Eppendorf Electrode Research Articles

Abstract A04: Is the lack of clinical success with antiangiogenic therapy due to vascular normalization not being a universal phenomenon?

Abstract Introduction: Pre-clinical studies have demonstrated that combining angiogenesis inhibitors (AIs) with conventional radiation and chemotherapy treatments can be an effective anti-tumor therapy. However, clinical benefits from such combinations have been more modest. A principal mechanism by which AIs are believed to increase tumor sensitivity to radiation and chemotherapy is vascular normalization, a process which should improve tumor blood perfusion and oxygenation. Here we review the universality of this concept by examining those pre-clinical studies that directly determined the effect of AI treatment on tumor oxygenation. Methods: This review was based on searching the PubMed database using the criteria angiogenesis inhibitors combined with one of each of the following: oxygenation, hypoxia, or normalization coupled with our own knowledge within the fields of tumor hypoxia and vascular targeting. A total of 51 pre-clinical studies were identified. The techniques used to monitor hypoxia/oxygenation included immunohistochemical analysis of hypoxia marker binding (pimonidazole and EF5) or hypoxia gene expression (CA9 and HIF-1alpha), direct oxygenation measurements (Eppendorf electrode, EPR oximetry, Oxylite probe or phosphorous quenching), or radiation response. The AIs included the two principal classes of inhibitors, those targeting VEGF (bevacizumab, DC101) and tyrosine kinase (vandetanib, sunitinib, sorafanib, axitinib, pazopanib, AG-013736, PTK787/ZK222584, SU5416), as well as a variety of other agents (TNP-470, suramin, endostatin, thrombospondin, thalidomide, anginex, arginine deiminase, nucleolin antagonist, nelfinavir). Results: 19 studies demonstrated significant improvements in tumor oxygenation status. These included a decrease in hypoxia marker labelling (bevacizumab, DC101, suramin, endostatin, nucleolin antagonist); an increase in oxygenation determined using the Eppendorf electrode (TNP-470 and an anti-VEGF antibody), EPR oximetry (thalidomide, vandetanib, SU5416), and Oxylite probe (bevacizumab); an increase in hypoxia regulated gene expression (endostatin and SU5416); and enhanced radiation response (thrombospondin). In contrast, 21 studies found a significant decrease in tumor oxygenation using hypoxic markers measured by immunohistochemistry (bevacizumab, DC101, sunitinib, pazopanib, AG-013736, nelfinavir) or PET activity (PTK787/ZK222584), and increased expression of HIF-1alpha (bevacizumab). Moreover, 9 studies reported no change in tumor oxygenation as measured with hypoxic markers (endostatin and SU5416), direct oxygen measurements (anti-VEGF antibody, DC101, arginine deiminase), or radiation response (DC101). Finally, two studies actually reported both increases and decreases when using direct oxygen measurements depending on the time after treatment with bevacizumab or DC101. Conclusions: While there is clear evidence that tumor oxygenation can improve following AI treatment, consistent with vascular normalization occurring, this is clearly not a universal finding. Importantly the effects on tumor hypoxia/oxygenation status show no relationship to either the specific AI or the assay used to monitor these parameters. These findings suggest that for the combination of AIs with conventional anti-cancer therapies to be most effective it will be important to establish the impact the AI has on the tumor vasculature and associated tumor pathophysiology in a given treatment setting. Citation Format: Michael R. Horsman, Dietmar W. Siemann. Is the lack of clinical success with antiangiogenic therapy due to vascular normalization not being a universal phenomenon? [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A04.

MO‐D‐141‐01: Quantification of Tumor Hypoxia Using [18F]‐Fluoromisonidazole Positron Emission Tomography and Tracer Kinetic Modeling

