Artificial tumor model suitable for monitoring ... - Wiley Online Library

Magnetic Resonance in Medicine 54:67–78 (2005)

Artificial Tumor Model Suitable for Monitoring 31P and 13 C NMR Spectroscopic Changes During ChemotherapyInduced Apoptosis in Human Glioma Cells Anthony Mancuso,1* Aizhi Zhu,1 Nancy J. Beardsley,1 Jerry D. Glickson,1 Suzanne Wehrli,2 and Stephen Pickup1 often contain cancerous cells (3). Very few clinical studies of therapeutic response of gliomas have been conducted with other nuclei, such as 31P and 13C, because of their low gyromagnetic ratios (4). However, with the clinical adoption of higher-field instruments (ⱖ3 Tesla) and phasedarray surface coils, studies with these nuclei will likely increase since in vivo and in vitro models have demonstrated that they provide metabolic information that cannot be detected with 1H spectroscopy (5,6). Exploratory clinical studies to identify useful metabolic markers for response to therapy would be very costly to conduct and constrained by ethical considerations. Far greater experimental flexibility can be achieved with artificial tumors that contain cells derived from human biopsied tissue. Such tumors can be perfused inside the sensitive volume of a spectrometer and be subjected to experimental treatments under well controlled conditions. The advantage of this approach over xenographic implantation of human cells into immune-deficient animals is that vascular and multiple organ complications are eliminated. However, in order to overcome the signal-to-noise ratio (SNR) limitations inherent in NMR, the cell density in the artificial tumor must be very high. This requirement has substantially hampered the widespread use of noninvasive NMR spectroscopy for in vitro studies of cells. Many approaches to culturing cells inside an NMR spectrometer have been examined experimentally (7). The ideal method for this purpose would provide a homogeneous environment so that an entire population of cells would be sustained in a single metabolic state. Heterogeneities can be minimized by using convective metabolite transport whenever possible and minimizing diffusion distances within the cell mass. An additional design consideration in monitoring therapeutic response of cultured cancer cells is that most chemotherapeutics induce apoptosis (8), and early in the apoptotic process anchoragedependent cells detach from their growth surfaces (9). Therefore, in order to study metabolic changes associated with apoptosis, some form of entrapment is necessary. Hydrogels (agarose, Matrigel™, or calcium alginate), cast as either beads or filaments, have been used to retain cells in standard NMR tubes during perfusion with oxygenated culture medium (7). Agarose has been used to entrap a number of different anchorage-dependent and -independent cell types in long cylindrical filaments approximately 500 ␮m in diameter (maximum diffusion distance ⫽ 250 ␮m) (10). However, agarose is not a suitable immobilization support for some anchorage-dependent cell types because it lacks the physical structure necessary for proper cellular attachment (10). Without proper attachment, nor-

An artificial tumor method was developed to study cells inside the sensitive volume of an NMR spectrometer during growth and apoptosis. The tumor was composed of a 50:50 mixture of tightly packed porous-collagen and nonporous-polystyrene microspheres. The porous collagen served as a growth surface for the tumor cells, and the nonporous polystyrene served as a structural support to limit compression of the packed bed during perfusion. The microspheres were held between two porous polyethylene discs that were tightly sealed inside the NMR perfusion chamber. The new method was evaluated with two cell types: a mouse mammary tumor line (EMT6/SF) and a human glioma line (SF188). The results indicate that for both lines, ⬃109 metabolically active cells could be sustained for at least 1 week in the 12-cm3 artificial tumor. Further, cells undergoing chemotherapy-induced apoptosis (which is known to cause detachment of cells from their surroundings) were retained in the artificial tumor. In preliminary 31P NMR studies, glioma cells treated with temozolomide (TMZ) exhibited reduced phosphocholine (PCh) levels relative to glycerophosphocholine (GPC) and diphosphodiester (DPDE) levels. They also exhibited sharply reduced oxygen consumption and TCA cycle 13C labeling, while they retained glycolytic activity. These metabolic changes are consistent with those that would be expected during mitochondrially-mediated apoptosis. Magn Reson Med 54:67–78, 2005. © 2005 Wiley-Liss, Inc. Key words: apoptosis; glioma; temozolomide;

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In current clinical practice, the response of brain tumors to therapy is generally monitored with imaging methods that detect morphological and vascular changes. Such changes occur slowly, and often many weeks pass before they can be quantified. Magnetic resonance spectroscopy (MRS) can detect metabolic changes within tumors that occur at much earlier time points, and thus may be very valuable in the clinical management of cancer (1,2). Single-voxel 1H MRS was recently adopted for clinical use in monitoring changes in brain tumors during therapy (3). Spatiallyresolved 1H MRS may prove to be even more useful for this purpose, since regions outside the MRI-detected lesion

1 Department of Radiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA. 2 NMR Core Facility, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. Grant sponsor: NIH; Grant numbers: R21-CA84380; 2RO1-CA51935. *Correspondence to: Anthony Mancuso, Ph.D., Molecular Imaging Laboratory, Department of Radiology/6021, University of Pennsylvania, B6 Blockley Hall, 423 Guardian Drive, Philadelphia, PA 19104-6021. E-mail: [email protected] Received 10 September 2004; revised 17 January 2005; accepted 14 February 2005. DOI 10.1002/mrm.20545 Published online in Wiley InterScience (www.interscience.wiley.com).

© 2005 Wiley-Liss, Inc.

