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.     

31P-nuclear magnetic resonance spectroscopy studies of the response of rat mammary tumors to endocrine therapy

We have used 31P-nuclear magnetic resonance spectroscopy to detect the metabolic changes that occur in estrogen-sensitive, N-methyl-N-nitrosourea-induced rat mammary tumors as they regress following ovariectomy. In untreated animals the spectra of the tumors showed a steady loss of high energy phosphates (phosphocreatine and nucleoside triphosphates) and an increase in inorganic phosphate. This was reversed after ovariectomy. Spectral changes occurred before detectable regression of the tumor. Estrogen-insensitive tumors, grown from implanted Rama 600 and 622 cells, did not regress in response to ovariectomy, and their high energy phosphates continued to fall; estrogen-sensitive tumors also failed to respond to sham ovariectomy. These effects are probably due to the reduction in cellular energy requirements that occurs when the hormonal stimulus to growth is removed. Because the nuclear magnetic resonance method is noninvasive, this technique should be applicable clinically as a means of predicting the response of a tumor to endocrine therapy.     

Effects of hydralazine-induced vasodilation on the energy metabolism of murine tumors studied by in vivo 31P-nuclear magnetic resonance spectroscopy

The effects of hydralazine on tumor energy metabolism and on some cardiovascular parameters were measured. Tumor energy metabolism was studied in C3Hf/Sed mice with isotransplants of a spontaneous murine fibrosarcoma (FSaII, congruent to 100 mm3 in volume) and 31P-NMR. Cardiovascular parameters were measured in anesthetized C3Hf/Sed mice via intracarotid catheter. Hydralazine doses of 0.25 mg/kg given ip caused an increase of the phosphocreatine to inorganic phosphate ratio (PCr: Pi) in 5 of 6 animals. These doses had minimal effects on mean arterial blood pressure, though there may have been an increased cardiac output due to a decreased afterload. Hydralazine doses greater than or equal to 2.0 mg/kg given ip were associated with a decrease in PCr, nucleotide triphosphate, and pH, and an increase in Pi (P less than .01 for control vs. 10 mg hydralazine/kg). This substantial decrease in high-energy phosphates was associated with a pronounced decrement in mean arterial blood pressure. These findings provide a rational basis for the study in experimental systems of hydralazine-induced enhancement of cell killing by hyperthermia and by agents toxic to hypoxic cells. Further, these results can be taken as a sign that hydralazine should be used with care in patients undergoing radiation treatment.     

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.     

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.     

FSaII mouse tumor metabolic changes with different doses of glucose measured by 31P nuclear magnetic resonance

Tumor acidosis and energy deprivation enhance thermal sensitivity. We have used in vivo 31P nuclear magnetic resonance spectroscopy to noninvasively monitor changes in pH and energy metabolism in FSaII mouse tumors after i.p. administration of glucose. A dose of 5 g/kg glucose induced a pH drop of 0.31 units without any statistically significant change in energy status. The pH changes resolved within 2 h. In contrast, administration of 10 g/kg glucose resulted in a severe acidosis (mean nadir pH of 6.19 corresponding to a mean pH drop of 0.96 units) and loss of energy, the latter most probably being due to an acidosis-induced inhibition of glycolysis during ischemic hypoxia. The resulting acidosis and energy loss were more persistent and resolved in 5.5-28 h. In contrast, after an identical dose of mannitol (10 g/kg), a pH drop of approximately only 0.1 units over 72 min was noted. The data suggest that both cleavage of glucose to lactic acid and blood flow inhibition are involved in glucose induced tumor acidosis. In vivo 31P nuclear magnetic resonance spectroscopy may be useful clinically to monitor therapeutic attempts at enhancing thermal sensitivity.     

Early metabolic response to tumor necrosis factor in mouse sarcoma: a phosphorus-31 nuclear magnetic resonance study

To investigate the effects of recombinant human tumor necrosis factor alpha (rHuTNF-alpha) on high-energy phosphate metabolism of cancer cells, 31P nuclear magnetic resonance (NMR) studies were performed on a murine methylcholanthrene-induced sarcoma. Injection of 15 micrograms of rHuTNF-alpha caused progressive depletion of ATP and phosphocreatine within 90 min, together with an increase in inorganic phosphate. Metabolic changes were correlated with the early histological appearance of thrombosis and hemorrhage. A spatially localized NMR technique demonstrated that these changes were specific for the tumor. Acute ischemia of the tumor produced similar metabolic changes; thus the metabolic effects of rHuTNF-alpha could be due to either a primary action on tumor biochemistry or a secondary action produced by ischemia. These findings indicate that rHuTNF-alpha has a very rapid onset of action, which can be detected by 31P NMR. Furthermore, the results suggest that 31P NMR spectroscopy will be extremely useful for detecting early biochemical changes produced by rHuTNF-alpha or other treatments in animal and human cancers.     

