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 14N nuclear magnetic resonance spectroscopy of tumors: detection of ammonium and trimethylamine metabolites in the murine radiation induced fibrosarcoma 1

The in vivo 14N nuclear magnetic resonance spectra of s.c. implanted murine radiation induced fibrosarcomas (RIF-1) display narrow resonances assignable to betaine and other trimethylamines and broad resonances due to amino acids and peptides. In 19 of the 41 tumors studied a distinct resonance from the ammonium ion is detectable. The accumulation of ammonium in the tumor to nuclear magnetic resonance detectable levels may result from glutaminolysis (a possible pathway for energy production in the tumor), from the degradation of peptides and proteins, or from the deamination of adenine nucleotides. Estimates of the tissue ammonium concentration were obtained from the in vivo tumor spectrum and the spectrum of the nonlabile trimethylamines in the perchloric acid extract. In the extract, the 14N resonances of betaine, carnitine, choline, phosphorylcholine, and glycerophosphorylcholine were resolved, and a relatively high level of tissue urea was observed. Spin-lattice relaxation times were obtained for the 14N nucleus of each of these metabolites in phosphate buffer.     

Effect of cancer on the in vivo energy state of rat liver and skeletal muscle

The effect of increasing tumor burden on host liver and skeletal muscle energy status was studied using P-31 nuclear magnetic resonance spectroscopy (NMR), in rats inoculated with a nonmetastasizing methylcholanthrene-induced sarcoma (TB), and compared to nontumor bearing (NTB) and pair-fed (PF) rats. During the 28-day study, serial measurements of body weight, food intake, and tumor volume were obtained. Using a 0.9-cm double-turn surface coil, weekly NMR measurements were obtained from liver and skeletal muscle. An increasing ratio of [Pi]/[ATP] was used as one measure of intracellular energy depletion. [Pi]/[ATP] in NTB rats remained constant over time at 0.78 +/- 0.10 in liver, and 0.30 +/- 0.10 in skeletal muscle. In TB rats, the [Pi]/[ATP] ratio increased significantly in liver (P = 0.00002) and skeletal muscle (P = 0.04) with increasing tumor burden. In PF rats, no significant change occurred in [Pi]/[ATP] in liver or skeletal muscle, indicating that declining food intake was not responsible for the change in [Pi]/[ATP] seen in TB rats. Surface-coil spectroscopy of liver and skeletal muscle permits serial measurement of visceral energy stores. Increasing tumor burden results in early, ongoing depletion of energy stores as reflected by increasing [Pi]/[ATP] in these organs.     

Direct relationship between high-energy phosphate content and blood flow in thermally treated murine tumors

In vivo 31P-nuclear magnetic resonance (NMR) spectroscopy and 133Xe clearance were used to monitor serially ATP content and blood flow, respectively, in C3H/HeJ mouse subcutaneous RIF-1 tumors treated with hyperthermia. There was a prompt decrease in ATP [measured spectroscopically and expressed as the ratio of ATP to Pi (ATP/Pi)]. The slope and magnitude of the change in ATP closely paralleled those of tumor blood flow. Close correlation between these two variables was seen when data were analyzed both by treatment group and by individual mouse. Ligated tumors showed qualitatively and quantitatively similar changes in ATP/Pi and blood flow. RIF-1 cells heated in vitro to a similar degree showed no decrease in ATP. The loss of ATP in subcutaneous RIF-1 tumors heated in vivo was primarily due to disruption of tumor blood flow. These data emphasize the importance of vascular factors to in vivo thermal effects. In vivo 31P-NMR spectroscopy can be used to monitor indirectly vascular effects from hyperthermia.     

Characterization of a 60,000-dalton Oncofetal Protein from the Plasma of Tumor-Bearing Rats

A tumor cell-associated protein, previously shown to be present in the circulation of carcinogen-treated and tumor-bearing animals and cancer patients, has now been identified in the cytosol of embryonic tissue. This oncofetal protein, which is absent from the plasma of normal animals, has been purified from the plasma of tumor-bearing rats by a series of steps including ammonium sulfate fractionation and chromatography on Sepharose CL-6B and on CM Affi-Gel Blue. The tumor and fetal-associated 60-kd rat factors appear to be identical based on their reactivity to polyclonal antibody produced against the tumor factor. The factor, assayed by its ability to induce the transport of RNA from isolated nuclei, is a phosphoprotein with a minimum molecular weight of 60,000, as determined by polyacrylamide gel electrophoresis. In its purified form it is phosphorylated in the presence of the catalytic subunit of heart muscle protein kinase and ATP but does not exhibit auto-phosphorylating activity. 32P-orthophosphate is also incorporated into the phosphoprotein in vivo.     