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mal cellular physiology is disrupted. For example, MDA-MB 231 breast cancer cells do not replicate in agarose. However, they do replicate in gels made of basement membrane proteins, such as Matrigel™ (BD Biosciences, San Jose, CA, USA) (11). Even cells with fastidious surface requirements, such as primary astrocytes and neurons, have been successfully immobilized in Matrigel™ for NMR studies (12). Filaments as small as 300 ␮m in diameter (maximum diffusion distance ⫽ 150 ␮m) have been used successfully. One of the disadvantages of using Matrigel™ is that diffusion of macromolecules, such as transferrin, may be hindered by the densely structured matrix (10). In addition, the total number of cells that can be studied with this method is limited by the difficulties associated with packing hydrogel filaments densely into an NMR tube. Calcium alginate has also been used to entrap many different cell types for NMR studies (13–15). Beads 1 mm in diameter (maximum diffusion distance ⫽ 500 ␮m) are commonly used. As with agarose, cells can be energetically stable for several days, but the gel does not provide a good surface for attachment and replication with some anchorage-dependent lines (16). An alternative to gel entrapment is to immobilize cells in preformed porous microcarriers. Such microcarriers provide a large surface area for growth, but have not been widely used for NMR studies because they are compressible, and when used in a packed bed they can only be perfused at low flow rates. This limits the delivery rate of nutrients, especially oxygen, which is only sparingly soluble in aqueous medium. Porous-collagen microcarriers have been used successfully in loosely packed beds to study neurons and astrocytes at relatively low densities (12). They have also been used to examine the response of radiation-induced fibrosarcoma (RIF-1) cells to 4-hydroxycyclophosphamide (16) and dexamethasone (17). Total cell numbers of ⬃6 ⫻ 107 in a 10-mm NMR tube have been reported (17). Porous microcarriers have been perfused in the extraluminal space of hollow-fiber bioreactors (HFBRs) (18 –20), which can prevent compression because the predominant convective flux is intraluminal. Another advantage of this approach is that the fibers can enhance retention of apoptotic bodies, due to their relatively small pore sizes. A disadvantage of this approach is that the space occupied by the fibers is not available for the microcarriers. In addition, very few HFBRs are currently available commercially, and those that are available are not well suited for NMR studies (7). In this work, a new artificial tumor method that is intended to retain cells undergoing apoptosis is described. Cells were immobilized in a tightly packed bed that was composed of a 50:50 (by volume) mixture of porous-collagen microcarriers and nonporous-polystyrene microspheres. The purpose of the nonporous microspheres was to prevent compression of the packed bed at moderately high perfusion rates. The capacity of the new method to support tumor cells with high metabolic rates was evaluated with a rapidly-growing mouse mammary tumor cell line (EMT6/SF). The results were compared with those obtained from the same cell line grown on nonporous microcarriers (21). To evaluate the retention of apoptotic cells within the artificial tumor, human glioma cells (SF188) grown inside collagen microcarriers were treated

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with temozolomide (TMZ; a newly approved DNA-alkylating agent for treating malignant brain tumors). The primary hypotheses were that 1) tumor cells could be grown to a high density inside the artificial tumor, and 2) TMZtreated glioma cells would remain entrapped in the porous collagen due to the convoluted structure of the pores. MATERIALS AND METHODS Cell Culture and Immobilization Cell lines were maintained in 25, 75, or 150 cm2 T-flasks (Fisher Scientific, Pittsburgh, PA, USA) that were incubated at 37°C in a 5% CO2, 95% air atmosphere. EMT6/SF cells (murine mammary tumor, obtained from Dr. Zaver Bhujwalla of the Johns Hopkins University) were cultured in Earle’s minimal essential medium (EMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 2 mM glutamine, 100 U/ml penicillin-G, and 100 ␮g/ml streptomycin sulfate. The doubling time for this organism in T-flasks was approximately 11 hr, and stationary phase cells had a mean diameter of 19 ␮m (21). SF188 cells (human glioma, grade 4, obtained from the UCSF Brain Tumor Research Center, San Francisco, CA, USA) were grown in Dulbecco’s modified eagle’s medium (DMEM), which contained 25 mM glucose, 6 mM glutamine, 50 ␮g/ml gentamicin sulfate, and 10% FBS. The growth rate and cell diameter for SF188 were determined with a Coulter Counter (model Z1; Beckman Coulter, Miami, FL, USA) that was equipped with a 100-␮m orifice. The doubling time for this cell line in T-flasks was approximately 28 hr, and stationary phase cells had a mean diameter of 17 ␮m. TMZ (3,4-dihydro-3methyl- 4-oxoimidazo[5,1-d]-as-tetrazine-8-carboxamide) was a generous gift from the Schering-Plough Corporation (Kenilworth, NJ, USA). The porous-collagen microcarriers (Cultispher威; Hyclone, Logan, UT, USA) had a mean diameter of 200 ␮m when they were fully hydrated. They were prepared for inoculation by the addition of 150 mg of the dry material to 30 ml of phosphate-buffered saline (PBS) in 1-liter Teflon威 or polypropylene bottles (Fisher Scientific). The bottles were autoclaved for 25 min at 121°C, and after the PBS was removed the microcarriers were washed with 30 ml of medium. Subsequently, they were resuspended in 30 ml of supplemented medium prior to inoculation. For the NMR experiments, cells grown to 50 – 80% confluency were detached from 150 cm2 T-flasks with 0.25% trypsin/0.53 mM EDTA 䡠 4Na. The microcarrier-containing bottles were inoculated with the cell suspension (⬃107 cells/wet-g) and then placed horizontally in a CO2 incubator. The cells were allowed to grow in the microcarriers without agitation for 24 –30 hr. For EMT6/SF, the microcarriers were transferred to a 2-liter spinner flask for 5 additional days of growth. The medium in the spinner flasks was DMEM, which was supplemented with the same components that were normally used with EMEM. After the first 3 days of growth, the medium was changed at 24-hr intervals. Growth in the microcarriers was monitored with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Observation under an inverted light microscope indicated that the cells filled much of the porous structure.

