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Diffusion and relaxation of water in C6 glioma and MLS human ovarian carcinoma spheroids were measured from 1D projections acquired using a 2D diffusion-relaxation correlation pulse sequence and processed by non-negative least-square (NNLS) analysis. Systematic underestimation of I(s) and ADC(s) were observed for I(s)/(I(s) + I(f)) < 0.001. In the presence of spheroids, two apparent diffusion coefficient (ADC) compartments were observed, where ADC(f), ADC(s), and I(f), I(s) are the respective ADCs and signal intensities of the fast and slow compartments. These compartments differed also in their T(2) relaxation (ADC(s) = 0.5-0.74 x 10(-5) cm(2)/s, T(2) = 36-45 ms; and ADC(f) = 2.2-2.8 x 10(-5) cm(2)/s, T(2) = 280-316 ms). The two ADC compartments and the slow T(2) compartment were consistent with slow exchange. The fast T(2) compartment showed a drift with diffusion weighting, suggesting that it represents water exchanging between compartments that differ in their ADC and T(2). Both ADC(s) and I(s) were markedly attenuated with increasing diffusion time (Delta) for Delta < 100 ms, and increased at longer Delta. These results are consistent with restricted diffusion and fast relaxation of intracellular water for short diffusion time (T(1)' = 46.6 ms), and with predominant extracellular contribution to ADC(s) at longer diffusion times. Magn Reson Med 46:68-77, 2001., (Copyright 2001 Wiley-Liss, Inc.)

Bold contrast MRI was applied for mapping vascular maturation in tumor- and wound-induced skin angiogenesis using the response of mature vessels to hypercapnia (inhalation of air vs. air 5% CO(2)) and the response of all vessels to hyperoxia (air 5% CO(2) vs. oxygen 5% CO(2) (carbogen)). MRI signal enhancement with hypercapnia was reduced in centered vs. linear phase encoding, suggesting increased blood flow. However, intravital microscopy demonstrated constriction of arterioles and reduced flux and density of red blood cells in mature capillaries with hypercapnia, with no change in the diameter of wound-induced neovasculature. The discrepancy in flow between MRI and intravital microscopy is consistent with increased plasma flow and reduced hematocrit. Hyperoxia resulted in increased blood oxygenation and constriction of all vessels. These results provide a hemodynamic explanation for the selective registration of MRI response to hypercapnia with mature vessels and the response to hyperoxia with total vascular function.

We describe here a strategy for photodynamic eradication of solid melanoma tumors that is based on photo-induced vascular destruction. The suggested protocol relies on synchronizing illumination with maximal circulating drug concentration in the tumor vasculature attained within the first minute after administrating the sensitizer. This differs from conventional photodynamic therapy (PDT) of tumors where illumination coincides with a maximal concentration differential of sensitizer in favor of the tumor, relative to the normal surrounding tissue. This time window is often achieved after a delay (3-48 h) following sensitizer administration. We used a novel photosensitizer, bacteriochlorophyll-serine (Bchl-Ser), which is water soluble, highly toxic upon illumination in the near-infrared (lambda max 765-780 nm) and clears from the circulation in less than 24 h. Nude CD1 mice bearing malignant M2R melanotic melanoma xenografts (76-212 mm3) received a single complete treatment session. Massive vascular damage was already apparent 1 h after treatment. Changes in vascular permeability were observed in vivo using contrast-enhanced magnetic resonance imaging (MRI), with the contrast reagent Gd-DTPA, by shortening spin-spin relaxation time because of hemorrhage formation and by determination of vascular macromolecular leakage. Twenty-four hours after treatment a complete arrest of vascular perfusion was observed by Gd-DTPA-enhanced MRI. Histopathology performed at the same time confirmed primary vascular damage with occlusive thrombi, hemorrhage and tumor necrosis. The success rate of cure of over 80% with Bchl-Ser indicates the benefits of the short and effective treatment protocol. Combining the sensitizer administration and illumination steps into one treatment session (30 min) suggests a clear advantage for future PDT of solid tumors.