Different energy metabolism in two human small cell lung cancer subpopulations examined by 31P magnetic resonance spectroscopy and biochemical analysis in vivo and in vitro.

Two human small cell lung cancer tumor lines, maintained as solid tumor xenografts on nude mice and as in vitro cell cultures, were studied by in vivo 31P magnetic resonance spectroscopy and by biochemical analysis of extracts of solid tumors and cell cultures. The tumor lines CPH SCCL 54A and CPH SCCL 54B are subpopulations from the same tumor. In solid tumors (n = 125), the ATP/Pi ratio was greater in 54A than in 54B. This was due to a higher ATP level in 54A, whereas there was no difference in Pi, ADP, and AMP. A decrease in ATP/Pi during growth was caused by a decline in ATP, whereas Pi remained unchanged. Small amounts of phosphocreatine were found in the xenografts and in tumor extracts, but not in the cell extracts; correspondingly, there was a low creatine kinase activity in solid tumors and no activity in the cell cultures. Thus, the phosphocreatine content of the solid tumors originated from the stroma. A difference in ATP content between 54A and 54B was also found in cell cultures; hence, the metabolic difference is an intrinsic quality of the malignant cells and is not caused by the host system.     

1H-NMR metabolic markers of malignancy correlate with spontaneous metastases in a murine mammary tumor model

End-products of glycolysis as well as phospholipid precursors and catabolites have been suggested as metabolic indicators of tumor progression. To test the hypothesis that increased levels of such indicators can distinguish metastatic phenotypes, we determined a limited cellular 1H-NMR metabolic profile of subpopulations of murine mammary 4T1 cells that differ in their metastatic potential. Subpopulations with differing metastatic phenotypes were identified by sorting for the expression of the cell surface adhesion oligosaccharide sialylated Lewis x (sLeX). The sLeX-negative subpopulation metastasizes to the lung of syngeneic mice more rapidly than the sLeX-positive subpopulations. The metabolic profile of the sLeX-negative subpopulation indicated higher levels of lactate and total choline metabolites than the sLeX-positive subpopulation, suggesting that altered metabolism is a critical component of the malignant phenotype. Analysis of shed cellular material from the sLeX-negative subpopulation displayed an increased ratio of phosphocholine to glycerophosphocholine when compared to the parental line and sLeX-positive subpopulation. Serum obtained from mice inoculated with either sLeX-negative or sLeX-positive tumor cells contained broader methylene resonances (P = 0.0002; P = 0.0003) and narrower methyl resonances (P = 0.0013; P < 0.0001) when compared to serum of naive mice. However, line widths of methylene and methyl resonances were not useful for distinguishing between the two tumor phenotypes. Results of this study further support the notion that metabolic indicators of malignancy can correlate with in vivo metastatic behavior.     

Growth of a human colonic adenocarcinoma cell line (HT 29) on microcarrier beads: metabolic studies by 31phosphorus nuclear magnetic resonance spectroscopy

A method allowing the growth of a human colon adenocarcinoma cell line (HT 29) on beaded polystyrene microcarriers has been developed by modifying the culture conditions used in monolayer cultures. Under optimized conditions, the cells became confluent 7 days after seeding and reached a density of 2.8 X 10(5) cells/cm2 of microcarrier (65% of the available area occupied). 31P NMR spectra were typically recorded on 300 X 10(6) cells continuously perfused at a flow rate of 15 ml/min in a specially designed NMR chamber in which the microcarrier beads were sequestered within the receiver coil volume. The in vivo spectrum displays a series of resonances assigned to nucleoside triphosphates (ATP and GTP), inorganic phosphate and various phosphomonoesters (mainly glucose-6-P and phosphorylcholine). Diphosphodiester resonances (DPDE, mainly UDP-N-acetyl-glucosamine and UDP-N-acetylgalactosamine) were not detected in the in vivo spectrum and were only apparent in the spectrum of the perchloric acid extract of the cells, indicating that these compounds have a restricted mobility in the intracellular compartment. The intracellular pH of HT 29 cells was 7.2 during the perfusion with a medium buffered at pH 7.3. The internal pH decreased slowly (2 X 10(-3) pH unit/min) during anoxic perfusion, but severe intracellular acidosis occurred after 40 min of ischemia (2.7 X 10(-2) pH unit/min). Sequential recording of 31P NMR spectra has shown that HT 29 cells are able to maintain their high energy phosphorylated compound levels (ATP) when subjected to 100 min of anoxia and 40 min of total ischemia.     

