor volume was 73 +/- 11 cm3 (range 1-192 cm3). The mean radiation dose administered concurrently with hyperthermia was 39 +/- 1 Gy (range 24-60 Gy, median of 40 Gy), in 15 fractions (range 8-25), over 32 elapsed days (range 15-43). The mean number of hyperthermia sessions administered was 5.4 +/- 0.5 (range 2-10). A battery operated pH meter and combination 21 ga recessed glass, beveled needle microelectrodes were used for tumor pH measurements. Calibration in pH buffers was performed before and after each pH measurement. The needle microelectrodes were 2.5 cm in length. RESULTS: A complete response (CR) was obtained in 20 of 26 patients (77%) and a partial response in six (23%). The mean extracellular tumor pH was 6.88 +/- 0.09 in CR patients while it was 7.24 +/- 0.09 in noncompletely responding (NCR) patients (p = 0.08). Logistic regression analysis indicated that the probability of obtaining a complete response was influenced by the tumor volume (p = 0.02), tumor depth (p = 0.05), and extracellular tumor pH (p = 0.08). Lesions in the pH range of 6.00-6.40 and lesions in the pH range of 6.41-6.80 exhibited a CR rate of 100%, while those lesions in the pH range of 6.81-7.20 exhibited a CR of 90% and those in the pH range of 7.21-7.52 exhibited a CR of 50% (p = 0.002). In lesions with depth < or = 1.5 cm, the CR rate was 100% when the tumor pH was < 7.15 and 75% when the tumor pH was > or = 7.15. In lesions with depth between 1.5 and 3 cm, the CR rate was 66% when the tumor pH was < 7.15 and 43% when the tumor pH was > or = 7.15 (p = 0.02). In small tumors, that is, < or = 20 cm3, tumor pH increased with volume, whereas in larger tumors, that is, > 20 cm3, tumor pH decreased as a function of tumor volume. CONCLUSION: Tumor extracellular pH may be useful as a prognostic indicator of tumor response to thermoradiotherapy.

pH distribution in human tumors.

The effectiveness of hyperthermia in tumor therapy may depend on a lower extracellular pH of tumor compared to that of normal tissue. A technique for measuring extracellular pH in human tumors has been devised to test the usefulness of this parameter as prognostic indicator of tumor hyperthermia response. In a preliminary study 50 of 53 pH readings from 14 human tumors (both heated and unheated) were below normal physiological pH. Tumor pH values ranged between 5.55-7.69 (average for unheated tumors 6.81 +/- 0.09, SEM, only one determination was above 7.40). Although there was considerable heterogeneity of pH within tumors, the accuracy and drift of the 21 gauge needle electrode were not a problem. Fifteen minutes were required for pH stabilization after insertion of an 18 gauge open-ended catheter, and less than 5 min for equilibration after electrode insertion into the catheter. A saline wheal was used for anesthesia to preclude modification of pH by anesthetics. Central portions of tumors were no more acidic than peripheral regions, but large tumors tended to be more acidic than small tumors. The pH of several tumors of various sizes and histologies was also determined immediately before subsequent treatment sessions. These measurements were made by reinsertion of catheters in approximately the same locations at each session. The trend appeared to be that pH increased with the number of treatment sessions. Measurements of pH were made in four patients immediately prior to and at the termination of a heating session (same locations since catheter remained in place during heating sessions). Three of the four tumors showed increased pH readings of 0.25-0.54 units during heating. However, none of the four tumors achieved temperatures exceeding 42 degrees C. The pH measurement technique developed provides a safe and relatively easy method for determining extracellular pH in human tumors. There appears to be a correlation of pH values with tumor size, treatment session, and possibly blood flow.

Thermal response and hyperthermic radiosensitization of scid mouse bone marrow CFU-C.

