In vivo 31P nuclear magnetic resonance spectroscopy of experimental murine tumours and human tumour xenografts: effects of blood flow modification

The effect of hydralazine on tumours appears to vary depending on tumour type. Blood flow and radiation sensitivity decrease more in murine tumours than human tumour xenografts. In this study a comparison between various tumour types has been made using in vivo 31P nuclear magnetic resonance spectroscopy (NMRS) to follow the metabolic responses occurring after clamping or intravenous administration of hydralazine (5 mg kg-1). Large increases in the Pi/total phosphate ratio were found with the murine sarcomas, KHT and RIF-1 implanted into C3H/He mice. However little or no effect was seen for the two human xenografted tumours, HX118 and HT29 implanted in MFI nu/nu/01a mice. An intermediate response was observed for KHT tumours grown in nu/nu mice. All tumours showed a large response to clamping. The anaesthetic Hypnorm/Hypnovel has a great influence on the response of the tumour metabolism to hydralazine appearing to both prolong and increase the changes induced. There is evidence to support the theory that the changes in 31P spectra are related to the oxygen status of the tumours.     

Characterization of a murine lymphoma cell line by 31P-NMR spectroscopy: In vivo monitoring of the local anti-tumor effects of systemic immune cell transfer

The intradermal ESb-MP murine T-cell lymphoma in syngeneic DBA/2 mice has been used as a model for adoptive immunotherapy (ADI). Cultured ESb-MP cells were characterized in suspension by ³¹P-NMR spectroscopy (MRS) at 11.7 T, and solid primary tumors were examined by ³¹P-MRS in vivo at 7.0 Tesla using surface-coil techniques. Growing tumors contained relatively high levels of phosphomonoesters (PME, predominantly phosphoethanolamine), nucleotides (NTP) and P₁, low levels of phosphodiesters (PDE) and no phosphocreatine. Mean tissue pH was found to be 6.7–6.9. The spectra of ESb-MP cells cultured in RPMI medium (containing choline but no ethanolamine) also showed low PDE and no phosphocreatine at an intracellular pH of 7.4; however, only a trace amount of phosphoethanolamine was detected and significant levels of nucleoside mono- and diphosphates were observed. The complete ADI treatment protocol involved low-dose irradiation (5 Gy) followed by i.v. transfer of immune spleen cells from allogeneic B10.D2 donors and resulted in 100% remission (responders); no treatment or incomplete ADI (irradiation or immune cell transfer alone) resulted in no remissions (non-responders). In vivo MRS could best discriminate between responders and non-responders on the basis of tissue pH, which increased in responders to 7.0 by day 5–6 after complete ADI. Following therapy, the sum of PME + P₁ (both absolute and as a percent of total phosphates) decreased significantly only for responders but only after a visible decrease in tumor volume was apparent. © 1996 Wiley-Liss, Inc.     

31P-NMR spectroscopy and histological studies of the response of rat mammary tumours to endocrine therapy

We have shown by 31P-NMR spectroscopy that ovariectomy, in N-methyl-N-nitrosourea induced mammary adenocarcinomas, increases signals from phosphocreatine (PCr) relative to nucleoside triphosphate (NTP) before measurable regression (2 days) and for at least a further 13 days. The present study correlates the NMR changes with histological changes in the regressing tumour. Mammary tumours were examined by NMR before, and 2 and 14 days after, ovariectomy or sham-ovariectomy. Sections were taken from five tumours at each time point after operation for histology and for immunocytochemical staining of myoepithelial cells, luminal cells and basement membrane material. The histology showed typical cribriform papillary type mammary adenocarcinomas. The luminal cell population had a high mitotic activity and there was a prominent myoepithelial layer. At 2 days post-ovariectomy no significant change in mitotic activity was observed and no cytological characteristics attributable to ovariectomy could be seen. At 14 days postovariectomy the tumour was indistinguishable from a tubular adenoma, had significantly reduced mitotic activity, a relative increase in myoepithelial cells and basement membrane material. The changes detected by NMR must reflect early metabolic events, perhaps related to the histological changes observed at 14 days after ovariectomy. 31P-NMR spectroscopy may permit early monitoring of endocrine therapy for mammary cancer.     