Tuberous sclerosis-associated neoplasms express activated p42/44 mitogen-activated protein (MAP) kinase, and inhibition of MAP kinase signaling results in decreased in vivo tumor growth.

PURPOSE: Tuberous sclerosis (TS) is a common autosomal disorder attributable to inactivation of the tumor suppressor genes tuberin and hamartin. To determine whether mitogen-activated protein (MAP) kinase signaling plays a role in the pathogenesis of TS, we stained human TS-associated neoplasms with antibodies directed against activated MAP kinase, and observed high-level expression. EXPERIMENTAL DESIGN: To determine whether MAP kinase is functionally important for the development of neoplasia in TS, we established a murine model of TS-associated neoplasia (Tsc2Ang1 cells) from a tumor arising in a mouse heterozygous for tuberin. Tsc2Ang1 cells demonstrate tumorigenesis in vivo and high-level expression of activated MAP kinase in vitro. The functionality of MAP kinase signaling was assessed by inactivating MAP kinase using a dominant-negative MAP kinase kinase in tsc2ang1 cells and assessing the effect of this intervention on in vivo tumorigenicity and production of the potent angiogenic factor vascular endothelial growth factor (VEGF). RESULTS: Human TS-related neoplasms demonstrate high-level expression of activated MAP kinase, as does a tumor arising in a mouse heterozygous for tuberin. The inhibition of MAP kinase signaling by the introduction of a dominant-negative MAP kinase kinase leads to the inhibition of tumor growth in vivo and decreased production of VEGF. CONCLUSIONS: MAP kinase is activated in TS-related neoplasia in mice and humans. Inhibition of MAP kinase leads to decreased tumor growth in vivo. Pharmacological inhibition of MAP kinase may be a therapeutic target in the prevention and treatment of TS-related tumors.     

Alteration of aliphatic lipid proton NMR linewidths by malignant tumors in guinea pigs

Water-suppressed proton nuclear magnetic resonance spectroscopy was used to observe plasma lipoprotein lipid methyl and methylene resonances from guinea pigs which had been injected with viable or heat-killed line 1 or line 10 tumor cells or sterile oil. It was shown that the widths of these resonances became significantly sharper as the number of tumor cells grew. Plasma from tumor-free control animals showed no change in the NMR linewidths. It is concluded that the changes observed reflect a specific host response to viable tumor cells, and in these models there is a reciprocal relationship between the number of viable tumor cells and the linewidths of plasma lipoprotein methyl and methylene resonances.     

In vivo 31P nuclear magnetic resonance spectroscopy of subcutaneous 9L gliosarcoma: effects of tumor growth and treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea on tumor bioenergetics and histology

In vivo 31P nuclear magnetic resonance spectroscopy was used to examine the bioenergetics of the rat 9L gliosarcoma during untreated growth and in response to chemotherapy with 1,3-bis(2-chloroethyl)-1-nitrosourea. Tumor growth was associated with a decline in the phosphocreatine and nucleoside triphosphate resonances, consistent with an increase in tumor hypoxia during untreated growth. Following chemotherapy with 1,3-bis(2-chloroethyl)-1-nitrosourea (10 mg/kg), tumor levels of phosphocreatine and nucleoside triphosphate rebounded while the level of inorganic phosphate in the tumor declined. Histological comparison of treated and untreated tumor sections 4 days posttreatment showed that the treated tumor had a lower proportion of necrotic cells, a higher proportion of viable cells, and a 5-fold higher level of interstitial space than the control tumor.     