Partial injury to the spinal cord can propagate itself, sometimes leading to paralysis attributable to degeneration of initially undamaged neurons. We demonstrated recently that autoimmune T cells directed against the CNS antigen myelin basic protein (MBP) reduce degeneration after optic nerve crush injury in rats. Here we show that not only transfer of T cells but also active immunization with MBP promotes recovery from spinal cord injury. Anesthetized adult Lewis rats subjected to spinal cord contusion at T7 or T9, using the New York University impactor, were injected systemically with anti-MBP T cells at the time of contusion or 1 week later. Another group of rats was immunized, 1 week before contusion, with MBP emulsified in incomplete Freund's adjuvant (IFA). Functional recovery was assessed in a randomized, double-blinded manner, using the open-field behavioral test of Basso, Beattie, and Bresnahan. The functional outcome of contusion at T7 differed from that at T9 (2.9+/-0.4, n = 25, compared with 8.3+/-0.4, n = 12; p<0.003). In both cases, a single T cell treatment resulted in significantly better recovery than that observed in control rats treated with T cells directed against the nonself antigen ovalbumin. Delayed treatment with T cells (1 week after contusion) resulted in significantly better recovery (7.0+/-1; n = 6) than that observed in control rats treated with PBS (2.0+/-0.8; n = 6; p<0.01; nonparametric ANOVA). Rats immunized with MBP obtained a recovery score of 6.1+/-0.8 (n = 6) compared with a score of 3.0+/-0.8 (n = 5; p<0.05) in control rats injected with PBS in IFA. Morphometric analysis, immunohistochemical staining, and diffusion anisotropy magnetic resonance imaging showed that the behavioral outcome was correlated with tissue preservation. The results suggest that T cell-mediated immune activity, achieved by either adoptive transfer or active immunization, enhances recovery from spinal cord injury by conferring effective neuroprotection. The autoimmune T cells, once reactivated at the lesion site through recognition of their specific antigen, are a potential source of various protective factors whose production is locally regulated.

Angiogenesis in the preovulatory follicle is confined to the theca cell layers, and penetration of capillaries through the basement membrane into the granulosa cell layers does not occur until after ovulation. However, elevated expression of the angiogenic growth factor (VEGF) has been reported in the cumulus cells surrounding the oocyte, which are expelled from the follicle during ovulation. This spatial and temporal discrepancy between VEGF expression and angiogenesis was studied here in the rat ovarian follicle, and we showed that cumulus cells secrete to the follicular fluid, in addition to VEGF, material with antiangiogenic activity that blocks endothelial cell proliferation, migration, and capillary formation in vitro. Hyaluronic acid produced by the cumulus cells can account for this antiangiogenic activity. Degradation of hyaluronic acid by hyaluronidase restored proliferation and migration of endothelial cells directed toward the cumulus. Inhibition of hyaluronic acid synthesis with 6-diazo-5-oxo-1-norleucine restored endothelial proliferation and migration in vitro, and it also resulted in early penetration of capillaries across the follicular basement membrane in vivo. These results support the role of hyaluronic acid produced by the cumulus cells as a high-molecular-weight, antiangiogenic shield that prevents premature vascularization of the preovulatory follicle by blocking endothelial cell migration and proliferation.

Magnetic resonance imaging (MRI) provides a range of non-invasive measures for visualization of tumor angiogenesis in the clinic as well as in experimental tumor models. MRI methods were developed for assessment of spatial and temporal changes in perfusion, blood volume fraction, vascular permeability, vascular function, vascular maturation, vessel diameter and tortuosity. Molecular targeted contrast agents were used for mapping specific markers of neovasculature. These approaches were applied for analysis of a number of regulatory mechanisms controlling tumor angiogenesis and for preclinical evaluation of tumor response to antiangiogenic agents.

A solid tumor presents a unique challenge as a system in which the dynamics of the relationship between vascularization, the physiological environment and metabolism are continually changing with growth and following treatment. Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) studies have demonstrated quantifiable linkages between the physiological environment, angiogenesis, vascularization and metabolism of tumors. The dynamics between these parameters continually change with tumor aggressiveness, tumor growth and during therapy and each of these can be monitored longitudinally, quantitatively and non-invasively with MRI and MRS. An important aspect of MRI and MRS studies is that techniques and findings are easily translated between systems. Hence, pre-clinical studies using cultured cells or experimental animals have a high connectivity to potential clinical utility. In the following review, leaders in the field of MR studies of basic tumor physiology using pre-clinical models have contributed individual sections according to their expertise and outlook. The following review is a cogent and timely overview of the current capabilities and state-of-the-art of MRI and MRS as applied to experimental cancers. A companion review deals with the application of MR methods to anticancer therapy.

