Anca Constantinescu

Non-invasive determination of tumor oxygen tension and local variation with growth

PURPOSE The objective was to develop and demonstrate a novel noninvasive technique of measuring regional pO2 in tumors. The method is based on measuring 19F nuclear magnetic resonance spin-lattice relaxation rate (R1 = 1/T1) of perfluorocarbon (PFC) emulsion discretely sequestered in a tumor. METHODS AND MATERIALS We have examined pO2 in the Dunning prostate tumor R3327-AT1 implanted in a Copenhagen rat. Oxypherol blood substitute emulsion was administered intravenously and became sequestered in tissue. Proton magnetic resonance imaging (MRI) showed tumor anatomy and correlated 19F MRI indicated the distribution of perfluorocarbon. Fluorine-19 spectroscopic relaxometry was used to measure pO2 in the tumor and repeated measurements over a period of 3 weeks showed the variation in local pO2 during tumor growth. RESULTS Perfluorocarbon initially resided in the vascularized peripheral region of the tumor: 19F nuclear magnetic resonance R1 indicated pO2 approximately 75 torr in a small tumor (approximately 1 cm) in an anesthetized rat. As the tumor grew, the sequestered PFC retained its original distribution. When the tumor had doubled in size the residual PFC was predominantly in the core of the tumor and the pO2 of this region was approximately 1 torr indicating central tumor hypoxia. CONCLUSION We have demonstrated a novel noninvasive approach to monitoring regional tumor pO2. Given the critical role of oxygen tension in tumor response to therapy this may provide new insight into tumor physiology, the efficacy of various therapeutic approaches, and ultimately provide a clinical technique for assessing individual tumor oxygenation.

Noninvasive investigation of blood oxygenation dynamics of tumors by near-infrared spectroscopy.

The measurement of dynamic changes in the blood oxygenation of tumor vasculature could be valuable for tumor prognosis and optimizing tumor treatment plans. In this study we employed near-infrared spectroscopy (NIRS) to measure changes in the total hemoglobin concentration together with the degree of hemoglobin oxygenation in the vascular bed of breast and prostate tumors implanted in rats. Measurements were made while inhaled gas was alternated between 33% oxygen and carbogen (95% O(2), 5% CO(2)). Significant dynamic changes in tumor oxygenation were observed to accompany respiratory challenge, and these changes could be modeled with two exponential components, yielding two time constants. Following the Fick principle, we derived a simplified model to relate the time constants to tumor blood-perfusion rates. This study demonstrates that the NIRS technology can provide an efficient, real-time, noninvasive means of monitoring the vascular oxygenation dynamics of tumors and facilitate investigations of tumor vascular perfusion. This may have prognostic value and promises insight into tumor vascular development.

Tumor oximetry: demonstration of an enhanced dynamic mapping procedure using fluorine-19 echo planar magnetic resonance imaging in the Dunning prostate R3327-AT1 rat tumor

PURPOSE We have developed an enhanced approach to measuring regional oxygen tension (pO(2)) dynamics in tumors. The technique is demonstrated in a group of 8 Dunning prostate rat tumors (R3327-AT1) with respect to respiratory challenge. METHODS AND MATERIALS Hexafluorobenzene was injected directly into the tumors of anesthetized rats. (19)F nuclear magnetic resonance echo planar imaging relaxometry was performed to obtain maps of regional tumor oxygenation under baseline conditions and when the inhaled gas was changed to oxygen or carbogen. RESULTS Sequential pO(2) maps required 8 min, with a typical precision of 1-3 torr at 30-100 individual regions across a tumor. When rats breathed 33% oxygen, distinct heterogeneity was observed for baseline oxygenation in each tumor with pO(2) values ranging from hypoxic to greater than 100 torr. Larger tumors showed significantly lower baseline pO(2). Respiratory challenge with oxygen or carbogen produced significant increases in tumor oxygenation with a close correlation between the response to each gas at individual locations. Regions of both small and large tumors responded to respiratory challenge, but the rate was generally much faster in initially well-oxygenated regions. CONCLUSIONS Regional pO(2) was assessed quantitatively and the response of multiple individual tumor regions observed simultaneously with respect to interventions.