31P-nuclear magnetic resonance spectroscopy in vivo of six human melanoma xenograft lines: tumour bioenergetic status and blood supply.

Six human melanoma xenograft lines grown s.c. in BALB/c-nu/nu mice were subjected to 31P-nuclear magnetic resonance (31P-NMR) spectroscopy in vivo. The following resonances were detected: phosphomonoesters (PME), inorganic phosphate (Pi), phosphodiesters (PDE), phosphocreatine (PCr) and nucleoside triphosphate gamma, alpha and beta (NTP gamma, alpha and beta). The main purpose of the work was to search for possible relationships between 31P-NMR resonance ratios and tumour pH on the one hand and blood supply per viable tumour cell on the other. The latter parameter was measured by using the 86Rb uptake method. Tumour bioenergetic status [the (PCr + NTP beta)/Pi resonance ratio], tumour pH and blood supply per viable tumour cell decreased with increasing tumour volume for five of the six xenograft lines. The decrease in tumour bioenergetic status was due to a decrease in the (PCr + NTP beta)/total resonance ratio as well as an increase in the Pi/total resonance ratio. The decrease in the (PCr + NTP beta)/total resonance ratio was mainly a consequence of a decrease in the PCr/total resonance ratio for two lines and mainly a consequence of a decrease in the NTP beta/total resonance ratio for three lines. The magnitude of the decrease in the (PCr + NTP beta)/total resonance ratio and the magnitude of the decrease in tumour pH were correlated to the magnitude of the decrease in blood supply per viable tumour cell. Tumour pH decreased with decreasing tumour bioenergetic status, and the magnitude of this decrease was larger for the tumour lines showing a high than for those showing a low blood supply per viable tumour cell. No correlations across the tumour lines were found between tumour pH and tumour bioenergetic status or any other resonance ratio on the one hand and blood supply per viable tumour cell on the other. The differences in the 31P-NMR spectrum between the tumour lines were probably caused by differences in the intrinsic biochemical properties of the tumour cells rather than by the differences in blood supply per viable tumour cell. Biochemical properties of particular importance included rate of respiration, glycolytic capacity and tolerance to hypoxic stress. On the other hand, tumour bioenergetic status and tumour pH were correlated to blood supply per viable tumour cell within individual tumour lines. These observations suggest that 31P-NMR spectroscopy may be developed to be a clinically useful method for monitoring tumour blood supply and parameters related to tumour blood supply during and after physiological intervention and tumour treatment. However, clinically useful parameters for prediction of tumour treatment resistance caused by insufficient blood supply can probably not be derived from a single 31P-NMR spectrum since correlations across tumour lines were not detected; additional information is needed.     

Use of high-resolution 31P-labeled topical magnetic resonance spectroscopy to monitor in vivo tumor metabolism in rats

A probe using a single-tuned solenoid coil has been constructed to study in vivo metabolism of rats in a wide-bore Bruker nuclear magnetic resonance spectrometer. Transplantable rat mammary adenocarcinomas (estrogen receptor negative) were implanted into the hind leg muscle of 8-week-old rats. The other leg without tumor was used as a control. Tumor metabolism could be distinguished from that of surrounding muscle by the appearance of inorganic phosphate and sugar phosphate resonances, reflecting tissue necrosis, and increased glycolysis. Tumor growth was accompanied by an increase in the size of these peaks, and the chemical shifts of the inorganic phosphate peak indicated that the intracellular pH became more acidic. Administration of methotrexate (i.v.) reversed these patterns and decreased tumor volume. Changes in the phosphocreatine peaks indicated changes in tumor volume rather than in tumor metabolism. These studies show that topical magnetic resonance not only can monitor the growth of tumors in vivo but can be also used to evaluate the efficacy of chemotherapeutic drugs.     

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.     

Effect of changing tumor oxygenation on glycolytic metabolism in a murine C3H mammary carcinoma assessed by in vivo nuclear magnetic resonance spectroscopy.