PURPOSE: Scid mice are severely immunodeficient as a result of a defective recombinase system. Mice with the scid mutation have been shown to have an increased sensitivity to ionizing radiation, presumably as a result of an inability to repair DNA damage. Little is known of the impact of this mutation on the thermal response and on hyperthermic radiosensitization. This investigation established the thermal response (42-44 degrees C), patterns of thermotolerance development, and the impact of hyperthermia (60 min at 40 degrees C or 42 degrees C) on the radiation response of bone marrow colony forming unit-culture cells (CFU-C) in scid mice. METHODS AND MATERIALS: Anesthetized scid mice (pentobarbital, 90 mg/kg) were killed by cervical dislocation and the nucleated marrow obtained from both tibia and femora by passing 2 ml of cold McCoy's 5A medium supplemented with 15% fetal bovine serum through each bone. Single cell suspensions of nucleated marrow were heated in 12 x 75 mm sterile tissue culture tubes at a concentration of approximately 5 x 10(6) cells/ml. Radiation, when used, was delivered immediately prior to hyperthermia by a 137Cs irradiator (dose rate of 1.20 Gy/min). Colony forming unit-culture were cultured in semisolid agar in the presence of colony stimulating factor (conditioned medium from L929 cells) for 7 days. RESULTS: The slope of the radiation dose-response curve for CFU-C in scid mice was biphasic, the Dos (+/- SE) were 0.29 +/- 0.03 Gy and 1.09 +/- 0.20 Gy, respectively. The Dos of the radiation dose-response curve for wild type marrow from CB-17 and Balb/c mice were 1.28 +/- 0.05 Gy and 1.47 +/- 0.15 Gy, respectively. The Dos of the hyperthermia dose-response curves for scid mice were 75 +/- 5, 10 +/- 1.4, and 4 +/- 0.2 min, respectively, for temperatures of 42 degrees, 43 degrees, and 44 degrees C. Thermotolerance development at 37 degrees C increased to a maximum at approximately 240 min after acute hyperthermia (15 min at 44 degrees C) and thereafter, decreased to control levels within 15 h. Thermotolerance did not develop in scid CFU-C during chronic hyperthermia at temperatures < 42.5 degrees C. Hyperthermia (60 min at 40 degrees or 42 degrees C) immediately after ionizing radiation did not significantly alter the terminal slope of the radiation dose-response curve of scid CFU-C (Do = 1.28 +/- 0.08 Gy). By contrast, hyperthermia following radiation of wild type CFU-C resulted in a decrease in the Do from 1.47 +/- 0.05 Gy (Balb/c, rad only) to 1.31 +/- 0.08 or 1.06 +/- 0.18 Gy for 60 min at 40 degrees or 42 degrees C, respectively. CONCLUSION: These results show that the thermal response and the pattern of thermotolerance development of scid CFU-C were similar to that of wild type Balb/c CFU-C, but that hyperthermia given immediately after ionizing radiation did not alter the radiation response of scid CFU-C. The scid mutation does not increase hyperthermic sensitivity or change the pattern of thermotolerance development of scid mouse CFU-C, implying that the scid mutation is not involved with thermal response, but does render the already radiation-sensitive scid cells incapable of thermal radiosensitization.

Human tumor extracellular pH as a function of blood glucose concentration.

PURPOSE: Mammalian cells are sensitized to hyperthermia when the extracellular pH (pHe) is acutely reduced to < pH 7.0-7.2. However, cells chronically adapted to low pHe may not demonstrate such sensitivity. Although much of the extracellular environment of human tumors is at lower than normal physiological pH, it may be necessary to acutely acidify tumors to cause a change in the therapeutic response to hyperthermia. The purpose of this study was to reduce extracellular pH in human tumors by elevation of blood glucose. METHODS AND MATERIALS: The change in tumor pHe was measured as a function of the change in blood glucose concentration after oral administration of 100 g glucose in 25 fasting, nondiabetic patients. pHe was determined by needle microelectrodes, and blood glucose determined by "Chemstrips" and a glucometer. In some patients blood glucose concentration rose with time after ingestion to a peak change of 50-100 mg/dL between 30-70 min and then began to decrease. In another group of patients glucose concentration increased by 100-200 mg/dL over 30-90 min and remained elevated as if the patients in this group were Type II diabetics. RESULTS: In 14 transient hyperglycemic patients (56%), as blood glucose increased tumor pHe decreased by a mean of -0.17 +/- 0.04 pH units (p < or = 0.0001, range of -0.41-(+)0.07). By contrast in eight persistent hyperglycemic patients, tumor pHe remained unchanged or actually increased an average of 0.03 +/- 0.04 pH units (range of -0.15-(-)0.14). Normal tissue pHe in five patients was unchanged by hyperglycemia, pHe = 7.33 +/- 0.03. Among all patients, 52% exhibited a pHe decrease > or = 0.1 pH unit, and 24% exhibited a pHe decrease > or = 0.2 pH unit. In five transient hyperglycemic patients whose preglucose tumor pHe was between 6.90 and 7.22, the average decrease in pHe induced by hyperglycemia was 0.25 +/- 0.05 pH unit. A linear relationship was observed between the change of pHe and the maximum change in blood glucose such that the greatest decrease in tumor pHe occurred when the glucose change was minimal. The slope was 0.0017 +/- 0.0005 pH units/mg/dL glucose (p < or = 0.005). The linear relationship included both tumors in transient hyperglycemic patients and in persistent hyperglycemia patients. CONCLUSION: Since patients who exhibited the lowest change in blood glucose exhibited the greatest decrease in tumor pHe, it may be that cells in these patients were better able to transport glucose intracellularly which in tumor cells would permit a more rapid production of lactic acid from aerobic and/or anaerobic glycolysis. These data may be helpful in predicting the response of individual patients to oral hyperglycemia as a clinical thermosensitizer.