Measurements of tumor tissue oxygen tension using a time-resolved luminescence-based optical oxylite probe: comparison with a paired survival assay

Recently, a system that measures tissue oxygen tension using time-resolved luminescence-based optical sensors has become available commercially (Oxford Optronix, Oxford, England). Two experiments were conducted using this system. First, the oxygen tension distribution was measured in two tumor lines: a spontaneous mouse fibrosarcoma, FSa-II, and a human squamous cell carcinoma xenograft, FaDu. The area in which the pO(2) was equal to or lower than 2.5 mmHg was defined as the hypoxic lesion, and the hypoxic cell fraction was taken as the fraction of these measurements in a tumor. The measured hypoxic cell fractions were compared with those determined by the paired cell survival assay for tumors of various sizes. Second, the tumor tissue pO(2) was measured continuously after administration of two different anesthetics to evaluate the effect of these drugs on tissue pO(2). Results indicated a good agreement between the hypoxic cell fractions measured by this system and those determined by the paired cell survival curve assay for tumors smaller than approximately 500 mm(3). For tumors larger than approximately 500 mm(3), the hypoxic cell fractions measured by the oxygen probe system were higher than those measured by the paired cell survival assay. This may suggest that the hypoxic cell fraction measured by the oxygen probes included both hypoxic and necrotic areas in large tumors where necrotic lesions occupied a significant portion of the tumor. Continuous measurements of pO(2) after anesthesia (Nembutal, or ketamine plus xylazine) showed a consistent rise in the pO(2) during the first 20-30 min of measurement. Subsequently, the pO(2) values became constant or continued to rise slowly. For comparison, the tumor cell survivals were assayed after a dose of 20 Gy given in air at 5, 20 and 60 min after anesthesia. The result showed a decrease in cell survival only in tumors irradiated 20 min after an injection of Nembutal.     

The effects of Efaproxyn (efaproxiral) on subcutaneous RIF-1 tumor oxygenation and enhancement of radiotherapy-mediated inhibition of tumor growth in mice

Efaproxiral, an allosteric modifier of hemoglobin, reduces hemoglobin-oxygen binding affinity, facilitating oxygen release from hemoglobin, which is likely to increase tissue pO(2). The purpose of this study was to determine the effect of efaproxiral on tumor oxygenation and growth inhibition of RIF-1 tumors that received X radiation (4 Gy) plus oxygen breathing compared to radiation plus oxygen plus efaproxiral daily for 5 days. Two lithium phthalocyanine (LiPc) deposits were implanted in RIF-1 tumors in C3H mice for tumor pO(2) measurements using EPR oximetry. Efaproxiral significantly increased tumor oxygenation by 8.4 to 43.4 mmHg within 5 days, with maximum increases at 22-31 min after treatment. Oxygen breathing alone did not affect tumor pO(2). Radiation plus oxygen plus efaproxiral produced tumor growth inhibition throughout the treatment duration, and inhibition was significantly different from radiation plus oxygen from day 3 to day 5. The results of this study provide unambiguous quantitative information on the effectiveness of efaproxiral to consistently and reproducibly increase tumor oxygenation over the course of 5 days of treatment, modeling the clinical use of efaproxiral. Also, based on the tumor growth inhibition, the study shows the efaproxiral-enhanced tumor oxygenation was radiobiologically significant. This is the first study to demonstrate the ability of efaproxiral to increase tumor oxygenation and to increase the tumor growth inhibition of radiotherapy over 5 days of treatment.     

Efaproxiral (RSR13) plus oxygen breathing increases the therapeutic ratio of carboplatin in EMT6 mouse mammary tumors