The aim of this study was to ascertain the potential usefulness of the antiangiogenic compound linomide for treatment of von Hippel-Lindau (VHL)-related tumors. Paraganglioma tissue fragments obtained at surgery from a VHL type 2a patient were transplanted s.c. to male BALB/c nu/nu (nude) mice: (a) 2-3-mm fragments for "prevention" experiments; and (b) 2-3-mm fragments allowed to grow to 1 cm for "intervention" studies. Both groups received either 0.5 mg/ml linomide in drinking water or acidified water and were followed until tumor diameter reached 3 cm or for 4 weeks. In both the prevention and intervention experiments, a significant diminution of tumor size and weight was observed in the drug-treated animals. In vivo nuclear magnetic resonance analysis of tumor blood flow in linomide-treated animals showed localization of blood vessels almost exclusively to the periphery of the poorly vascularized tumors with a significant reduction of both vascular functionality and vasodilation. Histological examination of tumors from linomide-treated animals revealed marked avascularity. Treated animals also displayed a 2.4-fold reduction of tumor vascular endothelial growth factor mRNA levels. Taken together, our data indicate that in VHL disease, therapy directed at inhibition of constitutively expressed VEGF induction of angiogenesis by VHL tumors may constitute an effective medical treatment.

One of the hallmarks of tumor neovasculature is the prevalence of immature vessels manifested by the low degree of recruitment of vascular mural cells such as pericytes and smooth muscle cells. This difference in the architecture of the vascular bed provides an important therapeutic window for inflicting tumor-selective vascular damage. Here we demonstrate the application of gradient echo magnetic resonance imaging (MRI) for noninvasive in vivo mapping of vascular maturation, manifested by the ability of mature vessels to dilate in response to elevated levels of CO2. Histological alpha-actin staining showed a match between dilating vessels detected by MRI and vessels coated with smooth muscle cells. Switchable, vascular endothelial growth factor (VEGF)-overexpressing tumors (C6-pTET-VEGF rat glioma s.c. tumors in nude mice) displayed high vascular function and significant vascular damage upon VEGF withdrawal. However, damage was restricted to nondilating vessels, whereas mature dilating tumor vessels were resistant to VEGF withdrawal. Thus, MRI provides in vivo visualization of vascular maturity and prognosis of vascular obliteration induced by VEGF withdrawal.

Halofuginone, an inhibitor of collagen alpha1(I) gene expression was used for the treatment of subcutaneously implanted C6 glioma tumors. Halofuginone had no effect on the growth of C6 glioma spheroids in vitro, and these spheroids showed no collagen alpha1(I) expression and no collagen synthesis. However, a significant attenuation of tumor growth was observed in vivo, for spheroids implanted in CD-1 nude mice which were treated by oral or intraperitoneal (4 microg every 48 hours) administration of halofuginone. In these mice, treatment was associated with a dose-dependent reduction in collagen alpha1(I) expression and dose- and time-dependent inhibition of angiogenesis, as measured by MRI. Moreover, halofuginone treatment was associated with improved re-epithelialization of the chronic wounds that are associated with this experimental model. Oral administration of halofuginone was effective also in intervention in tumor growth, and here, too, the treatment was associated with reduced angiogenic activity and vessel regression. These results demonstrate the important role of collagen type I in tumor angiogenesis and tumor growth and implicate its role in chronic wounds. Inhibition of the expression of collagen type I provides an attractive new target for cancer therapy.

The goal of this study was to monitor the vascular bed during the lag phase in growth of implanted spheroids as a model of tumor dormancy. Vascular development and tumor growth were followed up by magnetic resonance imaging in a model system of MLS ovarian carcinoma spheroids implanted subcutaneously in female nude mice. Apparent vessel density in a 1-mm rim surrounding the spheroid was evaluated by gradient echo imaging as a measure of the angiogenic potential of the tumor. Vascular functionality and maturation were assessed by signal intensity changes in response to hyperoxia (elevated oxygen) and hypercapnia (elevated carbon dioxide), respectively. Tumor growth was delayed by 12 to 57 days after implantation. During this long period in which tumor volume did not change, up to 6 cycles of vascular development and regression were observed. We propose here that dynamic remodeling of the vascular bed may precede exit of tumors from dormancy. The sustained oscillations in the angiogenic response to the implanted spheroid are consistent with hypoxic regulation of vascular endothelial growth factor (VEGF), combined with the role of VEGF as an essential survival factor for newly formed blood vessels. Vascular maturation, manifested by physiological vasodilatory response to carbon dioxide, may be important for conferring vascular stability and exit from dormancy.