Growth and Apoptosis in an Artificial Tumor

The same methods as those described above were used to culture SF188 cells; however, instead of a spinner flask, the inoculated microcarriers were cultured inside stationary 1-L polypropylene bottles (Fisher Scientific) for 9 –11 days. The bottles were incubated on their sides with 40 ml of supplemented DMEM, and approximately 120 cm2 was available for surface aeration. The medium was replaced once every 24 hr. MTT staining indicated that the cells initially grew as distinct colonies in the beads, and it took approximately 2 weeks to fill the pores. Total metabolic rates (combined 12C and 13C) were calculated from perfusate glucose and lactate levels that were determined with an immobilized enzyme analyzer (model 2300; YSI Life Sciences, Yellow Springs, OH, USA). Detection of Apoptotic Cells in Collagen Microcarriers With Confocal Microscopy SF188 cells grown in collagen microcarriers for 9 days were treated with either 160 ␮g/ml TMZ or 2 ␮g/ml actinomycin-D, an agent that rapidly induces apoptosis in nearly all cell types (positive control). One to three days after treatment, the cells within the microcarriers were fixed with 4% formaldehyde in PBS and stored in 70/30% ethanol/water at –20°C prior to analysis. Apoptosis was detected with a TUNEL assay kit (ApoAlert, BD Biosciences Clontech, Palo Alto, CA), which specifically labels DNA cleaved by endonucleases. Terminal deoxynucleotidyl transferase (TdT) was used to catalyze the incorporation of fluorescein-deoxyuridine triphosphate (FldUTP) at the free 3⬘-hydroxyl ends of fragmented DNA. Propidium iodide (PI), a nonspecific DNA intercalating agent, was used to detect total DNA levels. The labeled cells within the microcarriers were detected with a BioRad MRC 1024ES confocal microscope system (Biorad, Hercules, CA, USA). The slice thickness for the confocal images was 10 ␮m. Artificial Tumor Perfusion Apparatus The microcarriers were held in a screw-cap 20-mm NMR tube (Wilmad Glass, Buena, NJ, USA) between two 6-mmthick porous polyethylene discs (mean pore size ⫽ 80 ␮m diameter) that were machined in our laboratory. The distance between the two discs was 47 mm. FEP Teflon威 tubing (1.6 mm I.D. ⫻ 3.2 mm O.D.; Cole Parmer Vernon Hills, IL, USA) was used to introduce medium below the bottom disc and remove medium from above the top disc. A similar line was used to introduce the microcarriers into the volume between the filters. A fourth Teflon威 line, which was sealed at the bottom with silicone glue, contained a solution of phenol phosphonic acid (PPA; 0.6 M in 1M NaOH), which was used to determine the 90°-pulse time. Culture medium was circulated through the microcarrier bed with a peristaltic pump (Masterflex, Cole-Parmer) at a flow rate of 12 ml/min. Oxygen consumption rates were determined continuously with polarographic probes (Mettler-Toledo, Columbus, OH, USA) located in the inlet and outlet perfusion lines (22). The pH of the culture medium was monitored with an electrode (Mettler-Toledo) and was controlled at 7.25 ⫾ 0.1 by adjusting the CO2

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level in a gas exchange bottle (system lung). The temperature of the medium entering the NMR tube was controlled at 37°C. All probes were interfaced to a laptop computer equipped with a PCMCIA analog-to-digital interface card (model 1200; National Instruments, Austin, TX, USA). The computer was used to record oxygen, pH, and temperature, and to continuously calculate the oxygen consumption rate. During active cell growth, fresh medium was fed to and spent medium was removed from the system with a peristaltic pump (Minipuls 2; Gilson, Middleton, WI, USA). For 13C experiments, feed and product removal were discontinued and the system was operated as a closed loop. Further details regarding this system were described previously (21). The collagen microcarriers were somewhat compressible. At the perfusion rates necessary to meet cellular oxygen requirements, compression of the microcarrier bed was excessive. The pressure drop through the compressed bed was very high, and the upstream liquid pressure was sufficient to rupture the silicon tubing in the gas exchange bottle. Therefore, the collagen microcarriers were mixed with nearly incompressible polystyrene microspheres (250 ␮m diameter; Fisher Scientific) at a 50:50 volume ratio before they were used in the NMR tube. To ensure NMR spectral quality, the mixture was carefully packed in the NMR tube, and no liquid gaps or gas bubbles were left in the packing. NMR Spectroscopy NMR spectra were acquired with 400 MHz systems from either Bruker (Avance) or Varian (Inova). Both 9.4 Tesla magnets had 89-mm-diameter vertical bores, with 72 mm I.D. room temperature shims. A broadband 20-mm liquids probe (from either Bruker or Varian) was used to acquire 31 P and NOE-enhanced 13C spectra. 31P spectra were acquired with 60° pulses, a TR of 1000 ms, 4096 points, 1200 scans, and a spectral width of 15000 Hz. Free induction decays (FIDs) were zero-filled to 8192 points and apodized with exponential multiplication. 15 Hz line-broadening was used for quantitation of all resonances except phosphocholine (PCh). Because of the limited spectral resolution in the phosphomonoester region, the spectra were processed with 4 Hz line-broadening to quantify PCh. The signal intensities of the Fourier-transformed data were determined with the line-fitting subroutine of Nuts NMR (Acorn NMR, Freemont, CA, USA). T1 relaxation times for phosphorous metabolites were determined by progressive saturation (see Table 1). To calculate absolute concentrations, metabolite intensities were compared with the fully relaxed inorganic phosphate intensity, which was quantified while the NMR tube contained culture medium and no microcarriers. The 31P signal from the PPA of the reference capillary demonstrated that the 90° pulse time for the 31P coil was unchanged by the addition of the microcarriers. Hence, metabolite concentrations (mmol/L of NMR detected volume) could be calculated without corrections for coil sensitivity with the relationship: [X]⫽ {(1⫺cos␪e ⫺Tr/T 1)/sin␪ (1⫺e ⫺Tr/T 1)}{Sx,␪[0.94mM]/(SPi,0)}. In this equation [X] is the concentration of a metabolite, ␪ is the pulse angle for metabolite X, Tr is the relaxation delay, T1 is the spin-lattice relaxation time, Sx,␪ is the

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Table 1 Spin-Lattice (T1) Relaxation Times for Cells

RESULTS 31

P Metabolites in SF188

Metabolite

ppm

T1 (s)

PCh P1 (medium) GPC PCr ␥-NTP ␣-NTP DPDE-1 DPDE-2 ␤-NTP

6.30 5.07 2.98 0.00 ⫺2.45 ⫺7.51 ⫺8.29 ⫺9.87 ⫺16.09

2.34 ⫾ 0.25 8.50 ⫾ 0.42 4.43 ⫾ 0.39 2.88 ⫾ 0.31 0.50 ⫾ 0.01 0.49 ⫾ 0.01 1.34 ⫾ 0.04 1.35 ⫾ 0.11 0.42 ⫾ 0.02

signal intensity for metabolite X, 0.94 mM is the Pi concentration in the culture medium, and SPi,0 is the fully relaxed signal intensity following a 90°pulse for the inorganic phosphate. Cell numbers were estimated from 31P ␤-NTP levels as described previously (22). 13 C spectra were acquired with 60° pulses, a TR of 1200 ms, 4096 points, 900 scans, and a spectral width of 25000 Hz. Bilevel WALTZ-16 decoupling was used to produce nuclear Overhauser effect (NOE) enhancement and collapse 1H splittings. The FIDs were zero-filled to 16384 points and apodized with exponential multiplication (3 Hz linebroadening). The cells were initially fed DMEM with 10 mM unenriched glucose while background spectra were acquired. Subsequently, the unenriched medium was completely replaced with DMEM containing 10 mM [1,6-13C2] glucose. Metabolite concentrations were determined by comparing the peak areas with the peak area for the natural abundance 13C resonances of the N-[2-hydroxyethyl] piperazine- N⬘-[2-ethanesulfonic acid] (HEPES) buffer in the medium. The concentrations were corrected for T1 relaxation and NOE (21). Distortionless enhancement by polarization transfer (DEPT) was used to determine the functional groups for resonances in 13C spectra to confirm assignments. Glucose and lactate 13C metabolic rates were determined from spectra of the artificial tumor. The rates were only an approximation because the intra- and extracellular concentrations were not determined independently. However, any error introduced by this approach was likely small, since the cells occupied only a small fraction of the total NMR detectable volume.