Growth studies of subcutaneous rat tumours: comparison of 31P-NMR spectroscopy, acid extracts and histology

31P-NMP, surface coil spectra of three subcutaneously implanted rat tumours (Morris hepatoma 7777, GH3 prolactinoma, Walker carcinosarcoma) and an N-methyl-N-nitrosourea induced rat mammary adenocarcinoma at different stages of growth were obtained and compared with histological sections taken immediately after NMR acquisitions. Metabolite ratios (phosphocreatine (PCr)/beta nucleoside triphosphate (beta NTP), PCr/Pi, beta NTP/Pi) calculated from the NMR spectra were compared with ratios obtained from acid extracts of tumours of similar size. Measurements of creatine and ADP were also made. Three of the tumours showed positive correlations between increasing tumour size and decreasing metabolite ratios measured both by NMR and in extracts, whereas the Walker carcinosarcoma showed no correlation between size and any parameters measured. Phosphorus metabolite ratios, measured in extracts of skin overlying the tumours, indicated a fall in high energy phosphate when there was histological evidence of skin invasion by the tumour. Surface coil 31P-NMR spectra of subcutaneously grown or induced tumours in the rat represent a slowly changing steady state as the tumour increases in size. We conclude that increasing numbers of hypoxic tumour cells, rather than large areas of necrotic tissue, contribute largely to the NMR spectrum.     

Methodology for applied 4 MHz RF hyperthermia concomitant with 31P NMR spectroscopic monitoring of murine tumours

It has been generally found that solid tumours in vivo are more susceptible to destruction by heat than normal tissues. Hyperthermia has, thus, been employed in the treatment of cancer either applied alone or in combination with other modalities such as chemotherapy and radiotherapy. However, the critical mechanism(s) by which heat sensitizes and kills cells in the solid tumour remains poorly defined. Magnetic resonance spectroscopic monitoring of tumour metabolism during application of hyperthermia may provide important insight into the response to hyperthermic challenge. The implementation of dual antenna-coil methodology that provides for NMR spectroscopic monitoring (31P at 121?MHz) concomitant with applied 4?MHz RF hyperthermia in murine tumours is described herein, in some detail. This technology, which does not require advanced (and expensive) magnetic resonance imaging systems, should be readily adaptable by other laboratories with an interest in murine tumour models.     

Loss of high-energy phosphate following hyperthermia demonstrated by in vivo 31P-nuclear magnetic resonance spectroscopy

We have used in vivo 31P-nuclear magnetic resonance spectroscopy to study the changes in high-energy phosphates following hyperthermia. Immediately after heating, there is a fall in adenosine triphosphate and apparent intracellular pH and an increase in inorganic phosphate. Following sublethal heating (40 degrees for 15 min), these changes were partial, and they resolved over the subsequent 45 hr. With tumors given severe hyperthermia (47 degrees for 15 min), there was complete disappearance of adenosine triphosphate, with no recovery by 24 hr posttreatment. Qualitatively similar effects were seen after heating of normal leg muscle. The degree of fall of the adenosine triphosphate/inorganic phosphate concentration ratio was directly proportional to the heat dose and to thermal cell kill. 31P-Nuclear magnetic resonance spectroscopy may be useful in thermal dosimetry and treatment evaluation following hyperthermia.     

Methodology for applied 4 MHz RF hyperthermia concomitant with 31P NMR spectroscopic monitoring of murine tumours.

It has been generally found that solid tumours in vivo are more susceptible to destruction by heat than normal tissues. Hyperthermia has, thus, been employed in the treatment of cancer either applied alone or in combination with other modalities such as chemotherapy and radiotherapy. However, the critical mechanism(s) by which heat sensitizes and kills cells in the solid tumour remains poorly defined. Magnetic resonance spectroscopic monitoring of tumour metabolism during application of hyperthermia may provide important insight into the response to hyperthermic challenge. The implementation of dual antenna-coil methodology that provides for NMR spectroscopic monitoring (31P at 121 MHz) concomitant with applied 4 MHz RF hyperthermia in murine tumours is described herein, in some detail. This technology, which does not require advanced (and expensive) magnetic resonance imaging systems, should be readily adaptable by other laboratories with an interest in murine tumour models.     