Direct targeting of tumor cell F(1)F(0) ATP-synthase by radioiodine angiostatin in vitro and in vivo

Recent discoveries have identified endothelial cell-surface F(1)F(0) adenosine triphosphate (ATP) synthase as the key target for angiostatin (AST) action. As this enzyme is also present on tumor cells, we investigated whether radiolabeled AST may directly target cancer cell-surface ATP synthase in vitro and in vivo. METHODS: Cell-binding characteristics of (125)I-AST was evaluated on human umbilical vein endothelial (HUVE) and SNU-C5 colon carcinoma cells. The molecular target for binding was investigated with anti-ATP synthase antibodies and RGDyV. Biodistribution and imaging experiments were performed in mice xenografted with carcinoma and sarcoma tumors. Tumor localization of ATP synthase and exogenous AST was compared via double immunostaining. RESULTS: Both HUVE and SNU-C5 cells showed specific (125)I-AST binding that was dose-dependently inhibited by excess AST, with IC(50) values of 3.5 and 0.2 microM, respectively. Both cell types stained positive for ATP synthase and demonstrated an approximately 50% reduction in AST binding by antibodies against the alpha- and beta-subunit of the enzyme. (123)I-AST injected in mice allowed for the clear tumor visualization with significant tumor accumulation and uptake ratios. Immunostaining revealed a localization of exogenous AST to closely correlate to that of tumor-cell ATP synthase. CONCLUSIONS: AST can directly target tumor-cell ATP synthase, and AST imaging may thus be useful for monitoring tumor ATP synthase expression.     

Primary intracranial leiomyosarcoma in an immunocompetent patient: case report

We report a case of intracranial leiomyosarcoma (LMS) arising after resection of neurofibroma at the cerebellopontine angle. A 45-year-old immunocompetent woman presented with recurrence of a tumor 9 years after resection performed in another hospital. Magnetic resonance imaging demonstrated a heterogeneously enhancing, dura-based mass at the left cerebellopontine angle. The tumor was subtotally removed via lateral suboccipital craniotomy. LMS was diagnosed based on histological and immunohistochemical findings. Postoperatively, although the patient was treated using local radiotherapy, she died due to rapid regrowth of the tumor. Reevaluation of the specimen obtained in the first operation led to a diagnosis of neurofibroma. Both LMS and neurofibroma rarely occur intracranially. LMS is generally thought to arise from smooth muscle cells of the blood vessels or pluripotent mesenchymal cells. In this case, LMS might also have originated from smooth muscle cells of the vessels in the neurofibroma, possibly associated with mechanical and/or heat stimulation during the previous surgery.     

31P-NMR-spectroscopy measurements of energy metabolism of in vivo growing ascites tumours following addition of glucose

The cellular ATP content and the phosphorylation potential, defined as the ATP, ADP and inorganic phosphate (Pi) ratios, of exponentially growing Ehrlich ascites tumour cells were compared with cells at the plateau phase of growth. These phosphorus compounds were measured using 31P-NMR-spectroscopy immediately after removal of the cell material from the host and in their ascites fluid reflecting in vivo growth conditions. Reaching the plateau phase of growth, the ATP content and the phosphorylation potential decreased. Upon addition of glucose, the phosphorylation potential immediately increased. We concluded that the reduced phosphorylation potential was due to a limited availability of glucose in spite of the nearly normal blood glucose concentration found. An increasing diffusion distance from the host to all parts of the tumor is a possible reason for that.     

肿瘤体外三磷酸腺苷药敏试验

 目的　评价肿瘤细胞经药物处理后的生存情况 ,探讨将该方法用于卵巢癌药敏试验的可行性。方法　采用生物荧光法检测三磷酸腺苷 (ATP) ,对小鼠纤维瘤细胞株L92 9、人卵巢癌组织和腹水细胞进行抗癌药物处理后的细胞活性测定。结果　该法能测出不同浓度药物对细胞作用的剂量—效应关系 ,以及不同个体对不同浓度药物反应的差异 ,实验变异系数CV值为 1 2～ 15 8%。结论　ATP生物荧光法有稳定、重复性好、特别敏感的特点 ,用于卵巢癌体外药敏试验可行。     

Adult Extrarenal Wilms’ Tumor Mimicking Mixed Epithelial and Stromal Tumor in the Retroperitoneum: A Case Report with Immunohistochemical Study and Review of the Literature