Growth of EMT6/SF Murine Mammary Tumor Cells in Porous Collagen Initial studies were conducted with EMT6/SF cells to allow direct comparison of the results of this work with those we reported previously for the same cell line on nonporous-collagen-coated microcarriers (21). These cells have high oxygen consumption rates and exhibit complete TCA cycle activity. A typical 31P spectrum obtained for EMT6/SF cells in the artificial tumor is shown in Fig. 1. This spectrum was obtained in only 5 min. The SNR and (SNR)/T1/2 on the ␤-NTP resonance were 33:1 and 14.8 min–1/2, respectively. The high SNR was a result of the high cell number and the narrow linewidths. The intrinsic linewidth for uncoupled phosphorous metabolites (e.g., phosphocreatine (PCr)) was consistently less than 15 Hz (0.09 ppm). Resonances were also observed for ␣ and ␥-NTP, phosphomonoesters (including PCh), inorganic phosphate, phosphodiesters (including glycerophosphocholine (GPC)), PCr, and diphosphodiesters (DPDEs). The total NTP concentration in the NMR tube was ⬃1.16 mM, which corresponds to a total cell number of ⬃7.4 ⫻ 108. The oxygen consumption rate was high, at ⬃0.15 mmol/hr. All of these values are similar to those reported previously for EMT6/SF cells grown on nonporous microcarriers (21). To examine glycolytic and TCA cycle activity, the cells were perfused with medium containing 10 mM [1,613 C2]glucose for 4.5 hr. A 30-min carbon spectrum acquired at the maximum extent of labeling is shown in Fig. 2. The pathways by which label is transferred to metabolic intermediates were detailed previously (23). Both C-1 and C-6 of glucose are transferred to C-3 of pyruvate, which should be nearly 100% labeled. The only 12C for this carbon would be derived from the pentose phosphate shunt and the malate shunt, both of which would contain labeled and unlabeled carbon. In the spectrum, strong labeling was detected in C-3 lactate, C-3 alanine, and C-4 glutamate. Moderate labeling was detected for C-3 pyruvate, and C-3 and C-2 glutamate, and low levels were detected in C-2 and C-3 of aspartate. The C-3 glutamate resonance was a triplet composed of a doublet associated

Statistical Analysis Reaction rates were assumed to be zero-order and were determined by linear regression (Excel, Microsoft, Redmond, WA, USA) from concentration temporal profiles. The rates are reported as mean ⫾ standard error (SE). Nonlinear regressions to determine the 31PT1 relaxation times were performed with Sigmaplot 5.0 (Systat Software, Point Richmond, CA, USA). Statistical differences were evaluated with a Student’s t-test, and values of P ⬍ 0.05 were assumed to be significant. The SNR results from this work were compared with published results on an (SNR)/T1/2 basis, because noise in NMR spectroscopy increases with time1/2. The smoothed curves shown in the data graphs are not regression curves, but were drawn by hand (Powerpoint, Microsoft) to demonstrate general trends in the data.

FIG. 1. A typical 31P NMR spectrum of EMT6/SF cells grown in porous-collage microcarriers. This spectrum represents 300 scans that were acquired in just 5 min. The SNR on the ␤-NTP resonance was 33:1.

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FIG. 2. A typical 13C spectrum of metabolically active EMT6/SF cells during infusion of [1,6-13C2]glucose. The top spectra are expansions over the ranges of 26 –38 and 52– 58 ppm, and were processed with 5-Hz Gaussian apodization. Gluc ⫽ glucose; lac ⫽ lactate; glut ⫽ glutamate; asp ⫽ aspartate; F1,6DP ⫽ fructose 1,6-diphosphate; F6P ⫽ fructose-6-phosphate; H ⫽ natural abundance 13C in HEPES; Std ⫽ natural abundance 13C in the ethanol standard (in a capillary, which was used to optimize the pulse width for WALTZ16 decoupling). A background resonance was detected at 27.3 ppm, which overlapped with the pyruvate-3 resonance. A number of other small background resonances associated with natural abundance 13C in the collagen microcarriers, the culture medium, and the cells were also detected.

with the 13C-13C coupled carbons of [3,4-13C2]glutamate and singly labeled [3-13C]glutamate. A similar triplet was detected in our previous work with EMT6/SF (21). [3,413 C2]glutamate is derived from [3,4-13C2]citrate, which is formed by citrate synthase from [2-13C]acetate and [2-13C]oxaloacetate in the TCA cycle. Its presence alone confirms that complete TCA cycle activity exists in EMT6/SF cells. Label in C-2 of aspartate, which is in equilibrium with oxaloacetate, confirms that label was present in C-2 of oxaloacetate. A separate analysis of the extracellular medium with 13C NMR demonstrated that the labeled lactate, pyruvate, and alanine were both intra- and extracellular, but aspartate and glutamate were limited to the intracellular space. The total rates (labeled and unlabeled) for glucose consumption and lactate formation are compared with those observed with nonporous microcarriers (21) in Table 2. Also shown are the rates for 13C glucose and lactate determined with NMR. The time courses for labeling in C-4, C-3, and C-2 of glutamate are shown in Fig. 3. The initial rates of labeling were 5.4 ⫾ 0.2, 2.1 ⫾ 0.10, and 1.8 ⫾ 0.2 ␮mol/hr, respectively. For a 30-min acquisition, the maximum SNR for C-4 and C-2 of glutamate was 77:1, 14:1. In general, these results are comparable to those reported for EMT6/SF cells grown on nonporous microcarriers (21). However, the difference between the lactate formation rates (determined enzymatically) with the two different microcarriers was relatively large (24%). The reason for this difference was not apparent.