Cytokine production after whole body and localized hyperthermia

The levels of TNF, IL-1 and IL-6 in circulating blood female WAG/Ry rats were assessed in relation to treatment with localized hyperthermia of the right hind leg or with whole-body hyperthermia (WBH). After a localized treatment for 30 min at 43 or 44d`C no detectable increase in levels of IL-6 or TNF was obtained. Hyperthermia for 30 min at 45d`C led to an elevated level of IL-6 of 19.4 ± 5.2 U/ml above the control level 24 h after treatment. Levels of IL-1 were never higher than those in control animals that received only anaesthesia. Anaesthesia induced a peak level of approximately 131 U/ml IL-16 h after treatment. Serum levels of IL-1 and IL-6 are enhanced after WBH. IL-1 reaches a peak level already during WBH about 15 after reaching 41.5d`C. IL-6 levels were not enhanced during WBH but 1 h after WBH a clear peak was observed. Anaesthesia with sham WBH did not lead to enhanced IL-6 levels but enhanced IL-1 levels were clearly detected. We did not detect TNF in any sample after WBH. It is concluded from the present results that IL-6 is not induced by a ‘standard’ treatment of localized hyperthermia as used in oncotherapy (i.e. 60min at 43d`C) to such a high level locally that this is reflected in increased levels in circulating blood. WBH at clinically relevant temperatures leads to enhanced levels of IL-1 and IL-6. The difference in IL-6 response after WBH or localized hyperthermia probably is related to the fact that in WBH also the bone marrow is treated. This may lead to stimulation of this important stem cell compartment of the peripheral blood. The sequence of appearance of IL-1 and IL-6 after hyperthermia is akin to the sequence in an inflammatory response. However, the experiments with sham treatment show that IL-1 may appear in the circulating blood not followed by IL-6. These results indicate that enhanced IL-1 levels may reflect a stress reaction of the animal related to the (sham) treatment. Enhanced levels of IL-1 after WBH correlate with enhanced levels of ACTH in the circulating blood.     

Combination therapy of rat brain tumours using localized interstitial hyperthermia and intra-arterial chemotherapy

Several investigators have reported that a high concentration of drugs in a tumour can be achieved using intra-arterial (IA) chemotherapy. This treatment was highly effective, especially in brain tumours, but the actual therapeutic advantage is still unknown. There are also indications that human malignant gliomas can effectively be treated using interstitial hyperthermia. Therefore, a combined treatment of IA chemotherapy and interstitial hyperthermia should be very promising and this has been studied in a tumour model. Wistar rats with isotransplanted C 6 gliomas in the brain were treated with adriamycin (ADR, 1.0 mg/kg body weight) either infused via the carotid artery (i.a.) or via the tail vein (i.v.), with or without interstitial hyperthermia. Hyperthermia of the tumours was applied using a homemade radiofrequency antenna (RF-heating) and a heating device that maintained the tumour temperature above 40°C. Concentration of adriamycin in tumours after treatment was measured using HPLC. The effectiveness of treatment was determined by the survival time of the animals and histopathological examinations. The highest uptake of adriamycin in the rat C 6 glioma was obtained when the animals were treated with hyperthermia and i.a. ADR infusion ( p <0.01). These animals also showed significantly longer overall survival time (SF50=46 days) in comparison to the other treatments ( p < 0.05). The histological studies demonstrated a necrotic tumour; however, the surrounding normal brain tissue remained intact. Thus, a combination of IA chemotherapy with adriamycin and localized interstitial hyperthermia enhances considerably the efficacy of adriamycin and has a greater antitumour effect for malignant brain tumours. This method is suitable for clinical use, and may be a new strategy for treating gliomas not successfully treated today.     

In vivo applications of magnetic nanoparticle hyperthermia

Abstract

Hyperthermia is considered to be a promising tool for the treatment of tumours. Intensive research activities reveal a distinct impact not only on the cellular level but also on tumour physiology which favours the combination with the classical oncologic modalities radio- and chemotherapy. Different techniques have been established so far. Among them, magnetic hyperthermia exploits the intrinsic magnetic properties of iron oxide nanoparticles (magnetite and maghemite) which induce heating during the exposure to an alternating magnetic field. Beyond the advantage that heating is generated within the tumour and not from outside the body, the amounts of magnetic material and their intratumoral distribution patterns are key factors determining the therapeutic outcome. They can be influenced by the use of different application routes, which will be discussed in this paper.     