Pulsed arterial spin labeling was used for mapping ovarian perfusion and measurement of blood velocity in the ovarian artery. Arterial blood was tagged upstream by pulsed slice selective saturation, and saturation transfer due to perfusion was monitored within the rat ovary. The velocity of arterial blood was determined from the dependence of the saturation transfer on the thickness of the saturation slice and the delay between successive saturation pulses. This method allows for determination of arterial velocity, even when the artery itself is not identified in the images. The arterial velocity of blood to the ovary was 3.6+/-0.6 cm x s(-1). The mean ovarian perfusion was 8.7+/-3.5 ml x min(-1) x g(-1) during the surge of luteinizing hormone and 5.9+/-3.0 ml min(-1) x g(-1) during the luteal phase. Arterial labeling can thus be used for following vascular remodeling and angiogenesis during the ovarian cycle by MRI.

The goal of this study was to develop an experimental method for noninvasive analysis of angiogenesis, namely the sprouting of capillaries from existing blood vessels. Angiogenesis was assayed in the subcutaneous vasculature of nude mice in a region of 3 x 3 cm that included in its center a defined angiogenic stimulus. Angiogenic stimuli included agarose beads containing angiogenic growth factors, multicellular tumor spheroids, and dermal incisions. Highly significant correlation (r = 0.905, P = 0.0001) was found between the apparent vessel density determined by gradient echo MRI and the density of blood-containing vessels determined postmortem. The functionality of the neovasculature was demonstrated in mice breathing alternatingly carbogen or 95% air/5% CO2. Large signal enhancement with carbogen breathing corresponded to regions of high vessel density. The assay reported here can be applied for the study of dermal wound healing, primary vascularization of subcutaneous implants, and for measuring the activity of angiogenic and antiangiogenic agents.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen and migration factor for vascular smooth muscle cells (SMC), promoted neovascularization in vivo in the rabbit cornea. MRI demonstrated quantitatively the angiogenic effect of HB-EGF when introduced subcutaneously into nude mice. HB-EGF is not directly mitogenic to endothelial cells but it induced the migration of bovine endothelial cells and release of endothelial cell mitogenic activity from bovine vascular SMC. This mitogenic activity was specifically blocked by neutralizing anti-vascular endothelial growth factor (VEGF) antibodies. In contrast, EGF or transforming growth factor-alpha (TGF-alpha) had almost no effect on release of endothelial mitogenicity from SMC. In addition, RT-PCR analysis demonstrated that VEGF165 mRNA levels were increased in vascular SMC 4-10-fold by 0.35-2 nM of HB-EGF, respectively. Our data suggest that HB-EGF, as a mediator of intercellular communication, may play a new important role in supporting wound healing, tumor progression and atherosclerosis by stimulating angiogenesis.

We show here that elevated levels of gonadotropins (luteinizing hormone and follicle stimulating hormone), as found in menopause or after ovariectomy, promote growth of human ovarian carcinoma by induction of tumor angiogenesis. Human epithelial ovarian cancer tumors progressed faster in ovariectomized mice. This induced growth could be attributed to the elevated levels of gonadotropins associated with loss of ovarian function because direct administration of gonadotropins also was effective in promoting tumor progression in vivo. On the other hand, gonadotropins had no direct effect on the proliferation of human ovarian cancer cells in vitro. Using MRI, we demonstrated that ovariectomy significantly (P < 0.02) induces neovascularization of human ovarian carcinoma spheroids implanted in nude mice. Moreover, conditioned medium of gonadotropin-treated human ovarian carcinoma cells showed increased mitogenic activity to bovine endothelial cells, and this activity could be blocked by neutralizing antibodies against luteinizing hormone and against vascular endothelial growth factor. Accordingly, gonadotropin stimulation resulted in a dose-dependent-induced expression of vascular endothelial growth factor in monolayer culture as well as in the outer proliferating cells of human ovarian cancer spheroids. These results demonstrate the significance of the elevated levels of gonadotropins, as found in menopause and in all ovarian cancer patients, on the progression of ovarian cancer and could explain the protective effect of estrogen replacement therapy. Based on these results, we suggest that hormonal therapy aimed at lowering the circulating levels of gonadotropins may possibly prolong remission in ovarian cancer by extending tumor dormancy.