Carboplatin, a member of the platinum family of alkylating agents, is often used in combination with radiotherapy. Some studies, including a recent publication from our laboratory, have suggested that the cytotoxic effects of platinum compounds may be altered by changes in the post-treatment oxygenation. The study reported here assessed whether post-treatment changes in tumor oxygenation caused by oxygen breathing alone or in combination with efaproxiral (RSR13) altered the effects of carboplatin. Efaproxiral, which allosterically modifies hemoglobin-oxygen binding to increase tumor pO(2), has been shown to increase the effects of radiation in animal tumor models and is in a second, confirmatory phase III clinical trial as an adjuvant to radiotherapy. These studies with EMT6 tumors in BALB/c Rw mice used clonogenic assays to assess tumor cell survival and tumor growth studies to assess antineoplastic activity and treatment-related toxicity. Efaproxiral plus oxygen breathing for 5 hrs after carboplatin treatment significantly increased the antineoplastic effects of carboplatin. The increased antineoplastic effects of carboplatin produced by efaproxiral plus oxygen breathing occurred without a concomitant increase in host toxicity. These findings suggest that the increases in tumor oxygenation produced by Efaproxiral plus oxygen breathing increased the therapeutic ratio of carboplatin.     

Correlations between 31P-NMR spectroscopy and tissue O2 tension measurements in a murine fibrosarcoma

Size-dependent changes in therapeutically relevant and interrelated metabolic parameters of a murine fibrosarcoma (FSaII) were investigated in vivo using conscious (unanesthetized) animals and tumor sizes less than or equal to 2% of body weight. Tumor pH and bioenergetics were evaluated by 31P nuclear magnetic resonance spectroscopy (31P-MRS), and tumor tissue oxygen tension (pO2) distribution was examined using O2-sensitive needle electrodes. During growth FSaII tumors showed a progressive loss of phosphocreatine (PCr) and nucleoside triphosphate (NTP) with increasing inorganic phosphate (Pi) and phosphomonoester (PME) signals. Ratios for PCr/Pi, PME/Pi, NTP/Pi, and phosphodiester/inorganic phosphate (PDE/Pi) as well as pH determined by 31P-NMR (pHNMR) and the mean tissue pO2 progressively declined as the tumors increased in size. The only relevant ratio increasing with tumor growth was PME/NTP. When the mean tissue pO2 value was plotted against pHNMR, NTP/Pi, PCr/Pi, PME/Pi, and PDE/Pi for tumor groups of similar mean volumes, a highly significant positive correlation was observed. There was a negative correlation between mean tumor tissue pO2 values and PME/NTP. From these results we concluded that 31P-MRS can detect changes in tumor bioenergetics brought about by changes in tumor oxygenation. Furthermore, the close correlation between oxygenation and energy status suggests that the microcirculation in FSaII tumors yields an O2-limited energy metabolism. Finally, a correlation between the proportion of pO2 readings between 0 and 2.5 mmHg and the radiobiologically hypoxic cell fraction in FSaII tumors was observed. The latter finding might be of particular importance for radiation therapy.     

The oxygenation of murine tumor isografts and human tumor xenografts by nicotinamide.

The changes in pO2 caused by nicotinamide in the FSaII mouse tumor and three different xenografts of human tumors, HP-56, FaDu, and EO1, grown subcutaneously in the legs of mice were studied. The tumor pO2, as measured with microelectrodes, began to rise soon after the host mice were injected intraperitoneally with 500 mg/kg nicotinamide, and it increased continuously for 100-120 min. The rate and magnitude of the increase in tumor pO2 was dependent on the tumor line and also on the tumor size. In FSaII tumors, the increase in pO2 caused by nicotinamide was relatively small in the well-oxygenated small tumors (173 +/- 5 mm3) compared with that in the larger tumors (515 +/- 25 mm3). The blood perfusion in FSaII tumors as measured with the laser Doppler method was also increased by nicotinamide. The growth delay in FSaII tumors induced by X irradiation was enhanced significantly by nicotinamide. It was concluded that the enhancement of radiation damage in the experimental tumors in mice by nicotinamide, as observed in the present study and reported by others, is due to an increase in intratumor pO2, possibly as a result of an increase in blood perfusion.     