Purpose: Tumor hypoxia is correlated with treatment failure in radiotherapy. The hypoxic fraction of a tumor can be difficult to define using non‐invasive imaging methods such as Positron Emission Tomography (PET). The purpose of this study is to use tracer kinetic modeling techniques to increase the accuracy and precision of hypoxic volume (HV) quantification with 18F‐fluoromisonidazole (18F‐FMISO) PET imaging. Methods: Ten male BALB/c mice with EMT‐6 tumors were selected for imaging. Anaesthetized animals were injected with 18F‐FMISO and imaged using whole‐body 120‐min dynamic PET and Computed Tomography (CT). Data from dynamic 18F‐FMISO scans for 3 mice were fit to a 2‐compartment irreversible 3‐rate constant model (K1, k2, k3) and a PATLAK model (Ki). Different thresholds were applied to the resultant Ki and k3 rate constants generated on a voxel‐by‐voxel basis to calculate the HV for each tumor. These kinetic‐derived HVs were compared to HVs generated using conventional tumor‐to‐blood (TBR) ratios. Results: Kinetically‐derived HV (using the k3 tracer binding constant and the rate of tracer influx, Ki), calculated on a voxel‐by‐voxel basis, showed an increase in HV of up to 38% over the TBR method depending on the assumed threshold. Moreover, the PATLAK kinetic model using a Ki threshold may provide better delineation and calculation of the tumor HV than a 2‐compartment pharmacokinetic model. Conclusion: For PET imaging with 18F‐FMISO, pharmacokinetic modeling can improve the accuracy and precision of tumor hypoxia quantification over that obtained with tumor‐to‐blood ratios. In particular, the PATLAK method to define a tumor HV may be superior due to a reduction in noise and to better HV localization. Ongoing studies will correlate tumor HVs with Eppendorf electrode measurements and carbonic anhydrase IX staining of histologic sections. These studies could lead to improved techniques for identifying patients likely to benefit from therapies designed to overcome hypoxic radioresistance.

Imaging Hypoxia in Xenografted and Murine Tumors With 18F-Fluoroazomycin Arabinoside: A Comparative Study Involving microPET, Autoradiography, P o 2-Polarography, and Fluorescence Microscopy

Positron emission tomography (PET) allows noninvasive assessment of tumor hypoxia; however the combination of low resolution and slow tracer clearance from nonhypoxic tissue is problematic. The aim of this study was to examine the in vivo hypoxia selectivity of fluoroazomycin arabinoside ([18F]-FAZA), a promising tracer with improved washout kinetics from oxygenated tissue. Three squamous cell carcinomas and one fibrosarcoma with widely differing spatial patterns of vascularization, hypoxia, and necrosis were grown in mice and evaluated with PET and complementary methods. Eppendorf electrode measurements consistently demonstrated median PO2 values<1 mm Hg. In accordance with that, PET revealed that all tumors accumulated [18F]-FAZA in excess of reference tissue. Next the two-dimensional spatial distribution of [18F]-FAZA (from autoradiography) was compared with fluorescence images of the same tumor sections showing localization of the hypoxia marker pimonidazole and the perfusion marker Hoechst 33342. Pixel-by-pixel analysis of co-registered images showed a highly significant co-localization between the two hypoxia markers and an inverse correlation (except for the fibrosarcoma) between the distribution of [18F]-FAZA and Hoechst dye. Moreover intratumoral heterogeneity in tracer distribution was clearly visible on autoradiograms, with a [18F]-FAZA concentration approximately six times higher in poorly oxygenated areas than in vascular hot spots. The distribution of [18F]-FAZA is consistent with hypoxia as the key driving force for tracer tissue retention in a selection of tumors with widely differing physiology.

Comparison of Helzel and OxyLite Systems in the Measurements of Tumor Partial Oxygen Pressure (pO2)

Wen, B., Urano, M., Humm, J. L., Seshan, V. E., Li, G. C. and Ling, C. C. Comparison of Helzel and OxyLite Systems in the Measurements of Tumor Partial Oxygen Pressure (pO(2)). Radiat. Res. 168, 67-75 (2008). It has been demonstrated in both experimental and human malignancies that hypoxic tumor cells are linked with aggressive disease phenotype. One of the methods to identify these cells is by direct physical measurement of tumor pO(2). This study compared pO(2) values measured with two systems, the Helzel Hypoximeter (successor of the polarographic Eppendorf electrode) and the Oxford-Optronix OxyLite (fiber-optic probe), in R3327-AT and R3327-AT/tkeGFP tumors. Partial oxygen pressure was measured in individual tumors with either system or in the same tumor with both systems. The similarities and discrepancies in pO(2) measurements between the two systems were also investigated when tumor-bearing animals were breathing pure oxygen. Our data showed a considerable heterogeneity in pO(2) values in each tumor using both the Helzel and OxyLite systems. Similar results were obtained with both systems for the mean and median pO(2) values, and the distributions of pO(2) values within the interval 0 < pO(2) < 40 mmHg (the range important for defining tumor hypoxia) were found to be statistically equivalent. However, the frequencies of high pO(2) values (>40 mmHg) and zero values measured by the two systems were statistically significantly different.