Combination therapy of rat brain tumours using localized interstitial hyperthermia and intra-arterial chemotherapy.

Several investigators have reported that a high concentration of drugs in a tumour can be achieved using intra-arterial (IA) chemotherapy. This treatment was highly effective, especially in brain tumours, but the actual therapeutic advantage is still unknown. There are also indications that human malignant gliomas can effectively be treated using interstitial hyperthermia. Therefore, a combined treatment of IA chemotherapy and interstitial hyperthermia should be very promising and this has been studied in a tumour model. Wistar rats with isotransplanted C(6) gliomas in the brain were treated with adriamycin (ADR, 1.0 mg/kg body weight) either infused via the carotid artery (i.a.) or via the tail vein (i.v.), with or without interstitial hyperthermia. Hyperthermia of the tumours was applied using a homemade radiofrequency antenna (RF-heating) and a heating device that maintained the tumour temperature above 40 degrees C. Concentration of adriamycin in tumours after treatment was measured using HPLC. The effectiveness of treatment was determined by the survival time of the animals and histopathological examinations. The highest uptake of adriamycin in the rat C(6) glioma was obtained when the animals were treated with hyperthermia and i.a. ADR infusion (p <0.01). These animals also showed significantly longer overall survival time (SF50 =46 days) in comparison to the other treatments (p < 0.05). The histological studies demonstrated a necroti c tumour; however, the surrounding normal brain tissue remained intact. Thus, a combination of IA chemotherapy with adriamycin and localized interstitial hyperthermia enhances considerably the efficacy of adriamycin and has a greater antitumour effect for malignant brain tumours. This method is suitable for clinical use, and may be a new strategy for treating gliomas not successfully treated today.     

Comparative effects of thermosensitive doxorubicin-containing liposomes and hyperthermia in human and murine tumours

Purpose: In previous reports, laboratory-made lysolecithin-containing thermosensitive liposome encapsulating doxorubicin (LTSL-DOX) showed potent anticancer effects in FaDu human squamous cell carcinoma. To further study the spectrum of LTSL-DOX activity, the efficacy of its commercial formulation was re-examined in FaDu and compared in HCT116, PC3, SKOV-3 and 4T07 cancer cell lines. Factors that may influence differences in HT-LTSL-DOX efficacy were also examined.

Methods: Anticancer effect was measured using standard growth delay methods. We measured doubling time and clonogenic survival after doxorubicin exposure in vitro, and interstitial pH and drug concentrations in vivo.

Results: In all five tumour types, HT-LTSL-DOX increased median tumour growth time compared with untreated controls (p < 0.0006) and HT alone (p < 0.01), and compared with LTSL-DOX alone in FaDu, PC-3 and HCT-116 (p < 0.0006). HT-LTSL-DOX yielded significantly higher drug concentrations than LTSL-DOX (p < 0.0001). FaDu was most sensitive (p < 0.0014) to doxorubicin (IC₅₀ = 90 nM) in vitro, compared to the other cell lines (IC₅₀ = 129–168 nM). Of the parameters tested for correlation with efficacy, only the correlation of in vitro doubling time and in vivo median growth time was significant (Pearson r = 0.98, p = 0.0035). Slower-growing SKOV-3 and PC-3 had the greatest numbers of complete regressions and longest tumour growth delays, which are clinically important parameters.

Conclusions: These results strongly suggest that variations in anti-tumour effect of HT-LTSL-DOX are primarily related to in vitro doubling time. In the clinic, the rate of tumour progression must be considered in design of treatment regimens involving HT-LTSL-DOX.     