The preovulatory follicle provides a unique physiological example of rapid growth accompanied by neovascularization, two processes that are generally characteristic of pathologies such as wound repair or malignancy. During the hours preceding ovulation, follicular growth is accompanied by elevated levels of messenger RNA for vascular endothelial growth factor (VEGF). Angiogenic activity, mediated by VEGF, is manifested in the peripheral blood vessels surrounding the follicle, that show capillary sprouting and increased vascular permeability. Following ovulation, rapid infiltration of capillaries through the follicular wall is essential for the formation of the corpus luteum. In this review we compare the preovulatory follicle with a popular model of avascular solid tumour growth, namely the multicellular tumour spheroid, in particular the role of hypoxic stress in the regulation of angiogenesis in both systems.

Accumulation of ferritin, the iron storage protein, has been linked recently to aging and a number of pathologies. Noninvasive detection of iron storage by MRI relies on its extremely strong effect on water relaxation. The aim of this article is to characterize the effect of ferritin on transverse water relaxation in a high magnetic field, using an imaging Carr-Purcell Meiboom-Gill (CPMG) preparation sequence. Ferritin-induced water relaxation showed quadratic dependence on the iron loading factor, implying a paramagnetic mechanism. However, an additional zero order term was found, that could be due to the initial stages of the iron core loading. Significant enhancement of ferritin contrast was obtained at very short Tau CPMG durations. This approach for enhancing ferritin contrast was demonstrated by NMR microscopy of ferritin-injected Xenopus oocytes, thus showing the feasibility of ferritin detection in a high magnetic field, even in systems with short transverse relaxation.

The preovulatory rat follicle reaches a diameter of 1 mm with no internal blood vessels. Nutrient supply to the enclosed oocyte depends solely on passive diffusion across the follicular wall and the follicular fluid. Spin-echo and stimulated-echo NMR microscopy experiments were applied here for studying modulations in water diffusion during gonadotropin-induced maturation of perfused rat ovarian follicles (32 degrees C). Two diffusion compartments were observed for the follicular wall. The intracellular water diffusion coefficient, measured at a short diffusion time (9 ms) was 0.28 x 10(-5) cm2/s. Diffusion at long diffusion times was restricted to 16 microns, the size of cells in the follicular wall, and did not change during maturation. In the follicular fluid a transient 26% decrease in the diffusion coefficient was observed 4-7 h after gonadotropin stimulation, a change that is bound to affect the metabolic balance of the oocyte before ovulation.

Magnetic resonance imaging has been used to follow noninvasively tumor neovascularization and tumor growth in a model system of multicellular C6 rat glioma spheroids implanted s.c. in nude mice. By positioning a single spheroid approximately 1 cm from the site of incision both the vascularization of the tumor and the wound healing processes were spatially separated and could be simultaneously followed. The model proposed here provides defined initial conditions of tumor geometry and cell proliferative status and separation of initial tumor growth from neovascularization. Magnetic susceptibility relaxation provided an intrinsic marker for blood containing vessels. The implanted spheroid induced vessel growth within 4 days after implantation that was geometrically oriented toward the spheroid and distinct from wound healing at the site of incision. Volume measurements showed a corresponding 4-day lag in growth followed by Gompertz progression. Sham implantation of agarose beads of similar diameter showed no induction of vessel growth, ruling out a direct effect of wound healing. The new vessels penetrating the tumor were highly permeable to the contrast reagent gadolinium-diethylenetriaminepentaacetic acid. This permeability may be due to the action of vascular endothelial growth factor, a major angiogenic growth factor in this system, and a potent permeability factor.

Cyclocreatine, an analogue of creatine, inhibits tumor cell proliferation in vitro and in vivo. The effects of cyclocreatine in large C6 glioma multicellular spheroids were mapped here by magnetic resonance microscopy. Diffusion-weighted images of C6 glioma spheroids resolved the bright viable rim and the dark necrotic center. Sequential sets of diffusion images, following cyclocreatine administration, showed increasing self-diffusion coefficients of the intracellular water in the viable rim (0.49 x 10(-5) cm2/s for untreated spheroids, 0.62 x 10(-5) cm2/s after 48 h perfusion with 20 mM cyclocreatine). This fact correlated with cellular swelling apparent in histological sections. The radial distribution of cyclocreatine and soluble lipids across perfused C6 spheroids was measured by one-dimensional chemical shift imaging. Cyclocreatine accumulation was prominent throughout the viable cell layer, with no cyclocreatine accumulation in the necrotic center. In both cyclocreatine-treated and control spheroids the lipid signal was highest in the necrotic center and lower in the inner viable cell layer.

The geometric average of two spin-echo images obtained with opposite polarity diffusion gradients yields cross-term-free images that can be directly compared for diffusion anisotropy. This approach is demonstrated here for free water isotropic diffusion and anisotropic diffusion of water in the phloem system of celery (Apium graveolens).