Effect of irradiation fluence rate on the efficacy of photodynamic therapy and tumor oxygenation in meta-tetra (hydroxyphenyl) chlorin (mTHPC)-sensitized HT29 xenografts in nude mice

We present direct experimental evidence of the fluence-rate-dependent, radiation-induced variations in intratumor oxygen partial pressure (pO(2)) in HT29 human colon adenocarcinoma xenografts subjected to meta-tetra(hydroxyphenyl)chlorin (mTHPC)-based photodynamic therapy (PDT). The data establish a correlation between tumor oxygenation and treatment outcome. Tumor-bearing mice were injected with 0.3 mg/kg photosensitizer and subjected 72 h later to a 12 J/cm(2) red light dose administered at fluence rates of 5, 30, 90 and 160 mW/cm(2). A significant decrease in mean and median pO(2) was registered at approximately half of the total radiation fluence was delivered in tumors treated at rates of 160 and 90 mW/cm(2). Conversely, with the two lower fluence rates, intratumor pO(2) was maintained at levels comparable to those measured before illumination. Tumor oxygenation values registered shortly after every treatment protocol were at least equal to baseline levels, thus excluding the possibility of significant acute vessel damage during illumination. The tumor regrowth profile correlated with the pO(2) values monitored during irradiation. Tumors treated with fluence rates of 5 and 30 mW/cm(2) exhibited significantly longer tumor quadrupling times than those treated at 160 and 90 mW/cm(2). Improved tumor destruction could be expected by reducing the rate and the extent of oxygen depletion during meta-tetra(hydroxyphenyl)chlorin photodynamic therapy using low fluence rates.     

Perivascular oxygen tensions in a transplantable mammary tumor growing in a dorsal flap window chamber.

Fischer 344 rats with R3230 Ac mammary carcinomas implanted in dorsal flap window chambers served as a model to obtain measurements of perivascular and stromal oxygen tension in normal and tumor tissues using Whalen recessed-tip microelectrodes (3- to 6-microns tip). Perivascular measurements were made adjacent to vessels with continuous blood flow. Thus the measurements and models provided are reflective of conditions leading to chronic hypoxia. Perivascular oxygen tensions averaged 72 +/- 13 mmHg in normal tissue vessels adjacent to tumor, 26 +/- 5 mmHg in tumor periphery, and 12 +/- 3 mmHg in tumor central vessels. There was a significant trend toward lower perivascular oxygen tensions in the tumor center (Kruskal-Wallis test, P = 0.002). A similar tendency was seen with a limited number of stromal measurements. Krogh cylinder models, which incorporate these data for perivascular oxygen tension, along with morphometric data obtained from the same tumor model suggest that hypoxic regions will exist between tumor vessels in the tumor center unless O2 consumption rates are well below 0.6 ml/100 g/min. The low perivascular measurements observed near the tumor center combined with the theoretical considerations suggest, for this model at least, that tissue oxygenation may best be improved by increasing red cell velocity and input pO2 and reducing oxygen consumption. The low perivascular oxygen tensions observed near the center also suggest that conditions conducive to increased red cell rigidity exist, that drugs which can decrease red cell rigidity could improve tumor blood flow and oxygenation, and that the endothelium of those vessels may be susceptible to hypoxia-reoxygenation injury.     

Radiosensitization of murine tumors by Fluosol-DA 20%

The effect of perfluorochemicals in combination with carbogen breathing on the response of SCK tumors of mice to fractionated irradiation was investigated. The SCK tumors of A/J mice were irradiated twice a day at 3 Gy per fraction (6 Gy per day), with a total dose of 18 Gy over 3 days. When the host animals were treated with an intravenous (iv) injection of 12 ml/kg of Fluosol-DA 20% before the first daily tumor irradiation and carbogen breathing during every X irradiation with Fluosol-DA 20% injection without carbogen breathing. The hypoxic cell fraction, as determined by an in vivo-in vitro cloning assay, decreased significantly, and the intratumor pO2, as determined with microelectrodes, was markedly increased by Fluosol-DA 20% injection and carbogen breathing. It was concluded that oxygenation of hypoxic cells in SCK tumors during the course of fractionated irradiation was improved by the iv injection of Fluosol-DA 20% and carbogen breathing.     