We report an extremely rare case of adult extrarenal Wilms’ tumor (WT) in a 52-year-old woman who presented with fever and abdominal distension. Computed tomography revealed a well-defined mass lesion measuring 15.0 cm in the right retroperitoneum and that was in contact with the right kidney. The mass and kidney were surgically removed. Grossly, the mass was well-defined, measuring 16.3 × 11.0 × 9.8 cm, and appearing grayish-white in color. The border between the mass and the kidney was well-defined. Histologically, the tumor showed a triphasic pattern consisting of stromal, epithelial and blastemal components. The stromal component was predominant in the tumor and consisted both of spindle cells and smooth muscle cells. The epithelial component showed a mature glandular structure. Immunohistochemically, the stromal component was positive for vimentin, smooth muscle actin and desmin. The blastemal component was positive for vimentin, while the epithelial component was positive for cytokeratin (CK) 18, CK7 and vimentin. WT-1 was negative in the all three components, and the Ki-67 proliferation index was low. The postoperative histopathological diagnosis indicated extrarenal WT arising in the retroperitoneum. Although not treated by either chemotherapy or radiation therapy, she was free from disease recurrence for 30 months after surgery. To the best of our knowledge, this report is only the fourth case of adult extrarenal WT arising in the retroperitoneum. Furthermore, the present case showed predominant smooth muscle differentiation and a mature glandular structure, mimicking a mixed epithelial and stromal tumor.     

Energy Status Parameters, Hypoxia Fraction and Radiocurability Across Tumor Types

Under full nutrient in vitro conditions, the cellular adenylate energy charge of six different rodent and human tumor cell types was identical, i.e., 0.94 ± 0.01, suggesting the potential utility of this parameter as a cell (and tissue) independent marker of nutrient deprivation and hypoxia, across tumor types. The adenylate energy charge values of tumors, arising from these cells, was reduced and variable ranging from 0.72 to 0.91 for the various tumor types. However, neither the tumor adenylate energy charge, NTP/Pi, nor PCr/Pi ratios correlated with the radiobiologic hypoxic cell fractions across tumor types. The reduced adenylate energy charge in vivo suggests varying degrees of nutrient deprivation in the different tumor types, however, factors other than or in addition to hypoxia likely contribute to tumor energy status.     

Effects of oxygen on the metabolism of murine tumors using in vivo phosphorus-31 NMR

The effect of 100% inspired oxygen on in vivo tumor metabolism was examined using phosphorus-31 (31P) NMR spectroscopy. Isotransplants of two murine tumor histologies, designated MCaIV (C3H mammary adenocarcinoma) and FSaII (C3H fibrosarcoma), were used in syngeneic mice. Tumor volumes ranged from 30 to 1,800 mm3. Both tumor histologies are known to have a high hypoxic cell fraction when tumor volumes exceed 250 mm3. 31P nuclear magnetic resonance (NMR) spectra were obtained at 145.587 MHz, and the signal was detected using a 1.4 cm diameter, single loop coil designed to localize the signal from only the tumor. Spectral parameters for optimal signal-to-noise ratio (SNR) included a 60 degrees pulse and a 2-second recycle delay. Tumors were implanted in the hindfoot dorsum to assure that all detected mobile phosphates were of tumor origin. Phosphocreatine/inorganic phosphate (PCr/Pi) ratios of large tumors (greater than 250 mm3) were reduced compared with small tumors (less than 250 mm3) of the same histology. The increased PCr/Pi response to 100% inspired oxygen was greater for large tumors and for tumors with lower baseline PCr/Pi ratios. When host animals were given 10% oxygen for respiration, there was an increase in Pi and a decrease in both PCr and ATP. The response to 10% oxygen was observed in both large and small tumors of both tumor histologies studied. Resting skeletal muscle exhibited no alteration in the NMR spectrum during either 100 or 10% oxygen breathing. We conclude that the fractional increase in PCr/Pi ratio that occurs after 100% oxygen breathing is a sensitive, noninvasive method of detecting tumor hypoxia.     

Comparison of the growth properties in vitro and transplantability of continuous mouse mammary tumor cell lines and clonal derivatives.

Five continuous mouse mammary tumor cell lines and 12 clonal derivatives have been established from tumors arising in BALB/c or C3H mice either spontaneously or in response to viral (mammary tumor virus), hormonal (17 beta-estradiol), or chemical [7,12-dimethylbenz(alpha)anthracene] stimuli in vivo. The cell lines were examined for the following in vitro growth parameters:plating efficiency, saturation density, population-doubling times, colony formation on plastic surfaces, and anchorage-independent growth in methylcellulose. The majority of the mammary tumor cell lines were transplantable in syngeneic, immunocompetent mice and gave rise to tumors composed of epithelial cells. There was no growth parameter in vitro which invariably correlated with tumorigenicity in vivo. Most of the mammary tumor cell lines appeared to produce C-type virus. Only one, a C3H derivative, appeared to respond to stimulation by glucocorticoids with the enhanced production of B-type virus. Analysis of the growth properties exhibited in culture by transformed mammary epithelial cells revealed marked differences from those previously reported for transformed fibroblasts.