Tumor hypoxia and expression of c-met in cervical cancer

Hypoxia enhances malignant progression by promoting the development of metastases and increasing invasiveness. One key regulator that controls growth, invasion and metastasis in cancer cells is the growth factor receptor c-met. The aim of this study, therefore, was to investigate the expression of the c-met protooncogene in cervical cancers in relation to intratumoral hypoxia levels and to clinico-pathological parameters. 43 Patients with cervical cancer were subjected to intratumoral pO(2) measurement with the Eppendorf electrode and biopsies were taken. The tissue was subsequently analyzed by immunohistochemistry with an anti-c-met antibody. c-met was expressed in 72% of cervical cancers. There was a significantly stronger expression in poorly differentiated tumors (r=0.4, p=0.008). Furthermore, c-met expression was significantly associated with a spray-like pattern of invasion (p=0.008). However, there was no significant relationship between c-met expression and intratumoral hypoxia, pT stage, FIGO stage, lymphovascular space involvement, tumor size or overall survival. Although c-met has been shown to be hypoxia-induced in vitro, our results suggest that it is not the mediator of deleterious effects of hypoxia on clinical outcome in cervical cancer.

Loss of Apaf-1 expression and resistance to hypoxia-induced apoptosis in cervical cancer

5046 Background: Clinical observations suggest that intratumoral hypoxia may increase the aggressiveness of tumors through clonal selection of cancer cells that have lost their apoptotic potential. We have shown previously that patients with hypoxic cervical cancers displaying a low apoptosis rate had a significantly worse prognosis compared to all other patients. The aim of this study, therefore, was to investigate the expression of the proapoptotic protein Apaf-1 in cervical cancers and to analyze its relation to intratumoral hypoxia and apoptosis. Methods: 56 patients with cervical cancer (FIGO stage IB to IV) were subjected to pretherapeutical intratumoral pO2 measurement with the Eppendorf electrode after informed written consent was given by each patient. The study was approved by the Ethics Committee of the University of Leipzig. Cervical cancer tissue from these patients was taken by needle core biopsy and analyzed by TUNEL assays as well as by immunohistochemistry with an anti-Apaf-1-antibody. For statistical analysis, Fisher’s exact test was used. Results: Apaf-1 was expressed in 86% of cervical cancers. The median apoptosis rate was 1.0% (range: 0.3– 3.4%). There was no direct correlation between Apaf-1 expression and intratumoral hypoxia. However, in the group of hypoxic cervical cancers (pO2 &lt;10 mmHg) with a low apoptosis rate (&lt;1.0%), 43.8 % (95% CI: 19.8–70.1%) of tumors showed a negative Apaf-1 expression compared to only 2.5% (95% CI: 0.06–13.1%) in non-hypoxic cancers and hypoxic cancers with a high apoptosis rate (p = 0.0003, Fisher’s exact test). Conclusions: While Apaf-1 is expressed in the vast majority of cervical cancers, a significant proportion of tumors with low apoptosis rates despite intratumoral hypoxia showed a loss of Apaf-1 expression. This finding suggests a mechanism by which hypoxic cervical cancers acquire resistance to apoptosis. No significant financial relationships to disclose.

Hypoxia and expression of the proapoptotic regulator BNIP3 in cervical cancer

Hypoxia plays a major role in the malignant progression of tumors. Here, we investigate the expression of Bcl-2/adenovirus E1B 19 kd-interacting protein 3 (BNIP3), a proapoptotic Bcl-2 family member, and its relationship to hypoxia in cervical cancer cell lines and clinical samples of cervical cancer. Cervical cancer cell lines were grown under hypoxia or normoxia, and BNIP3 mRNA expression was examined by Northern blot analysis. In 50 patients with cervical cancer, intratumoral oxygen measurement with the Eppendorf electrode and needle biopsies of the tumor were performed. The obtained tissue was subsequently analyzed by immunohistochemistry with an anti-BNIP3 antibody. Cervical cancer tissue collected upon surgery was used for Northern blot analysis ofin vivoBNIP3 mRNA expression. BNIP3 mRNA is strongly induced under hypoxic conditions in all cervical cancer cell lines investigated. Furthermore, Northern blot analysis revealed that BNIP3 mRNA is expressed in cervical cancer tissue. Using immunohistochemistry, we demonstrated that BNIP3 protein is expressed in 82% of the investigated cervical cancers and that more advanced tumor stages showed significantly stronger BNIP3 expression. However, we observed no correlation between BNIP3 expression and intratumoral hypoxia. In conclusion, BNIP3 is expressed in different cervical cancer cell lines as well as in clinical samples of cervical cancer. Although BNIP3 is clearly hypoxia-induciblein vitro, our results suggest additional mechanisms of BNIP3 regulationin vivo.Our findings therefore highlight a discrepancy betweenin vitromodels of tumor hypoxia and the complexity of human cancer.