Measurement of tumor oxygenation: in vivo comparison of a luminescence fiber-optic sensor and a polarographic electrode in the p22 tumor

Hypoxia is important in tumor biology and therapy. This study compared the novel luminescence fiber-optic OxyLite sensor with the Eppendorf polarographic electrode in measuring tumor oxygenation. Using the relatively well-oxygenated P22 tumor, oxygen measurements were made with both instruments in the same individual tumors. In 24 air-breathing animals, pooled electrode pO(2) readings lay in a range over twice that of sensor pO(2(5min)) values (-3.2 to 80 mm Hg and -0.1 to 34.8 mm Hg, respectively). However, there was no significant difference between the means +/- 2 SE of the median pO(2) values recorded by each instrument (11.0 +/- 3.3 and 8.1 +/- 1.9 mm Hg, for the electrode and sensor respectively, P = 0.07). In a group of 12 animals treated with carbon monoxide inhalation to induce tumor hypoxia, there was a small but significant difference between the means +/- 2 SE of the median pO(2) values reported by the electrode and sensor (1.7 +/- 0.9 and 2.9 +/- 0.7 mm Hg, respectively, P = 0.009). A variable degree of disparity was seen on comparison of pairs of median pO(2) values from individual tumors in both air-breathing and carbon monoxide-breathing animals. Despite the differences between the sets of readings made with each instrument from individual tumors, we have shown that the two instruments provide comparable assessments of tumor oxygenation in groups of tumors, over the range of median pO(2) values of 0.6 to 28.1 mm Hg.     

Optical sensor-based oxygen tension measurements correspond with hypoxia marker binding in three human tumor xenograft lines

Hypoxia has a negative effect on the outcome of radiotherapy and surgery and is also related to an increased incidence of distant metastasis. In this study, tumor pO(2) measurements using a newly developed time-resolved luminescence-based optical sensor (OxyLitetrade mark) were compared with bioreductive hypoxia marker binding (pimonidazole). Single pO(2) measurements per tumor were compared to hypoxia marker binding in tissue sections using image analysis. Both assays were performed in the same tumors of three human tumor lines grown as xenografts. Both assays demonstrated statistically significant differences in the oxygenation status of the three tumor lines. There was also a good correlation between hypoxia marker binding and the pO(2) measurements with the OxyLitetrade mark device. A limitation of the OxyLitetrade mark system is that it is not yet suited for sampling multiple sites in one tumor. An important strength is that continuous measurements can be taken at the same position and dynamic information on the oxygenation status of tumors can be obtained. The high spatial resolution of the hypoxia marker binding method can complement the limitations of the OxyLitetrade mark system. In the future, a bioreductive hypoxic cell marker for global assessment of tumor hypoxia may be combined with analysis of temporal changes in pO(2) with the OxyLitetrade mark to study the effects of oxygenation-modifying treatment on an individual basis.     

The role of oxygen monitoring during photodynamic therapy and its potential for treatment dosimetry.

Understanding of the biology of photodynamic therapy (PDT) has expanded tremendously over the past few years. However, in the clinical situation, it is still a challenge to match the extent of PDT effects to the extent of the disease process being treated. PDT requires drug, light and oxygen, any of which can be the limiting factor in determining efficacy at each point in a target organ. This article reviews techniques available for monitoring tissue oxygenation during PDT. Point measurements can be made using oxygen electrodes or luminescence-based optodes for direct measurements of tissue pO2, or using optical spectroscopy for measuring the oxygen saturation of haemoglobin. Imaging is considerably more complex, but may become feasible with techniques like BOLD MRI. Pre-clinical studies have shown dramatic changes in oxygenation during PDT, which vary with the photosensitizer used and the light delivery regimen. Better oxygenation throughout treatment is achieved if the light fluence rate is kept low as this reduces the rate of oxygen consumption. The relationship between tissue oxygenation and PDT effect is complex and remarkably few studies have directly correlated oxygenation changes during PDT with the final biological effect, although those that have confirm the value of maintaining good oxygenation. Real time monitoring to ensure adequate oxygenation at strategic points in target tissues during PDT is likely to be important, particularly in the image guided treatment of tumours of solid organs.     