Nuclear magnetic resonance microimaging measurements of the self-diffusion coefficient of water in large (greater than 2 mm) EMT-6 multicellular spheroids were performed in order to elucidate diffusion mechanisms in tumors. Pulsed gradient spin echo-imaging methods were developed for measuring diffusion in an intravoxel multicompartment system. The self-diffusion coefficient (at 22 degrees C) for water in the medium (Dm) consisted of only a single diffusion compartment [Dm = 1.99 +/- 0.03 (SE) x 10(-5) cm2/s]. Similarly, the spheroid necrotic center showed a single water diffusion compartment with a self-diffusion coefficient (Dc) significantly lower than that of the medium (Dc = 1.54 +/- 0.05 x 10(-5) cm2/s). The spheroid viable rim region showed two distinct compartments of approximately equal volume, one with a large diffusion coefficient (1.70 +/- 0.12 x 10(-5) cm2/s) and a second with a significantly smaller diffusion coefficient (0.25 +/- 0.01 x 10(-5) cm2/s). We propose that these two experimentally distinguishable compartments correspond to the extra- and intracellular regions, respectively, of the viable rim of the spheroid. Although the diffusion coefficients were significantly different in the medium, the necrotic center, and the viable rim, the activation energy for diffusion was the same in the three regions (0.20 eV). Studies of perfused spheroids at 37 degrees C show the same dependence of the diffusion coefficients on the diffusion filter as observed for unperfused spheroids at 22 degrees C. These results demonstrate the ability of nuclear magnetic resonance microimaging to investigate diffusion at the cellular level, which will lead to a better understanding of microenvironmental regulation in tumors.

We have measured the 31P nuclear magnetic resonance spectra of EMT6/Ro multicellular tumor spheroids over a wide range of sizes under constant nutrient conditions which matched those used for culturing the spheroids. The amount of nucleotide triphosphate per cell decreased with spheroid growth, roughly in proportion to the decrease in cell volume. There was no correlation between the intracellular pH, the nucleotide triphosphate:Pi ratio, or the phosphocreatine:Pi ratio and either the spheroid cellularity, the mean cell volume, the S-phase fraction, the clonogenic capacity, or the amount of central necrosis. The phosphoryethanolamine:phosphorylcholine ratio also increased with increasing spheroid size. There was a negative correlation between the phosphoryethanolamine:phosphorylcholine ratio and the S-phase cell fraction or the mean cell volume; this ratio was positively correlated with the extent of central necrosis. The membrane degradation components glycerophosphorylcholine and glycerophosphorylethanolamine showed no significant changes with increasing spheroid size. These results imply that spheroid necrotic areas induced by chronic nutrient deficiencies are "invisible" to 31P nuclear magnetic resonance and that the development of cellular quiescence in spheroids is not caused by a decrease in the steady-state level of high-energy phosphates or a reduced intracellular pH. Together, these data support a model in which cells maintain normal steady-state levels of high energy phosphates until they are very close to necrotic cell death. This implies that the deterioration of 31P nuclear magnetic resonance spectra of tumors with increasing size is not caused by chronic nutrient deficiencies resulting from cells outgrowing the capillary supply, but rather is more related to transient nutrient deprivation phenomena.

We report proton NMR images obtained at microscopic (less than 30 microns) resolution of EMT6/Ro and HT1080 multicellular tumor spheroids 1.2-1.7 mm in diameter. T1-weighted images showed little contrast across a slice through the spheroid. There was also no difference between the inner and outer spheroid regions when signal intensity was measured as a function of the repetition time (TR), showing that T1 was the same across the spheroid. Conversely, T2-weighted and multi-echo images clearly revealed the central necrosis that occurs as the spheroids develop. Measurements of the thickness of the viable cell zone made on NMR images agreed with those made on standard histology sections for two different cell lines. The basis for the NMR discrimination of the necrotic region from the viable rim cells was found to be a shortened apparent T2 in the necrotic region (132 +/- 17 ms) with respect to that in the viable cells (173 +/- 9 ms). These results illustrate the applicability of NMR microscopy to assaying conditions inside intact tumor spheroids and suggest that this technology will allow the use of spheroids to investigate several important questions in tumor biology and pathophysiology.