The rate of conversion of D-[1-(13)C]glucose into [3-(13)C]lactate (apparent glycolytic rate) has been determined in C3H murine mammary carcinomas in vivo using tumor-selective (13)C nuclear magnetic resonance spectroscopy with (1)H-(13)C cross-polarization. Under conditions of acute hypoxia induced by breathing carbon monoxide at 660 ppm, the apparent glycolytic rate was 0.0239 +/- 0.0019 min(-1). The proportion of (13)C label incorporated into [4-(13)C]glutamate (measured in tumor extracts) was 25-fold lower than that incorporated into [3-(13)C]lactate, reflecting a very limited oxidative metabolism during this hypoxic episode. For animals breathing air or carbogen (95% O(2) + 5% CO(2)), the calculated glycolytic rates were correspondingly lower (0.0160 +/- 0.0021 min(-1) and 0.0050 +/- 0.0011 min(-1), respectively). Although (13)C labeling of glutamate at C4 was still an order of magnitude lower than that for lactate at C3 (11-fold for air and 9-fold for carbogen), these ratios did show a greater degree of oxidative metabolism than that seen in animals breathing carbon monoxide at 660 ppm. The marked difference in apparent glycolytic rate for this tumor model between well-oxygenated and hypoxic conditions demonstrates a substantial Pasteur effect (inhibition of glycolysis by oxygen). Dynamic (13)C nuclear magnetic resonance spectroscopy provides a noninvasive estimate of tumor glycolysis that can be used to evaluate the relationship between oxygenation and energy metabolism, and this has potential consequences for the sensitivity of hypoxic cells to treatment and their ability to promote angiogenesis.     

The response of spontaneous and transplantable murine tumors to vasoactive agents measured by 31P magnetic resonance spectroscopy.

31P magnetic resonance spectroscopy has been used to compare the effects of the vasoactive agents hydralazine and flunarizine on the oxygenation of the transplantable tumors, SCCVII/Ha and 16C, and a range of spontaneous mammary tumors arising in the breeding stock in the Genetics Division at the Radiobiology Unit. The vasodilator hydralazine, previously shown to increase the radiobiological hypoxic fraction of transplantable murine tumors, increased inorganic phosphate to total phosphate (Pi/total) in SCCVII/Ha and 16C tumors. However, only two spontaneous tumors responded to this agent (2/12). The calcium antagonist flunarizine, which sensitizes the SCCVII tumor to X rays, consistent with a reduction in hypoxic fraction, reduced Pi/total in this and the 16C tumor. Further, most spontaneous tumors tested (8/10) responded to this agent, as measured by a reduction in Pi/total. These results point to fundamental differences between transplantable and spontaneously arising tumors in mice in their response to vasoactive agents.     

In vivo assessment of RAS-dependent maintenance of tumor angiogenesis by real-time magnetic resonance imaging

New blood vessel formation is a prominent feature of human cancers and tumor progression and is frequently accompanied by the acquisition of an angiogenic phenotype associated with a switch in the balance of proangiogenic and antiangiogenic molecules. This study was designed to investigate the role of activated H-RAS on the angiogenic phenotype of melanoma that arises in the inducible Tyr/Tet-RAS Ink4a/Arf(-/-) model using in vivo imaging with histopathologic correlation. We show that loss of RAS activity in fully established melanomas led to a reduction in tumor volume, which was preceded by impairment of vascular function as determined by in vivo magnetic resonance imaging. This correlated with activation of apoptosis in host-derived endothelial cells as well as in tumor cells. Thus, real-time in vivo imaging provided evidence that maintenance of tumor angiogenesis requires activated RAS in this model system, and that loss of vascular integrity upon inactivation of RAS is an active process rather than a consequence of loss of tumor cell viability.     

31P-nuclear magnetic resonance studies of the effect of recombinant human interleukin 1 alpha on the bioenergetics of RIF-1 tumors

The effect of a single injection of human recombinant interleukin 1 alpha (IL-1 alpha) on s.c. RIF-1 tumors in mice was studied by in vivo 31P nuclear magnetic resonance spectroscopy. Spectra were obtained before and up to 24 h after IL-1 alpha. At 2, 4, 6, and 8 h after IL-1 alpha injection, RIF-1 tumors exhibited a reduction in bioenergetic status compared to untreated controls. The Pi to beta-nucleoside triphosphate and the phosphomonoester to beta-nucleoside triphosphate ratios increased, while the phosphocreatine to Pi and phosphodiester to phosphomonoester ratios decreased. Tumor blood flow, estimated by 86RbCl uptake, decreased within 30 min after IL-1 alpha treatment. Minimum perfusion was detected at 4 h, with recovery between 6 and 12 h after IL-1 alpha treatment. Histological sections of the RIF-1 tumors revealed intravascular congestion by 2 h, extravascular hemorrhage by 4 h, and necrosis by 12 h after treatment with IL-1 alpha. The time course of bioenergetic changes in RIF-1 tumors determined by 31P-NMR spectroscopy was found to parallel the reduction and subsequent recovery of tumor blood flow.     