The effect of angiotensin II on blood flow in tumours during localized hyperthermia

The selective enhancement of drug delivery to tumours is an important factor in the effectiveness of thermochemotherapy as well as in standard normothermal chemotherapy. We have attempted to clarify experimentally using AH 100B tumour-bearing rats whether or not a selective increase in blood flow in tumours can be produced under specific conditions of local hyperthermia by administration of angiotensin (AGT II). AGT II (2 micrograms/kg/min) produced an elevation of blood pressure (ca. 150 mm Hg) when local hyperthermia, at 41, 43, and 45 degrees C, was induced. Furthermore, at 41 and 43 degrees C a selective increase in blood flow in tumours resulted from the AGT II-induced hypertension. By contrast, a decrease in blood flow was observed at 45 degrees C both in tumour and in muscle. These results indicate that AGT II-induced hypertension and the resultant selective increase in drug delivery to tumours during the initial phase of heating may result in an augmentation of the anticancer effects of thermochemotherapy.     

Dietary fat modulation of murine mammary tumor metabolism studied by in vivo 31P-nuclear magnetic resonance spectroscopy

The effect of dietary fat concentration and saturation on high energy phosphate metabolites and phospholipid turnover in transplanted line 168 murine mammary tumors was studied using surface coil 31P-nuclear magnetic resonance spectroscopy. Female BALB/c mice were fed one of five diets each containing at least the minimum of essential fatty acids (EFA). Four diets contained additional safflower or palm oil for a total fat concentration of 5 or 20% by weight. The growth rate of tumors from mice fed the high safflower oil diet was significantly greater than the growth rate of tumors for mice fed all other diets including the one which contained the minimal EFA. 31P-nuclear magnetic resonance-observable phosphate metabolite ratios. ATP/Pi, ATP/phosphomonoester (ATP/PME), and PME/Pi, and tumor pH of line 168 tumors decreased with increasing tumor volume, indicating a shift from active to inactive tumor metabolism. The rates of those decreases with progressive tumor growth differed significantly among tumors of mice fed the different diets. Decreases in ATP/Pi, ATP/PME, and pH were the most rapid in the tumors of mice fed the high safflower oil diet and significantly faster than tumors of mice fed the diet containing minimum EFA. In addition, the decrease in the PME/Pi ratio of tumors was significantly greater in mice fed the high fat (high palm oil and high safflower oil) diets than mice fed the diet containing the minimum of EFA. The rate of decline of ATP/Pi and ATP/PME with progressive tumor growth was directly correlated with levels of linoleic acid as well as total unsaturated fat. High levels of a polyunsaturated fat had a significant effect on mammary tumor metabolism particularly during early stages of tumor growth. Differences in high energy phosphate metabolite dynamics relative to dietary fat were present in tumors of equal volume. Thus, dietary fat influences on mammary tumorigenesis may be related to high energy phosphate metabolites.     

Laser Doppler flowmetry in subepidermal tumours and in normal skin of rats during localized ultrasound hyperthermia

Laser Doppler flowmetry has been applied to normal skin and to subepidermal tumours during localized ultrasound hyperthermia in the rat. In normal skin, 40°C hyperthermia only induced a marginal increase in the red blood cell flux. Significant increases occurred after 20 min at 42°C and after 4 min at 44°C. During 44°C hyperthermia maximum fluxes were reached after 24 min. Thereafter, the flow declined and finally approached preheating values. In contrast, in subepidermal tumours 40°C hyperthermia on the average induced a slight decrease of the flux. During 42°C hyperthermia a significant flow decrease was found after 40 min of heating. Following a transient increase in the laser Doppler flow during the heating-up period, 44°C hyperthermia led to a significant impairment of the flux after 24 min. A total shutdown of RBC flux was observed in about 30 per cent of the tumours at 44°C. Upon elevated tissue temperatures, pronounced inter-tumour variabilities in the time- and temperature-dependent changes of RBC flux were observed. Rhythmic oscillations of the RBC flux were found in some subepidermal tumours (0.40 ± 0.05 cycles/min). Upon heating, these periodic flow variations slowed down significantly (0.20 ± 0.04 cycles/min), whereas in normal skin the frequency of the flow fluctuations increased.     