A mathematical model of tumor oxygen and glucose mass transport and metabolism with complex reaction kinetics

Hypoxia imparts radioresistance to tumors, and various approaches have been developed to enhance oxygenation, thereby improving radiosensitivity. This study explores the influence of kinetic and physical factors on substrate metabolism in a tumor model, based on a Krogh cylinder. In tissue, aerobic metabolism is assumed to depend on glucose and oxygen, represented by the product of Michaelis-Menten expressions. For the base case, an inlet pO(2) of 40 mmHg, a hypoxic limit of 5 mmHg, and a tissue/capillary radius ratio of 10 are used. For purely aerobic metabolism, a hypoxic fraction of 0.16 and volume-average pO(2) of 8 mmHg are calculated. Reducing the maximum oxygen rate constant by 9%, decreasing the tissue cylinder radius by 5%, or increasing the capillary radius by 8% abolishes the hypoxic fraction. When a glycolytic term is added, concentration profiles are similar to the base case. Using a distribution of tissue/capillary radius ratios increases the hypoxic fraction and reduces sensitivity to the oxygen consumption rate, compared to the case with a single tissue/capillary radius ratio. This model demonstrates that hypoxia is quite sensitive to metabolic rate and geometric factors. It also predicts quantitatively the effects of inhibited oxygen metabolism and enhanced mass transfer on tumor oxygenation.     

Increase in tumor oxygenation and radiosensitivity caused by pentoxifylline.

The effects of pentoxifylline (PTX), a drug commonly used for vascular disorders in humans, on the pO2 in SCK tumors of A/J mice and FSa-II tumors of C3Heb/FeJ mice as well as on the radioresponse of SCK tumors were investigated. When the host mice were injected intraperitoneally (ip) with 5 mg/kg PTX, the tumor pO2 increased slowly, peaked 20-50 min postinjection, and returned to its original level in 70-90 min. The magnitude of the increase in tumor pO2 varied markedly depending on the site and tumors. The magnitude of the changes in tumor pO2 after an ip injection of 25 or 50 mg/kg PTX was similar to that caused by 5 mg/kg PTX, but the pO2 tended to remain elevated longer with the higher dose of PTX. When the A/J mice bearing SCK tumors in the legs were injected ip with 50 mg/kg PTX and the tumors were X-irradiated 20 min later, the radiation-induced growth delay of the tumors was greater than that caused by X irradiation alone. The present study demonstrated that PTX is potentially useful for increasing the pO2 and the radioresponse of human tumors.     

Regional tumor oxygenation and measurement of dynamic changes.

We recently described a novel approach to measuring regional tumor oxygen tension using (19)F pulse burst saturation recovery (PBSR) nuclear magnetic resonance (NMR) echo planar imaging (EPI) relaxometry of hexafluorobenzene. We now compare oxygen tension measurements in a group of size-matched R3327-AT1 Dunning prostate rat tumors made using this new method with those using a traditional polarographic method: the Eppendorf histograph. Similar oxygen tension distributions were found using the two methods, and both techniques showed that tumors with volume greater than 3.5 cm(3) were significantly (P < 0.0001) less well oxygenated than smaller tumors (volume less than 2 cm(3)). Using the (19)F EPI approach, we also examined the response to respiratory challenge. Increasing the concentration of inspired oxygen from 33% to 100% O(2) produced a significant increase (P < 0.0001) in tumor oxygenation for a group of small tumors. In contrast, no change was observed in the mean pO(2) for a group of large tumors. Consideration of individual tumor regions irrespective of tumor size showed a strong correlation between the maximum pO(2) observed when breathing 100% O(2) compared with mean baseline pO(2). These results further demonstrate the usefulness of (19)F EPI to assess changes in regional tumor oxygenation.     