Bone phenotype of the aromatase deficient mouse

Estrogens are important for normal bone growth and metabolism. The mechanisms are incompletely understood. Thus, we have undertaken characterization of the skeletal phenotype of aromatase (ArKO) deficient mice. No abnormalities have been noted in skeletal patterning in newborns. Adult ArKO mice show decreased femur length and decreased peak Bone Mineral Density (BMD) with accelerated bone loss by 7 months of age in females. Magnetic resonance microscopy (MR) and microCT (microCT) imaging disclosed decreased cancellous connectivity and reduced cancellous bone volume in ArKO females. Bone formation rate (BFR) is increased in ArKO females and decreased in ArKO males. Estradiol therapy reverses these changes. This anabolic effect of estradiol in the male skeleton is supported by 18-F- Positron Emission Tomography (PET) imaging, which clearly demonstrates decreased spinal uptake, but marked increase after estradiol therapy. Serum IGF-1 levels are high in young female ArKO mice but low in young ArKO males. The reduced BMD in ArKO females, despite the presence of elevated serum IGF 1, suggests that other mechanism(s) are operative. There is increased B-cell lymphopoiesis in adult female ArKO bone marrow cells. These results show that ArKO mice show the effects of estrogen deficiency on bone growth, mass, metabolism, microarchitecture and the hematopoietic microenvironment.

Enhancing hepatocyte adhesion by pulsed plasma deposition and polyethylene glycol coupling.

Decreased hepatocyte adhesion to polymeric constructs limits the function of tissue engineered hepatic assist devices. We grafted adhesion peptides (RGD and YIGSR) to polycaprolactone (PCL) and poly-L-lactic acid (PLLA) in order to mimic the in vivo extracellular matrix and thus enhance hepatocyte adhesion. Peptide grafting was done by a novel technique in which polyethylene glycol (PEG)-adhesion peptide was linked to allyl-amine coated on the surface of PCL and PLLA by pulsed plasma deposition (PPD). Peptide grafting density, quantified by radio-iodinated tyrosine in YIGSR, was 158 fmol/cm(2) on PLLA and 425 fmol/cm(2) on PCL surfaces. The adhesion of hepatocytes was determined by plating 250,000 hepatocytes/well (test substrates were coated on 12 well plates) and quantifying the percentage of adhered cells after 6 h by MTT assay. Adhesion on PCL surfaces was significantly enhanced (p < 0.05) by both YIGSR (percentage of adhered cells = 53 +/- 7%) and RGD (53 +/- 12%) when compared to control surfaces (31 +/- 8%). Hepatocyte adhesion on PLLA was significantly (p < 0.05) enhanced on PLLA-PEG-RGD surfaces (76 +/- 14%) compared to control surfaces (42 +/- 19%) and more (68 +/- 25%) but not statistically significant (p = 0.15) on PLLA-PEG-YIGSR surfaces compared to control surfaces. These results indicate that hepatocyte adhesion to PCL and PLLA based polymeric surfaces can be enhanced by a novel adhesion peptide grafting technique using pulsed plasma deposition and PEG cross-linking.

Interplay of tumor vascular oxygenation and tumor pO2 observed using near-infrared spectroscopy, an oxygen needle electrode, and 19F MR pO2 mapping

This study investigates the correlation of tumor blood oxygenation and tumor pO(2) with respect to carbogen inhalation. After having refined and validated the algorithms for calculating hemoglobin concentrations, we used near-infrared spectroscopy (NIRS) to measure changes of oxygenated hemoglobin concentration (delta[HbO(2)]) and used an oxygen needle electrode and (19)F MRI for pO(2) measurements in tumors. The measurements were taken from Dunning prostate R3327 tumors implanted in rats, while the anesthetized rats breathed air or carbogen. The NIRS results from tumor measurements showed significant changes in tumor vascular oxygenation in response to carbogen inhalation, while the pO(2) electrode results showed an apparent heterogeneity for tumor pO(2) response to carbogen inhalation, which was also confirmed by (19)F MR pO(2) mapping. Furthermore, we developed algorithms to estimate hemoglobin oxygen saturation, sO(2), during gas intervention based on the measured values of delta[HbO(2)] and pO(2). The algorithms have been validated through a tissue-simulating phantom and used to estimate the values of sO(2) in the animal tumor measurement based on the NIRS and global mean pO(2) values. This study demonstrates that the NIRS technology can provide an efficient, real-time, noninvasive approach to monitoring tumor physiology and is complementary to other techniques, while it also demonstrates the need for an NIR imaging technique to study spatial heterogeneity of tumor vasculature under therapeutic interventions.