31 P NMR Spectroscopy of Untreated Human Glioma Cell Growth

The porous-collagen microcarriers were inoculated with SF188 cells (⬃1 ⫻ 107/wet-gm microcarrier) and cultured in an incubator for approximately 10 days. Subsequently, they were perfused in an NMR tube while 31P spectra were acquired. A typical 31P spectrum acquired after cell levels had reached a steady state is shown in Fig. 4a. The detected resonances are the same as those for EMT6/SF, but the relative intensities are somewhat different. A second spectrum acquired 72 hr later (Fig. 4b) was very similar, indicating that the culture was very stable. The full time courses for five of the major resonances (PCh at 6.3 ppm, GPC at 2.9 ppm, PCr at 0 ppm, DPDE-2 at –9.9 ppm, and ␤-nucleoside triphosphates (␤-NTP) at –17 ppm) are shown in Fig. 5a and b. The ␤-NTP resonance increased linearly by approximately threefold over the first 60 hr of the experiment. The NTP level peaked at ⬃1.15 mM and subsequently leveled off at ⬃1.0 mM. These values correspond to 1.0 ⫻ 109 and 8.9 ⫻ 108 cells, respectively, for a mean diameter of 17 ␮m. The PCr resonance did not increase as much as the NTP resonance, and declined only slightly over the last 60 hr of the experiment. GPC levels did not increase linearly, and the initial rate (0 –15 hr) was significantly higher than the rate between 20 and 80 hr. The maximum in the GPC level occurred approximately 40 hr after that for beta NTP. Both the second DPDE resonance (DPDE-2) and the PCh resonance also exhibited a

Table 2 Absolute Metabolic Rates for EMT6/SF

Microcarrier

Porous collagen Nonporousa a

Oxygen consumption (mmol/hr) 0.15 ⫾ 0.01 0.17 ⫾ 0.01

[1,6-13C2]

Total

[3-13C]

Total

Glutamate labeling [4-13C] (␮mol/hr)

0.215 ⫾ 0.003 0.230 ⫾ 0.004

0.208 ⫾ 0.005 0.20 ⫾ 0.02

0.318 ⫾ 0.002 0.29 ⫾ 0.01

0.342 ⫾ 0.007 0.26 ⫾ 0.02

5.4 ⫾ 0.2 4.6 ⫾ 0.1

Glucose consumption (mmol/hr)

Data are for experiments described previously (21).

Lactate formation (mmol/hr)

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FIG. 3. Time courses for labeling in glutamate during infusion with [1,613 C2]glucose. Label in C-4 of glutamate precedes and is much stronger than C-3 and C-2, which suggests that fluxes into the TCA cycle other than that through pyruvate dehydrogenase are relatively high. Replacement of the [1,6-13C2]glucose medium with medium containing unenriched glucose produced the marked drop in labeling at 4 hr.

very high rate initially followed by a slower rate, with peak levels occurring at approximately the same time as that for ␤-NTP. A preliminary assessment of the extent of oxygen limitation in the cell mass was conducted with a culture FIG. 5. 31P metabolite concentrations during untreated growth over the course of a 6-day experiment. NTP ⫽ nucleoside triphosphates; PCr ⫽ phosphocreatine; PCh ⫽ phosphocholine; GPC ⫽ glycerophosphocholine; and DPDE-2 ⫽ diphosphodiesters (–9.87 ppm).

grown to 8.0 ⫻ 108 cells. When the oxygen concentration in the inlet medium was gradually reduced from 0.26 mM to 0.06 mM (130 to 30% of air saturation) over a 4-hr period, no reduction was observed in either the NTP or the PCr level. These results indicate that excess oxygen was available in the perfusate to sustain the SF188 cells. Response of Human Glioma Cells to a Single Dose of TMZ

FIG. 4. 31P response of SF188 cells to TMZ. a: Spectrum acquired for untreated cells grown inside the spectrometer for 58 hr. b: The 31 P spectrum is relatively unchanged 72 hr later. c: In a parallel experiment, cells grown for 20 hr inside the spectrometer were treated with a single dose of TMZ (160 ␮g/ml). The spectrum shown was acquired 100 hr after treatment. d: Results similar to those shown in spectrum c were observed for a culture treated twice with 125 ␮g/ml of TMZ. The spectrum shown was acquired 90 hr after the second treatment.

A culture grown in microcarriers to a level of 4 ⫻ 108 cells was treated with 160 ␮g/ml of TMZ. The 31P NMR results are shown in Figs. 4c and 6. Immediately following treatment, the NTP and PCr levels continued to increase (Fig. 6). Approximately 40 hr later, both NTP and PCr began to decline. PCh was the first metabolite to show a marked reduction, which began 25 hr after treatment. Reductions in GPC and DPDE-2 were observed ⬃30 hr later. The maximal values for ␤-NTP, PCr, and DPDE-2 were somewhat lower than those for untreated growth. GPC and PCh levels both peaked at levels similar to those for untreated growth (Fig. 5b). PCr reached undetectable levels 100 hr after treatment, and PCh also declined to very low levels. The percentage reductions (from the maximum to the level at 140 hr) were largest for PCr (100%) and PCh (87%), and significantly smaller reductions were observed for GPC (39%) and DPDE-2 (57%). The spectrum acquired 100 hr after treatment (Fig. 4c) shows that the reductions for both

Growth and Apoptosis in an Artificial Tumor

FIG. 6. Effects of a single treatment with TMZ. Cells grown in the NMR tube to a level of ⬃4 ⫻ 108 cells were treated once with 160 ␮g/ml TMZ at the time indicated by the arrow. The dashed gray line indicates the time point when oxygen consumption dropped below detectable levels.

of the DPDE resonances were similar, as were the reductions for all three of the NTP resonances. The confocal microscopy results for detection of apoptosis are shown in Fig. 7. The microcarriers were treated with either TMZ (160 ␮g/ml) or actinomycin-D (2 ␮g/ml). The images are from a single 10-␮m-thick slice through each microcarrier. The bottom row of images shows PI labeling (total DNA), and the middle row shows Fl labeling (apoptotically cleaved DNA). The top row shows the combined results of the middle and bottom rows; nonapoptotic cells are red and apoptotic cells are yellow. The effects of actinomycin-D are shown in the first column, and the effects of TMZ are shown in the second column. For actinomycin-D, essentially all of the cells labeled with PI were also labeled with fluorescein 24 hr after treatment. This result indicates that the retention of apoptotic cells in the microcarriers was excellent. The rapid development of apoptosis was expected since this agent acts by inhibiting RNA synthesis and does not require DNA synthesis. With TMZ treatment, very few apoptotic cells were detected within the first 24 hr (data not shown), but many were detected within 72 hr. Thus, as with most alkylating agents, DNA synthesis must occur before apoptosis is initiated. The microcarriers in the last column of the figure were untreated, and no apoptotic cells were detected. Effects of Repeated Doses of TMZ In normal clinical use, TMZ is administered once a day for 5 successive days. To examine the effects of repeated