In vivo selection and characterization of a murine mammary tumor subline with high potential for spontaneous lymph node metastasis

A transplantable mammary adenocarcinoma, grown in Balb/c mice, with a marked enhancement in its draining lymph node metastatic ability (MM3LN), was obtained through an in vivo procedure from a variant tumor moderately metastatic to lymph nodes (MM3). Both MM3 and MM3LN presented a similar latency and tumor growth rate and reached the same tumor mean diameter at death. MM3LN tumor-bearing mice exhibited a larger mean survival time. The new variant showed a 2.5-fold higher incidence of tumor-draining lymph node metastases than MM3 line, with no differences in the incidence of lung metastases. Morphology as well as cytogenetic and in vitro adhesion properties were studied in order to characterize the new subline. This murine tumor model has potential application in the study of the metastatic process in lymphoid tissue.     

环磷酰胺治疗小鼠S180肉瘤的体内31P磁共振波谱研究

目的 观察环磷酰治疗小鼠Ｓ１８０肉瘤的＾３１ＰＭＲＳ参数变化,以及这些参数的变化是否早于常规观察的瘤体积变化。方法 利用表面线圈＾３１ＰＮＭＲ方法,并研究了小鼠皮下接种Ｓ１８０肉瘤的体积。结果：对照组的ＰＣｒ／Ｐｉ和＋β－ＮＴＰ／Ｐｉ比值降低,而环磷酰胺给药组升高,且给药后２４,４８,７２ｈ时显著高于对照组（Ｐ〈０．０５）,对照组的ＰＭＥ／β－ＮＴＰ比值继续升高,而给药组ＰＭＥ／β－ＮＴＰ比值在给     

In vivo imaging of tumor response to therapy using a dual-modality imaging strategy

In vivo assessment of the outcome of cancer therapy is hampered by the paucity of imaging probes that target tumors specifically and noninvasively. The importance of such probes increases with the continuous development of chemotherapeutics and the necessity to evaluate their effectiveness in a clinical setting. We have recently reported on a dual-modality imaging probe specifically targeting the underglycosylated mucin-1 tumor-specific antigen (uMUC-1), which is one of the early hallmarks of tumorigenesis in a wide variety of tumors. This probe consists of crosslinked superparamagnetic iron oxide nanoparticles (CLIO) for MR imaging, modified with Cy5.5 dye (for near infrared optical fluorescence imaging (NIRF)), and has peptides (EPPT), specifically recognizing uMUC-1, attached to the nanoparticle's dextran coat. In the present study, we demonstrated that this probe could not only detect orthotopically implanted preclinical models of adenocarcinomas but could also track tumor response to chemotherapy in vivo in real time. Considering the high cost associated with the development and testing of new cancer therapeutics and the need for accurate, noninvasive assessment of their effectiveness, we believe that the developed probe represents a valuable research tool relevant to clinical discovery.     

In vivo magnetic resonance imaging of the effects of photodynamic therapy

Nuclear magnetic resonance (NMR) proton imaging and measurements of the parameters T1 and T2, have been carried out in vivo on the murine mammary tumour T50/80. Tumours had been treated 24 h previously by photodynamic therapy (PDT, using haematoporphyrin derivative and 630 nm laser light). Proton images clearly demarcated a high signal-intensity region on the side of the tumour closest to the incident light beam, while the parts of the tumour more remote from the beam resembled the images from untreated controls. Both T1 and T2 values were raised in the high-intensity region. This high-intensity region was shown to correspond to PDT-induced histological necrosis, the low-intensity region to histologically intact tumour. Linear regression analysis of the relationship of depth of necrosis measured histologically and 'depth of necrosis' measured from the NMR images, yielded a slope of 0.93 (r2 = 0.95).     

The In Vitro mammary gland response to mammotropic hormones in mice with different mammary tumorigenesis

Mammary gland DNA synthesis estimated by the in vitro incorporation of [³H]thymidine in response to mammotropic hormones was compared between high and low mammary tumor strains of virgin mice (SHN and SLN). In SHN, mammary gland DNA synthesis when cultured in the medium containing insulin (I), aldosterone (A), estradiol-17β (E), progesterone, prolactin (PRL) and growth hormone (GH) showed a peak on day 2 of culture and declined thereafter. Quite the opposite was the case in SLN mammary glands. There was little strain-difference in mammary gland DNA synthesis when cultured for 6 days in the medium containing complete hormone mixture. However, DNA synthesis of SHN mammary glands cultured in the medium deficient in PRL was less than one-third of the control, whereas that of SLN glands was two-thirds of the control. Moreover, mammary gland DNA synthesis was decreased significantly by deficiency in GH or E in SHN strain only. In both strains, mammary gland DNA synthesis declined with an increasing dose of PRL when cultured in the medium containing I, A and PRL, which was associated with an activated secretory function. However, the changes were much more marked in SHN than in SLN. The results have demonstrated the higher dependency of SHN mammary glands than SLN glands upon mammotropic hormones, especially PRL. They further indicate that mammary gland potential for both growth and function is well reflected by mammary gland sensitivity to PRL.