Regional tumor oximetry : 19F NMR spectroscopy of hexafluorobenzene

PURPOSE An accurate method for monitoring oxygen tension (pO2) of individual tumors could be valuable for optimizing treatment plans. We have recently shown that 19F nuclear magnetic resonance (NMR) spin-lattice relaxometry of hexafluorobenzene (HFB) provides a highly sensitive indicator of tumor oxygenation. We have now refined the methodology to provide enhanced precision, and applied the method to investigate dynamic changes in tumor oxygenation. METHODS AND MATERIALS Dunning prostate adenocarcinoma R3327-AT1 was grown in the form of pedicles on the foreback of male Copenhagen rats. When the tumors reached approximately equal to 1 cm diameter, HFB (20 microl) was administered, either centrally or peripherally, by direct intratumoral (i.T) injection. Local pO2 was determined using pulse-burst saturation recovery (PBSR) 19F NMR spectroscopy on the basis of the spin-lattice relaxation rate, R1. RESULTS Interrogation of the central region of tumors provided typical values in the range pO2 = 1.4-6.4 mmHg, with a typical stability of +/-2 mmHg over a period of 20 min, when rats breathed 33% O2. Altering the inhaled gas to oxygen or carbogen (95% O2/5% CO2) produced no significant change. In contrast, interrogation of tumor periphery indicated baseline pO2 in the range 7.9-78.9 mmHg. Altering inspired gas produced significant changes (p < 0.0001) with O2 or carbogen, although the change was generally greater with carbogen. In each case, pO2 returned to baseline within 16 min of returning the inhaled gas to baseline. CONCLUSION We believe this method provides a valuable new approach with the requisite precision and accuracy to investigate tumor pO2.

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.

Dynamic response of breast tumor oxygenation to hyperoxic respiratory challenge monitored with three oxygen-sensitive parameters

The simultaneous measurement of three oxygen-sensitive parameters [arterial hemoglobin oxygen saturation (SaO2), tumor vascular-oxygenated hemoglobin concentration ([HbO2]), and tumor oxygen tension (pO2)] in response to hyperoxic respiratory challenge is demonstrated in rat breast tumors. The effects of two hyperoxic gases [oxygen and carbogen (5% CO2 and 95% O2)] were compared, by use of two groups of Fisher rats with subcutaneous 13762NF breast tumors implanted in pedicles on the foreback. Two different gas-inhalation sequences were compared, i.e., air-carbogen-air-oxygen-air and air-oxygen-air-carbogen-air. The results demonstrate that both of the inhaled, hyperoxic gases significantly improved the tumor oxygen status. All three parameters displayed similar dynamic response to hyperoxic gas interventions, but with different response times: the fastest for arterial SaO2, followed by biphasic changes in tumor vascular [HbO2], and then delayed responses for pO2. Both of the gases induced similar changes in vascular oxygenation and regional tissue pO2 in the rat tumors, and changes in [HbO2] and mean pO2 showed a linear correlation with large standard deviations, which presumably results from global versus local measurements. Indeed, the pO2 data revealed hetergeneous regional response to hyperoxic interventions. Although preliminary near-infrared measurements had been demonstrated previously in this model, the addition of the pO2 optical fiber probes provides a link between the noninvasive relative measurements of vascular phenomena based on endogenous reporter molecules, with the quantitative, albeit, invasive pO2 determinations.

Regional tumor oxygen dynamics: 19F PBSR EPI of hexafluorobenzene

We demonstrate a novel approach to measuring regional tumor oxygen tension using 19F pulse burst saturation recovery echo planar imaging (EPI) relaxometry of hexafluorobenzene. Hexafluorobenzene offers exceptional sensitivity to changes in oxygen tension, and has a single resonance making it ideal for imaging studies. By combining a pulse burst saturation recovery preparation sequence with EPI, the relaxation experiments were performed in approximately 20 min facilitating measurements of dynamic changes in pO2 accompanying interventions. Direct intratumoral administration of hexafluorobenzene permitted labeling of specific regions of interest, and imaging provided maps of pO2, confirming distinct intra tumoral heterogeneity. For a group of three Dunning prostate adenocarcinoma R3327-AT1 tumors interrogation of the central tumor region showed skewed pO2 distributions with considerable radiobiological hypoxia (approximately 90% voxels had pO2 < 15 torr) when rats breathed 33% O2. Altering the inspired gas to pure oxygen caused distributions to shift towards increased pO2 with significant increases in mean oxygen tension (p < 0.05) in two cases. Interrogation of both central and peripheral regions in a fourth tumor showed bimodal distribution for tumor oxygenation including approximately 75% voxels with pO2 > 15 torr. EPI allows the fate of individual voxels to be traced: upon altering the inspired gas to pure oxygen those voxels with baseline pO2 > 30 torr showed significant changes (p < 0.05), whereas those with pO2 < 16 torr showed minimal response. The precision of the measurements, together with the ability to simultaneously examine dynamic changes in multiple regions should provide a useful technique for investigating tumor hypoxia with respect to therapy.