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doses, a culture was grown to a much higher cell level (9 ⫻ 108) prior to treatment. Two 125 ␮g/ml doses, with a delay of 24 hr between treatments, were administered. The full time series for the 31P metabolic changes are shown in Fig. 8. A small increase in the NTP level was observed after the first treatment. The exact time course for the increase was not determined, because 31P spectra were not acquired until approximately 4 hr after treatment. Following a small decline, a much larger increase in NTP was observed after the second treatment. In separate studies, detailed examinations of the acute effects of TMZ have demonstrated that increases in NTP occur within minutes of each administration of the drug (24). This will be described in detail in a forthcoming manuscript. Within 6 hr after the second treatment, the NTP level began to decline rapidly. PCr levels were not increased acutely by TMZ, but did decline 25 hr after the second treatment. The GPC level increased briefly after the first treatment, and the maximum level was slightly lower than that in the untreated control experiment. The GPC level began to decline before the second treatment. PCh levels dropped slowly at first, but 60 hr after the second treatment they dropped very rapidly. The DPDE-2 level dropped more slowly than the level for any other metabolite. The percentage reductions (from the maximum to the level at 175 hr) were largest for PCr (100%) and PCh (83%). Significantly smaller reductions were observed for GPC (58%) and DPDE-2 (54%). The final 31 P spectrum acquired 90 hr after the second treatment (Fig. 4d) was very similar to that for the lower density culture that was treated once with TMZ (Fig. 4c). Changes in Metabolic Fluxes Following Treatment of SF188 Cells With TMZ Metabolic rates for TMZ-treated and untreated SF188 cells were determined with 13C NMR spectroscopy during infusion of [1,6-13C2] glucose. The detected resonances were similar to those observed for EMT6/SF, with the exception that C-2 and C-3 of aspartate were not observed (spectrum not shown). The C-3 resonance for glutamate was a triplet due to the presence of [3,4-13C2]glutamate. In addition, shoulders were present on the C-4 glutamate resonance due to the same 13C-13C couplings. These shoulders were not detected with EMT6/SF because the spectral resolution was slightly worse. The absolute metabolic rates determined from the time course of 13C spectra are summarized in Table 3a. The NTP-normalized results are given in Table 3b. Also shown are rates determined with offline glucose and lactate analyses of samples of the perfusate. Oxygen consumption for SF188 cells was moderately high, but not as high as that for EMT6/SF cells on an NTP normalized basis (9.5 vs. 11.4 mmol/mmol NTP-hr). The rate of labeling in C-4 glutamate was also reduced. The yields of lactate from glucose for SF188 were similar to those for EMT6/SF. The final lactate levels observed during the SF188 experiments with [1,6-13C2]glucose ranged from 10 to 15 mM, which is comparable to the values of up to 11 mM that have been reported in clinical studies for malignant gliomas (25). With TMZ treatment, oxygen consumption initially declined in parallel with the reduction in the NTP level. However, for both single and double TMZ treatment experiments, oxygen consumption

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Mancuso et al.

FIG. 7. Confocal microscopy of cells in collagen microcarriers. SF188 cells grown inside microcarriers were stained with PI (total DNA) and fluorescein (TUNEL assay for apoptosis). The image at the top is for a dualpass filter and shows cells that were labeled with both fluorescein and PI. Actinomycin-D treated cells served as a positive control; essentially all cells that were detected were apoptotic. Apoptosis following treatment with TMZ developed more slowly than with actinomycin-D. The negative control microcarriers were not treated with any therapeutic agent, and no apoptosis was observed.

dropped below detectable levels, while a significant amount of NTP remained. With one TMZ treatment, oxygen consumption could not be detected 84 hr after drug administration, while the NTP level was ⬃0.3 mM. With two treatments, oxygen consumption could not be detected 90 hr after the second treatment, while the NTP level was ⬃0.2 mM. Consistent with this observation, 13C labeling in C-4 of glutamate was barely detectable 90 hr after the second TMZ treatment (Table 3). No label was detected in either C-3 or C-2 of glutamate. These results suggest that a marked reduction in mitochondrial function occurred subacutely after TMZ treatment. Glucose consumption and lactate formation per unit of NTP were also reduced, but not nearly to the same extent as oxygen consumption and C-4 glutamate labeling (on a percentage basis). Cell Shedding From the Artificial Tumor

FIG. 8. Response to two doses of TMZ given 24 hr apart. Cells grown to 9 ⫻ 108 were treated with 125 ␮g/ml TMZ twice at the times indicated by the arrows. Oxygen consumption dropped below the detectable limit at the time indicated by the dashed line.

A small percentage of cells were released from both treated and untreated tumors into the perfusate. These cells readily passed through the polyethylene filters in the NMR tube and settled in the perfusate recirculation bottle. They were removed aseptically once daily. For the culture represented by Fig. 5, the loss of metabolically active cells (determined with trypan-blue exclusion and a hemacytometer) per day was 3 (⫾0.6) ⫻ 107 at the end of the NMR experiment ([NTP] ⫽ ⬃1.0 mM). This corresponds to approximately 3% of the total number of living cells present in the tumor. For the TMZ-treated culture represented by Fig. 8, the loss of trypan-blue excluding cells was 2 (⫾0.4) ⫻ 107 at the same time point ([NTP] ⫽ ⬃0.5 mM). The difference between the treated and untreated cultures was not statistically significant. Some of these trypan-blue

Growth and Apoptosis in an Artificial Tumor

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Table 3 Absolute Metabolic and NTP Normalized Rates for SF188 Cells 120 Hours After Therapy TMZ Treatments

Oxygen consumption (mmol/hr)

Glucose consumption (mmol/hr) 13

Lactate formation (mmol/hr) 13

[1,6- C2]

total

[3- C]

total

0.204 ⫾ 0.004 0.031 ⫾ 0.003

0.181 ⫾ 0.003 0.025 ⫾ 0.003

0.309 ⫾ 0.010 0.037 ⫾ 0.003

0.334 ⫾ 0.004 0.053 ⫾ 0.009

19.8 ⫾ 0.4 10 ⫾ 1**

17.6 ⫾ 0.3 8 ⫾ 1**

30 ⫾ 1 12 ⫾ 1**

32.5 ⫾ 0.4 17 ⫾ 3*

Glutamate labeling (␮mol/hr) [4-13C]

a

Absolute metabolic rates 0 0.098 ⫾ 0.005 2 ⬍0.0009 NTP normalized rates 0 9.5 ⫾ 0.5 2 ⬍0.3**