We have developed a system for the perfusion of a stirred suspension of multicellular spheroids during nuclear magnetic resonance spectroscopy. Measurement of the medium temperature, pH, oxygen tension, and glucose and lactate concentrations demonstrated that the macroenvironmental conditions around the spheroids during perfusion matched those in standard spinner culture flasks. Spheroids cultured in the NMR perfusion chamber for up to 48 h were virtually identical to spheroids cultured under standard conditions in terms of volume and cell number growth, the extent of central necrosis, cellular clonogenicity, and proliferative status. To avoid problems in interpreting the NMR spectra, we have used a medium containing 10% of the normal inorganic phosphate concentration; comparative growth and NMR studies showed that this medium had no effect on the results reported. 31P NMR spectroscopic analysis demonstrated that the mean pH, nucleotide triphosphate (NTP) to inorganic phosphate (Pi) ratio, the total amount of NTP, and the total energy charge were essentially constant over 8 h of analysis. Stopping the stirring of the spheroid culture during analysis resulted in depletion of the nucleotide phosphate pool in 30 min, with an accumulation of Pi and a shift to a more acid intracellular pH. This effect could be reversed if stirring was resumed within 30 min. Stopping the perfusion while maintaining stirring resulted in a deterioration of the 31P spectra until no high energy phosphates remained at 120 min and the pH fell to approximately 6. This effect was also partially reversible after 30 min of reperfusion, with recovery to a normal 31P spectrum requiring 10 h. The combination of the spheroid model system with 31P NMR spectroscopic analysis will provide a powerful tool for investigating basic questions about the regulation of tumor cell energy metabolism and viability.

The early changes in the energetics of T47D-clone 11 human breast cancer cells, following treatment with adriamycin and several other anti-cancer drugs were characterized by 31P- and 13C-NMR spectroscopy. Treatment of the cells with cytotoxic doses of either adriamycin (10(-5) M), daunomycin (10(-5) M) or actinomycin-D (2 x 10(-6) M) induced an immediate increase in the content of the nucleoside triphosphate (NTP) pool. A maximum increase of 30 to 50% was reached 6 to 8 h after treatment, and was followed by a gradual decrease, in accord with the decline in cell number due to cell death. High-performance liquid chromatography measurements indicated that the adriamycin-induced build-up of the NTP pool was mainly due to a specific increase in ATP and GTP. Treatment with cytotoxic doses of cytosine arabinofuranoside (10(-4) M) and cis-platin (10(-4) M) and with the antiestrogen tamoxifen at a dose which inhibited growth (2 x 10(-6) M) did not induce an early increase in the NTP content. Adriamycin and actinomycin-D did not alter significantly the rates of glucose consumption and lactate production via glycolysis during the first 4 to 8 h of treatment. Both drug, however, caused during this time interval a 50% inhibition in the rate of glutamate synthesis via the Krebs cycle. Complementary flow cytometry studies have indicated that within 4 h of treatment with either adriamycin or actinomycin-D there is no detectable change in cell cycle distribution. Treatment for longer time periods indicated that each drug affects the cell cycle distribution in a different manner. Thus, the early increase in NTP can not be associated with a specific cell cycle distribution. The results suggest therefore that drugs of the anthracycline and actinomycin type exert a similar specific and early metabolic induction which may affect the energy state of the cells. This induction may relate to the cytotoxic mechanism and could potentially serve as an early marker for response to treatment.

The effects of 17 beta-estradiol treatment versus tamoxifen on the metabolism of human breast cancer T47D-clone 11 cells were studied by noninvasive 31P and 13C nuclear magnetic resonance techniques. 31P nuclear magnetic resonance spectra revealed differences between estrogen and tamoxifen treated cells. The steady state content of phosphorylcholine and of the nucleoside diphosphates was higher in the tamoxifen treated cells by 33 and 140%, respectively, relative to estrogen treated cells. The intracellular pH of 7.2 and the content of the nucleoside triphosphates, Pi, phosphocreatine, glycerolphosphorylcholine, and glycerolphosphorylethanolamine and uridine diphosphoglucose remained the same in both treatments. Glucose utilization and subsequent lactate, glutamate, alanine, and glycerol 3-phosphate synthesis were monitored on line following administration of specifically labeled [13C]glucose. In estrogen treated cells the rate of lactate production via glycolysis was 560 fmol/cell/h and the initial rate of 13C labeling of the glutamate pool via the Krebs cycle was 6.8 fmol/cell/h. In the tamoxifen treated cells these rates were 2-fold lower, at 250 and 2.9 fmol/cell/h for lactate and glutamate labeling, respectively. In estrogen treated cells, the calculated content of glutamate (19 fmol/cell), alanine (11 fmol/cell), and glycerol 3-phosphate (8 fmol/cell) was higher than in tamoxifen treated cells, where only glutamate labeling was detected (13 fmol/cell). The observed differences in the in vivo kinetics of glucose metabolism may provide a sensitive measure for detecting the response of human breast cancer cells to estrogen versus tamoxifen treatments.