Hypoxia and expression of the proapoptotic regulator BNIP3 in cervical cancer

Hypoxia plays a major role in the malignant progression of tumors. Here, we investigate the expression of Bcl-2/adenovirus E1B 19 kd-interacting protein 3 (BNIP3), a proapoptotic Bcl-2 family member, and its relationship to hypoxia in cervical cancer cell lines and clinical samples of cervical cancer. Cervical cancer cell lines were grown under hypoxia or normoxia, and BNIP3 mRNA expression was examined by Northern blot analysis. In 50 patients with cervical cancer, intratumoral oxygen measurement with the Eppendorf electrode and needle biopsies of the tumor were performed. The obtained tissue was subsequently analyzed by immunohistochemistry with an anti-BNIP3 antibody. Cervical cancer tissue collected upon surgery was used for Northern blot analysis of in vivo BNIP3 mRNA expression. BNIP3 mRNA is strongly induced under hypoxic conditions in all cervical cancer cell lines investigated. Furthermore, Northern blot analysis revealed that BNIP3 mRNA is expressed in cervical cancer tissue. Using immunohistochemistry, we demonstrated that BNIP3 protein is expressed in 82% of the investigated cervical cancers and that more advanced tumor stages showed significantly stronger BNIP3 expression. However, we observed no correlation between BNIP3 expression and intratumoral hypoxia. In conclusion, BNIP3 is expressed in different cervical cancer cell lines as well as in clinical samples of cervical cancer. Although BNIP3 is clearly hypoxia-inducible in vitro, our results suggest additional mechanisms of BNIP3 regulation in vivo. Our findings therefore highlight a discrepancy between in vitro models of tumor hypoxia and the complexity of human cancer.

105 Using 18F-fluoroazomycin arabinoside to non-invasively image hypoxia in human tumour xenografts: A comparative assessment with the eppendorf electrode and pimonidazole binding

105 Using 18F-fluoroazomycin arabinoside to non-invasively image hypoxia in human tumour xenografts: A comparative assessment with the eppendorf electrode and pimonidazole binding

Comparison between pimonidazole binding, oxygen electrode measurements, and expression of endogenous hypoxia markers in cancer of the uterine cervix

Although tumor hypoxia has been associated with a more aggressive phenotype and lower cure rate, there is no consensus as to the method best suited for routine measurement. Binding of the chemical hypoxia marker, pimonidazole, and expression of the endogenous hypoxia markers HIF-1alpha and CAIX were compared for their ability to detect hypoxia in tumor biopsies from 67 patients with advanced carcinoma of the cervix. Two biopsies were taken one day after administration of pimonidazole and were analyzed for pimonidazole binding using flow cytometry or immunohistochemistry. CAIX and HIF-1alpha expression and degree of colocalization were measured in sequential antibody-stained sections. Patient subsets were examined for tumor oxygen tension using an Eppendorf electrode, S phase DNA content, or change in HIF-1alpha expression over the course of treatment. Approximately 6% of the tumor area stained positive for pimonidazole, HIF-1alpha, or CAIX. The CAIX positive fraction correlated with the pimonidazole positive fraction (r = 0.60). Weaker but significant correlations were observed between pimonidazole and HIF-1alpha (r = 0.31) and CAIX and HIF-1alpha (r = 0.41). Taking the extent of marker colocalization into consideration increased the confidence that all markers were identifying hypoxic regions. Over 65% of stained areas showed a high degree of colocalization with the other markers. Oxygen microelectrode measurements and S phase fraction were not correlated with the hypoxic fraction measured using the three hypoxia markers. HIF-1alpha levels tended to decrease with time after the start of therapy. Endogenous hypoxia marker binding shows reasonable agreement, in extent and location, with binding of pimonidazole. CAIX staining pattern is a better match to the pimonidazole staining pattern than is HIF-1alpha, and high CAIX expression in the absence (or low levels) of HIF-1alpha may indicate a different biology.