Laser Doppler flowmetry in subepidermal tumours and in normal skin of rats during localized ultrasound hyperthermia

Laser Doppler flowmetry has been applied to normal skin and to subepidermal tumours during localized ultrasound hyperthermia in the rat. In normal skin, 40 degrees C hyperthermia only induced a marginal increase in the red blood cell flux. Significant increases occurred after 20 min at 42 degrees C and after 4 min at 44 degrees C. During 44 degrees C hyperthermia maximum fluxes were reached after 24 min. Thereafter, the flow declined and finally approached preheating values. In contrast, in subepidermal tumours 40 degrees C hyperthermia on the average induced a slight decrease of the flux. During 42 degrees C hyperthermia a significant flow decrease was found after 40 min of heating. Following a transient increase in the laser Doppler flow during the heating-up period, 44 degrees C hyperthermia led to a significant impairment of the flux after 24 min. A total shutdown of RBC flux was observed in about 30 per cent of the tumours at 44 degrees C. Upon elevated tissue temperatures, pronounced inter-tumour variabilities in the time- and temperature-dependent changes of RBC flux were observed. Rhythmic oscillations of the RBC flux were found in some subepidermal tumours (0.40 +/- 0.05 cycles/min). Upon heating, these periodic flow variations slowed down significantly (0.20 +/- 0.04 cycles/min), whereas in normal skin the frequency of the flow fluctuations increased.     

The effect of blood flow modification on intra- and extracellular pH measured by 31P magnetic resonance spectroscopy in murine tumours.

Intra- and extracellular pH (pHi and pHe) were measured simultaneously by 31P magnetic resonance spectroscopy (MRS) in CaNT tumours before and after blood flow modification. Before modification, pHi was 7.1 +/- 0.09 (n = 11) and pHe [measured with an MRS-visible extracellular marker, 3-aminopropyl phosphonate (3-APP)] was 6.7 +/- 0.05 (n = 8). Chemical shift imaging and localised MRS experiments showed that the 3-APP signal was only from the tumour, not surrounding tissue. After modification by vascular occlusion, independent of whether tumours were maintained at room temperature (22-24 degrees C) or kept warm (33-35 degrees C), there was a decrease in pHi and pHe with pHi decreasing to a greater extent. Qualitatively similar results were found using flavone acetic acid (FAA) as a blood flow modifier; only four out of nine tumours responded to FAA. Concomitant with the reduction of the pH gradient after modification was a decrease in the phosphorylation state of the adenine nucleotides measured either as ATP/Pi by MRS or [ATP]/[ADP][P(i)] in tumour extracts. These results indicate that the intracellular uptake of chemotherapeutic drugs which are dependent on the transmembrane pH gradient will not be enhanced in cells made ischaemic as a result of vascular shutdown.     

Response-specific adriamycin sensitivity markers provided by in vivo 31P nuclear magnetic resonance spectroscopy in murine mammary adenocarcinomas

The in vivo phosphorus-31 nuclear magnetic resonance (NMR) spectra of Adriamycin (ADR)-sensitive murine mammary adenocarcinomas (17/A) and an ADR-resistant subline of this tumor which has been isolated in vivo (17/A/ADR) were compared both before and after i.v. administration of 12 mg/kg ADR. Significant differences between ADR-sensitive and -resistant tumors for the changes observed 1 day after treatment (prior to significant decreases in tumor size) included: the pH increased to greater than 7.3 in response to treatment (or pH remained elevated) in ADR-sensitive tumors only; the inorganic phosphate to nucleoside triphosphates peak height ratio decreased to less than 1 in response to treatment only in ADR-sensitive tumors; glycerophosphocholine to nucleoside triphosphates peak height ratio decreased in response to treatment in ADR-sensitive tumors only; and the phosphocholine to nucleoside triphosphates peak height ratio decreased in response to treatment in ADR-sensitive tumors only. These differences are evidence in support of the hypothesis that in vivo 31P-NMR provides response-specific markers of ADR sensitivity. Because 31P-NMR can be applied to humans, these differences may be of prognostic value in the clinical management of human breast cancer if they are present after treatment with lower, nontoxic doses of ADR.     

Metallothionein levels in ovarian tumours before and after chemotherapy

The metallothionein content of ovarian tumours is considerably higher than that found in normal ovaries (greater than 100-fold differences in mean values, P less than 0.001). There was no difference between the metallothionein content of tumours from patients who had completed chemotherapy, usually with a regimen containing a platinum drug, and tumours from untreated patients. Similarly, the level of metallothionein was not influenced by response to therapy, age, stage, histology, or tumour cell differentiation state. These data do not support the hypothesis that metallothionein content is a major determinant of tumour sensitivity in ovarian cancer.