The scores on 22 items of the Purdue Perceptual-motor Survey, administered to 567 normal school children of mean IQ 103 and mean age of 116 mo., were subjected to R-factor analysis by the method of principal factoring with orthogonal varimax rotation. The analysis led to the definition of 10 factors: J, ocular control; K, dynamic balance; L, visual-motor fluency; M, chalkboard; N, unilateral-bilateral coordination; O, upper-lower body differentiation; P, form perception; Q, developmental maturation; R, sex; and S, spatial body perception. The results provide cross-validation for previous studies on the perceptual-motor attributes of normal school children.

The metabolic states and the uptake and metabolism of [1-(13)C]glucose, [2-(13)C]glycine, and [(15)N]glycine in intact Nicotiana tabacum L. (cv Xanthi) mesophyll protoplasts were measured by (13)C and (15)N nuclear magnetic resonance spectroscopy. Changes in the concentration of metabolites during the first two days of culture in darkness were followed. Protoplasts isolated in 0.55 molar mannitol medium showed a drop in the concentration of all the intracellular metabolites during the first 28 hours of culture. Uptake of glucose and synthesis of glucose-derived metabolites were observed, indicating activity of glycolysis and the tricarboxylic acid cycle. Addition of glycine caused the accumulation of serine in dark cultured protoplasts, via the photorespiratory pathway. Glutamate dehydrogenase and glutamine synthetase activities in photorespiratory NH(4) (+) assimilation were observed. Glucose uptake and metabolism and cell division were inhibited by 3 millimolar glycine, suggesting that the accumulating serine or the release of ammonia during serine synthesis had toxic effects in this system.

The 4-fluoro analogue of 17 beta-estradiol was investigated for estrogenic action in rats by determination of uterine hyperemia and [3H]uridine incorporation and was found to be an active estrogen. Antitumor activity of this analogue was demonstrated against 7,12-dimethylbenz[a]anthracene (DMBA) induced rat mammary adenocarcinoma. Its 13C NMR spectrum was determined, and all signals were assigned. A detailed X-ray diffraction investigation of 4-fluoro-17 beta-estradiol was carried out, and it crystallized in triclinic space group P1 with two steroids and one methanol in a unit cell of a = 7.367 (1), b = 9.363 (6), c = 12.531 (1) A, alpha = 89.31 (3), beta = 93.38 (1), gamma = 109.62 (3) degrees, V = 812.8 A3, and Z = 2. The structure was solved and refined to an R index of 0.062 using 3045 reflections measured on an automated diffractometer. The oxygen atoms at C(3) and C(17) at either ends link the molecules together in a head to tail fashion. The hydroxy groups of the solvent molecules also take part in linking the molecules sideways through the hydroxy groups.

The concentration of phosphates and the kinetics of phosphate transfer reactions were measured in the human breast cancer cell line, T47D, using 31P-NMR spectroscopy. The cells were embedded in agarose filaments and perifused with oxygenated medium during the NMR measurements. The following phosphates were identified in spectra of perifused cells and of cell extracts: phosphorylcholine (PC), phosphorylethanolamine (PE), the glycerol derivatives of PC and PE, inorganic phosphate (Pi), phosphocreatine (PCr), nucleoside triphosphate (primarily ATP) and uridine diphosphate glucose. The rates of the transfers: PC----gamma ATP (0.2 mM/s), Pi----gamma ATP (0.2 mM/s) and the conversion beta ATP----beta ADP (1.3 mM/s) were determined from analysis of data obtained in steady-state saturation transfer and inversion recovery experiments. Data from spectrophotometric assays of the specific activity of creatine kinase (approx. 0.1 mumol/min per mg protein) and adenylate kinase (approx. 0.4 mumol/min per mg protein) suggest that the beta ATP----beta ADP rate is dominated by the latter reaction. The ratio between the rate of ATP synthesis from Pi and the rate of consumption of oxygen atoms (4 X 10(-3) mM/s) was approx. 50. This high value and preliminary measurements of the rate of lactate production from glucose, indicated that aerobic glycolysis is the main pathway of ATP synthesis.