3.3 ⫾ 0.5 0.15 ⫾ 0.02 0.32 ⫾ 0.05 0.049 ⫾ 0.007**

A t-test was used to determine the significance of the observed reduction in metabolic rates associated with TMZ treatment. *P ⬍ 0.05; **P ⬍ 0.005.

excluding cells were smaller than normal and may have been early-phase apoptotic cells released from the outer surface of the microcarriers. Both treated and untreated artificial tumors also shed ⬃2 ⫻ 107 nontrypan excluding cells per day. DISCUSSION Artificial Tumor Function and NMR Sensitivity The most significant findings of this work are that human glioma cells can be grown to very high densities in an NMR-compatible artificial tumor, and that following treatment with TMZ, apoptotic cells are retained in the porouscollagen microcarriers. These microcarriers provide an excellent surface for cell growth, and the polystyrene microspheres limit their compression during perfusion. Total cell numbers as high as ⬃1 ⫻ 109 were observed, which resulted in very high SNR spectra. The retention of cells with endonuclease cleaved DNA (which is known to occur late in apoptosis) demonstrates that the temporal dependence of apoptotic changes can be monitored. This will probably be very important because spectroscopic changes that occur during early apoptosis will likely differ substantially from those that occur late in apoptosis (26). During untreated growth, the EMT6/SF metabolic patterns were very similar to those observed previously with cells grown on the surface of nonporous microcarriers, where diffusion distances are only one to two cell diameters (21). This finding suggests that EMT6/SF metabolism in the porous collagen was not limited by the rate of oxygen diffusion, despite the high oxygen consumption rate of this cell line. Moreover, when the effect of inlet oxygen concentration on a dense SF188 culture was examined, levels as low as 0.06 mM (30% of air saturation) did not affect the levels of any 31P metabolite including PCr (results not shown). The lack of oxygen limitation can be attributed to the relatively short diffusion distances (maximum diffusion distance ⫽ 100 ␮m) and the fact that oxygen can diffuse through the regions of the collagen matrix that do not contain cells. In vivo, the maximum diffusion distance in non-necrotic regions of tumors is also approximately 100 ␮m (27). The 31P (SNR)/T1/2 in this work for the EMT6/SF ␤-NTP resonance was 14.8 min–1/2. This value is nearly identical to the value of 15.2 min–1/2 we observed for the same cell line with collagen-coated nonporous microcarriers (21). It is also much higher than the values observed with most

other cell immobilization methods (21) and is comparable to the values reported for HFBRs (28,29). The only significantly higher ␤-NTP (SNR)/T1/2 was observed with rat glioma cells (C-6) in collagen microcarriers that were perfused in an HFBR (18). The ␤-NTP (SNR)/T1/2 of 21 min– 1/2 observed in this work is likely the highest value ever reported (7). However, an evaluation of the availability of oxygen with this configuration was not presented. For 13C spectroscopy with EMT6/SF, the (SNR)/T1/2 for C-4 and C-2 of glutamate were 14.1 and 2.6 min–1/2, respectively. These values are slightly lower than the values we reported previously (16.5 and 3.3 min–1/2) for EMT6/SF on nonporous microcarriers, but they are substantially higher than those observed with other methods (7). An important factor in producing the high SNR was the narrowness of the spectral lines (as low as 5 Hz without apodization), which reflects the high degree of magnetic-field homogeneity in the packed bed. The artificial tumor used in this work has several other attractive features. First, the tight packing of the microcarriers allows the accurate determination of intracellular diffusion rates with pulsed-gradient NMR spectroscopy. With loosely packed beds, the microcarriers are free to move randomly under the effects of perfusion, which makes it difficult to obtain diffusion measurements. Second, the cells form 3D structures with extensive cell– cell contacts in the pores of the microcarriers. Such cell– cell contacts can make a population of cells more resistant to chemotherapy, just as they are in vivo (30). Third, the cells are protected from fluid shear, which may be significant for cells grown on the outside of nonporous microcarriers. Fourth, the design is flexible enough to allow the examination of reduced oxygen concentrations. The inlet oxygen concentration can be reduced and the perfusion rate can be increased to maintain a low oxygen level along the length of the bed. If necessary, the ratio of solid microspheres to porous microcarriers can be increased to reduce the compressibility of the bed and allow higher perfusion rates. This would also reduce the total cell number, but for some investigations this may be a reasonable trade-off. Finally, the stability of the system allowed the response to therapy to be monitored continuously for many days. Because of problems associated with the long-term use of anesthesia, this would not be possible in xenographic animal models.

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Response of

Mancuso et al. 31

P Metabolites in SF188 to TMZ

The initial response to therapy was consistently an increase in the three NTP resonances, which occurred within minutes and persisted well beyond the end of TMZ treatment (24). The subsequent gradual decline in NTP for the cultures may indicate a loss of metabolically active cell mass in the artificial tumor. However, it may also indicate a reduction in the NTP level per cell. Complementary studies to determine the total volume of cells, as done previously for cells grown in porous-collagen microcarriers (31), would help to clarify this issue. The difference in the delay before the onset of NTP reduction following the first TMZ treatment for the cultures represented by Figs. 6 and 8 may have been due to the differences in growth rate at the time of treatment. The culture for Fig. 8 was much closer to saturation. The subacute reduction in the ratio of PCh to GPC with either single or double TMZ treatment is consistent with findings for many other cell types in response to chemotherapeutics (5,32). High PCh is commonly observed in rapidly proliferating cells, and reductions in PCh are often observed with chemotherapy-induced apoptosis (2). Some types of therapy are known to cause increased GPC (2). However, in this work only the GPC/NTP ratio was increased, and the levels of all the metabolites eventually declined. The data in Figs. 6 and 8 clearly demonstrate that the temporal changes in both PCh and GPC are complex. Further investigation will be necessary to rigorously establish the relationship between these two metabolites in cultured glioma cells during response to therapy. Significance of the