Improvement of tumor oxygenation by mild hyperthermia

There is now abundant evidence that oxygenation in rodent, canine and human tumors is improved during and for up to 1-2 days after heating at mild temperatures. An increase in tumor blood perfusion along with a decline in the oxygen consumption rate appears to account for the improvement of tumor oxygenation by mild hyperthermia. The magnitude of the increase in tumor pO(2), determined with oxygen-sensitive microelectrodes, caused by mild hyperthermia is less than that caused by carbogen breathing. However, mild hyperthermia is far more effective than carbogen breathing in increasing the radiation response of experimental tumors, probably because mild hyperthermia oxygenates both (diffusion-limited) chronically hypoxic and (perfusion-limited) acutely hypoxic cells, whereas carbogen breathing oxygenates only the chronically hypoxic cells. Mild hyperthermia is also more effective than nicotinamide, which is known to oxygenate acutely hypoxic cells, in enhancing the radiation response of experimental tumors. The combination of mild hyperthermia with carbogen or nicotinamide is highly effective in reducing the hypoxic cell fraction in tumors and increasing the radiation response of experimental tumors. A primary rationale for the use of hyperthermia in combination with radiotherapy has been that hyperthermia is equally cytotoxic toward fully oxygenated and hypoxic cells and that it directly sensitizes both fully oxygenated and hypoxic cells to radiation. Such cytotoxicity and such a radiosensitizing effect may be expected to be significant when the tumor temperature is elevated to at least 42-43 degrees C. Unfortunately, it is often impossible to uniformly raise the temperature of human tumors to this level using the hyperthermia devices currently available. However, it is relatively easy to raise the temperature of human tumors into the range of 39-42 degrees C, which is a temperature that can improve tumor oxygenation for up to 1-2 days. The potential usefulness of mild hyperthermia to enhance the response of human tumors to radiotherapy by improving tumor oxygenation merits continued investigation.     

The effect of darbepoetin alfa on growth, oxygenation and radioresponsiveness of a breast adenocarcinoma

Tumor hypoxia is associated with poor clinical outcome in a variety of tumors, including cervical, head/neck and breast cancer. Administration of erythropoietic factors has been suggested as a means of improving tumor oxygenation (pO2). This study randomized rats to treatment with low-dose or high-dose darbepoetin alfa or a placebo to determine the effect of darbepoetin alfa on the pO2, growth and response to radiation therapy of R3230 mammary adenocarcinoma. Rats received 3 microg/kg (high dose) or 0.2 microg/kg (low dose) darbepoetin alfa or placebo for eight doses prior to either (1) pO2 measurement and pimonidazole staining or (2) irradiation or sham irradiation on post-transplant day 20. In the animals randomized to radiation treatment, placebo or darbepoetin alfa administration at a reduced dose was continued for 9 weeks or until the tumor diameter exceeded 15 mm (defined as failure for survival analysis). Treatment with high-dose and low-dose darbepoetin alfa produced hematocrits of 68 and 56% compared to 44 and 45% in their respective control groups (both P < 10(-5)). At 18 days post-transplant, tumor volume was not different for either darbepoetin alfa group compared to the placebo group. Tumor oxygenation, as measured by the fraction of pO2 measurement <10 mmHg and the intensity of pimonidazole staining, was significantly improved in the high-dose group (P = 0.046 and 0.03, respectively, compared with controls) but not in the low-dose group. Growth delay curves after irradiation did not differ significantly for high- or low-dose darbepoetin alfa compared to placebo. In this nonanemic animal model of mammary adenocarcinoma, darbepoetin alfa does not significantly alter tumor growth or radioresponsiveness, even though it improves oxygenation when administered at high doses.     

Response to radioimmunotherapy correlates with tumor pO2 measured by EPR oximetry in human tumor xenografts

The efficacy of radiation treatment depends upon local oxygen concentration. We postulated that the variability in responsiveness of tumor xenografts to a fixed dose of radioimmunotherapy might be related to the tumor pO2 at the time that radioimmunotherapy was administered. We evaluated the growth of xenografts of CALU-3 tumors, a non-small cell lung carcinoma, in response to an 8.9-MBq dose of 131I-RS-7-anti-EGP-1 and correlated tumor growth rate with initial tumor pO2 measured by EPR oximetry. The greatest growth delay in response to radioimmunotherapy had the highest initial pO2, and the fastest-growing tumors had the lowest initial pO2. We then determined the dynamic effect of radioimmunotherapy on tumor pO2 by serial measurements of pO2 for 35 days after radioimmunotherapy. This information could be important for ascertaining the likelihood that a tumor will respond to additional doses as part of a multiple dose scheme. Serial tumor pO2 measurements may help identify a window of opportunity when the surviving tumor regions will be responsive to a second round of radioimmunotherapy or a second therapeutic modality such as chemotherapy or an anti-vascular agent. After radioimmunotherapy, there was an increase in tumor pO2 followed by a decrease below initial levels in most mice. Thus defined times may exist when a tumor is more or less radiosensitive after radioimmunotherapy.     

Targeted gadolinium-loaded dendrimer nanoparticles for tumor-specific magnetic resonance contrast enhancement

A target-specific MRI contrast agent for tumor cells expressing high affinity folate receptor was synthesized using generation five (G5) ofpolyamidoamine (PAMAM) dendrimer. Surface modified dendrimer was functionalized for targeting with folic acid (FA) and the remaining terminal primary amines of the dendrimer were conjugated with the bifunctional NCS-DOTA chelator that forms stable complexes with gadolinium (Gd III). Dendrimer-DOTA conjugates were then complexed with GdCl3 followed by ICP-OES as well as MRI measurement of their longitudinal relaxivity (T1 s(-1) mM(-1)) of water. In xenograft tumors established in immunodeficient (SCID) mice with KB human epithelial cancer cells expressing folate receptor (FAR), the 3D MRI results showed specific and statistically significant signal enhancement in tumors generated with targeted Gd(III)-DOTA-G5-FA compared with signal generated by non-targeted Gd(III)-DOTA-G5 contrast nanoparticle. The targeted dendrimer contrast nanoparticles infiltrated tumor and were retained in tumor cells up to 48 hours post-injection of targeted contrast nanoparticle. The presence of folic acid on the dendrimer resulted in specific delivery of the nanoparticle to tissues and xenograft tumor cells expressing folate receptor in vivo. We present the specificity of the dendrimer nanoparticles for targeted cancer imaging with the prolonged clearance time compared with the current clinically approved gadodiamide (Omniscan) contrast agent. Potential application of this approach may include determination of the folate receptor status of tumors and monitoring of drug therapy.     

Monitoring photodynamic therapy of solid tumors online by BOLD-contrast MRI

Antivascular photodynamic therapy (PDT) of tumors with palladium-bacteriopheophorbide (TOOKAD) relies on in situ photosensitization of the circulating drug by local generation of cytotoxic reactive oxygen species, which leads to rapid vascular occlusion, stasis, necrosis and tumor eradication. Intravascular production of reactive oxygen species is associated with photoconsumption of O(2) and consequent evolution of paramagnetic deoxyhemoglobin. In this study we evaluate the use of blood oxygenation level-dependent (BOLD) contrast magnetic resonance imaging (MRI) for real-time monitoring of PDT efficacy. Using a solid tumor model, we show that TOOKAD-PDT generates appreciable attenuation (25-40%) of the magnetic resonance signal, solely at the illuminated tumor site. This phenomenon is independent of, though augmented by, ensuing changes in blood flow. These results were validated by immunohistochemistry and intravital microscopy. The concept of photosensitized BOLD-contrast MRI may have intraoperative applications in interactive guidance and monitoring of antivascular cancer therapy, PDT treatment of macular degeneration, interventional cardiology and possibly other biomedical disciplines.     

Oxygen depletion during and after mTHPC-mediated photodynamic therapy in RIF1 and H-MESO1 tumors

During photodynamic therapy (PDT), low oxygenation levels, induced both by oxygen consumption and by vascular occlusion, can lead to an inefficient photochemical reaction that may compromise the efficacy of PDT. In the present studies, tumor oxygenation was measured before, during and after meta-tetrahydroxyphenylchlorin (mTHPC)-mediated PDT of murine RIF1 tumors and human mesothelioma xenografts (H-MESO1). Tumor pO2 was measured in real time with Eppendorf polarography, and the extent of relative hypoxia at specific times was measured by immunohistochemical staining. Significant decreases in median pO2 values, as well as an increase in the number of values below 2.5 mmHg, were seen during and after PDT in RIF1 tumors, although there was a large intertumoral variation. Tumor pO2 values did not change significantly in H-MESO1 tumors. Staining with antibodies against the hypoxia marker EF3 showed significant increases in relative hypoxia after PDT in both tumor types compared with separate groups of untreated controls. Our results are consistent with PDT-induced oxygen depletion (reduced pO2) leading to an increase in relative hypoxia in RIF1 tumors. Extensive necrosis in the H-MESO1 tumors may have prevented the detection of PDT-induced hypoxia using the Eppendorf polarographic needle, whereas immunohistochemistry did reveal increases in relative hypoxia.