13

C and Oxygen Consumption Findings

Our observation that oxygen consumption declined to undetectable levels following treatment with TMZ may prove to be very important. The marked low levels of 13C labeling in glutamate from [1,6-13C2]glucose are consistent with the lack of oxygen consumption, and this may have direct clinical applications. The cause of the marked reduction in oxygen consumption, even while NTP levels were readily detectable, was not determined in this work. However, in many cell types undergoing apoptosis, cytochrome-c is released from the space between the inner and outer mitochondrial membranes (33). This may interfere with functioning of the electron transport chain and hence impact both oxygen consumption and 13C-glutamate labeling. In a recent study with subcutaneous RIF-1 tumors, 13Clabeling experiments demonstrated that TCA cycle activity was increased 24 hr after treatment with cyclophosphamide (6). Increased NADH levels (detected with 3D optical redox scanning) and decreased tumor oxygen levels were also observed (with an Eppendorf electrode). All of these findings are consistent with increased aerobic metabolism. The mechanism underlying this change was not determined. It may not be directly related to apoptosis, which was not assessed in this study. Cyclophosphamide treatment can cause vascular changes, which may have impacted these results (6). Detection of Response to Therapy in Other Types of Cultured Cells Very few NMR studies of response to therapy have been reported for cells grown in porous microcarriers. In a pre-

vious study of RIF-1 cells in collagen microcarriers, 2 ⫻ 107 cells were sustained in a 10-mm probe, but the exact perfusion configuration was not described (17). Dexamethasone (4 ␮M) treatment of these cells induced only a small amount of apoptosis as detected by a TUNEL assay (17). The only change observed in 31P spectra was a small increase in the PCr-to-NTP ratio. In another study with RIF-1 cells, 4 ⫻ 107 cells were sustained in a loosely packed porous-collagen bed (16). Treatment with 50 ␮M 4-hydroperoxycyclophosphamide (an activated form of cyclophosphamide) caused a growth arrest, as demonstrated by a lack of increase in NTP levels. No other 31P changes were reported, and apoptosis was not assessed. Other studies have demonstrated that NTP levels are acutely increased by chemotherapeutics. A significant acute increase in NTP, but not PCr, was observed when T74D-clone 11 human mammary cells (on nonporous microcarriers) were treated with adriamycin, actinomycin-D, or daunomycin (34). The changes occurred over a time scale of several hours. Cis-platin did not have any effect on NTP levels of T74D-clone 11 cells. However, cis-platin was found to increase NTP levels of human ovarian carcinoma and rat lymphoma cells (embedded in agarose) (35). These findings indicate that not all cell types exhibit the same response to a given chemotherapeutic agent. In contrast to these findings, reduced NTP levels have been reported for 31P cell extracts of L1210 leukemia cells treated with mechlorethamine, and SW620 colorectal cells treated with doxorubicin (26). The reduced NTP was associated with a significant increase in fructose-1,6-diphosphate (F-1,6-DP) 3 hr after treatment in both cell lines. The increase was hypothesized to be caused by inhibition of glycolysis at the level of glyceraldehyde-3-phosphate dehydrogenase, possibly as a consequence of NAD depletion following poly(ADP-ribose) polymerase activation. Large increases in F-1,6-DP were also detected in 31P extract studies of human promyelocytic leukemia cells (HL-60) treated with several drugs that induced apoptosis (20). Significantly smaller changes were observed with Chinese hamster ovary cells. In HL-60 cells, the increase in F1,6-DP levels was associated with reduced NAD(H) levels, which is consistent with the findings of Ronen et al. (26). A 31P increase in F-1,6-DP was not observed in metabolically active SF188 cells treated with TMZ. Further investigations with cell extracts are needed to confirm a lack of increase, since F-1,6-DP resonates in the poorly resolved PME region of the spectrum. Detection of Response to Chemotherapy in Clinical Studies Very few clinical MRS studies of response to therapy have been reported (3,5). However, the results from some case studies are encouraging. In a recent report, the response of a patient with gliomatosis cerebri (a rare infiltrating glioma) to TMZ was described (36). Single-voxel 1H spectroscopy demonstrated that total choline and creatine were markedly reduced with just three monthly treatment cycles (200 mg/m2 for 5 consecutive days each month). The spectral changes preceded a reduction in the extent of edema detected with T2-weighted MRI. Small-scale 31P NMR studies of the response of human brain tumors to

Growth and Apoptosis in an Artificial Tumor

therapy have been inconclusive (4). However, given the increased use of higher-field instruments (ⱖ3T) and surface coil arrays, the feasibility of obtaining useful data with 31P NMR should improve substantially. The use of 13C NMR should also increase with improvements in clinical spectroscopy. An advantage of 13C NMR over 31P NMR is that the magnetization can be detected with greatly enhanced sensitivity through the attached 1H nuclei. With this method of detection, C-4 labeling in glutamate from [1-13C]glucose can be detected in 22 cm3 voxels of healthy human gray matter in ⬃10 min (4.1 Tesla, volume coil) (37). Significantly higher resolution should be possible with the use of [1,6-13C2]glucose and surface coil arrays. This approach may be especially well suited for superficial tumors, such as meningiomas. The detection of glutamate in deeper brain tumors will depend on how dependent the cells are on aerobic metabolism. Clinical 18F-FDG and 15O2 PET studies of grade IV human gliomas have demonstrated that the molar ratio of oxygen consumption to glucose consumption can vary widely (from 0.6 to 4.7) (38). The value observed was at the lower end of this range. Because this value varies widely, preand post-treatment 13C labeling rates will have to be determined before such measurements can be clinically useful. Limitations A limitation of this work is that the collagen microcarriers did not provide an absolute barrier for the retention of cells, and some cells were shed from both treated and untreated tumors. However, in general, the percentage of cells lost per day was very small. In addition, because some of these cells may have been postapoptotic, their loss from the tumor would be desirable since in vivo they would be consumed by phagocytosis in neighboring glioma cells, microglia, and normal astrocytes (39). CONCLUSIONS The results of this work demonstrate that high numbers of anchorage-dependent cancer cells (⬃109) can be sustained in an artificial tumor composed of porous-collagen and nonporous-polystyrene microspheres. The spectral quality was very high and was comparable to that observed for nonporous microcarriers. Human glioma cells grew well in the artificial tumor. Following treatment with TMZ, apoptotic cells were retained in the porous collagen, which allowed prolonged examination with NMR spectroscopy. 31 P spectra indicated that the PCh levels were reduced relative to the GPC and DPDE levels. 13C spectra and oxygen consumption measurements indicated that, subacutely a marked reduction in aerobic metabolism occurred, which is consistent with the belief that mitochondrial dysfunction commonly occurs during apoptosis. ACKNOWLEDGMENTS We thank Allen Bonner and David S. Nelson, and Drs. Edward J. Delikatny, Matthew Milkevitch, and William Lee of the University of Pennsylvania for many helpful conversations about resolving experimental problems.

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