Effect of intratumoral heterogeneity in oxygenation status on FMISO PET, autoradiography, and electrode P o2 measurements in murine tumors

To explore conflicting results obtained when tumor hypoxia is assessed with Eppendorf electrode Po(2) measurements and with positron emission tomography (PET) by use of [(18)F]fluoromisonidazole (FMISO). We compared the 2 methods in conjunction with 2-[(18)F]fluoro-2-deoxy-D-glucose (FDG) PET, dual-tracer ex vivo autoradiography (FMISO and 2-deoxy-D-[1-(14)C]glucose (2DG)), and histology in 2 murine tumor models, the C3H mammary carcinoma and the SCCVII squamous cell carcinoma. 2-[(18)F]fluoro-2-deoxy-D-glucose (FDG)-PET showed tumor-to-reference tissue ratios of 3.5 in both tumor models after 2 hours. C3H mammary carcinoma reached an FMISO PET ratio of 11 after 3.5 hours. Autoradiography showed large confluent areas of FMISO and 2DG uptake. Median Po(2) was 7 mm Hg and necrotic fraction was 10% to 30%. SCCVII squamous-cell carcinoma reached an FMISO PET tumor-to-reference tissue ratio of 2 after 2.5 hours. Autoradiography showed homogeneous 2DG uptake and scattered foci of high FMISO uptake. Median Po(2) was 1 mm Hg and necrotic fraction was below 5%. Ex vivo dual-tracer autoradiography documented the ability of in vivo FMISO PET to distinguish between confluent areas of either viable tissue or necrosis. Electrode Po(2) measurements could not be ascribed to specific areas in the tumors. Less uptake of FMISO in SCCVII squamous-cell carcinoma than in C3H mammary carcinoma could be caused by scattered foci versus confluent areas of viable hypoxic tissue in the 2 tumors, respectively.

Expression of Apaf-1 in cervical cancer correlates with lymph node metastasis but not with intratumoral hypoxia

Objectives The aim of this study was to investigate the expression of the proapoptotic protein Apaf-1 in cervical cancers. Moreover, we studied its correlation to intratumoral pO 2 and to clinico-pathological parameters. Methods 86 patients with cervical cancer were subjected to intratumoral pO 2 measurement with the Eppendorf electrode. From these patients, cervical cancer tissue was used for immunohistochemistry with an anti-Apaf-1 antibody. Results Apaf-1 is expressed in cervical cancer. Cervical cancers with strong or moderate Apaf-1 expression had significantly less lymph node metastases at time of surgery than tumors with weak or negative Apaf-1 expression ( P = 0.022). There was no significant correlation between Apaf-1 expression and intratumoral pO 2, pT stage, FIGO stage, lymphovascular space involvement, and grade. Conclusions Loss of Apaf-1 expression may represent a marker of aggressive tumor behavior since it correlates significantly with the occurrence of lymph node metastasis in cervical cancer.

Preclinical studies on how to deal with patient intolerance to nicotinamide and carbogen

Background and purpose Accelerated radiation carbogen nicotinamide (ARCON) therapy is generally well tolerated, but some patients experience intolerance to nicotinamide and carbogen (95% O 2+5% CO 2). This study investigated the effect of reducing both the nicotinamide dose and carbogen CO 2 content on radiation response. Materials and methods A C3H mouse mammary carcinoma grown in the right rear foot of female CDF1 was used and treated when at 200 mm 3. Nicotinamide was intraperitoneally injected 20 min prior to irradiation. Carbogen (CO 2 content of either 2 or 5%, balance O 2) breathing was started 5 min before, and continued during, additional treatments. Radiation was given locally to tissues of restrained non-anaesthetised mice either as a single or fractionated (10 fractions in 12 days) schedule. The endpoints were local tumour control at 90 days, development of moist desquamation 11–23 days after treatment of normal foot skin, or tumour oxygenation measured with the Eppendorf electrode. Results The TCD50 values in this tumour following single or fractionated radiation treatment were 52 and 71 Gy, respectively. Carbogen (5% CO 2 content) breathing with every radiation treatment in the fractionated schedule significantly (Chi-squared test; P<0.05) enhanced radiation response (ER 1.25). Significant enhancements were also seen with nicotinamide given either as 10×120 mg/kg (ER 1.25), 6×120 mg/kg (ER 1.11) or 10×90 mg/kg (ER 1.18), although the 6×120 schedule was significantly less effective than 10×120. Combining nicotinamide with carbogen resulted in ERs of 1.39–1.44, and these were independent of the nicotinamide treatments. There was also no significant difference in the enhancement of tumour radiation response or improved tumour oxygenation status if the CO 2 content of the gas breathing was varied from 0% (i.e. 100% O 2) to 2 or 5% (balance O 2), although a CO 2 content of 2% did give a smaller enhancement of radiation-induced normal skin damage than 5%. Conclusions Both the nicotinamide dose, but not the frequency, and carbogen CO 2 content may be reduced in patients experience intolerance without any significant loss of sensitisation.

Polarographic electrode study of tumor oxygenation in clinically localized prostate cancer

PurposeTo describe the oxygenation of clinically localized prostate cancer. Methods and materialsIntraprostatic oxygen tension was measured using the Eppendorf electrode in 55 unanesthetized men with localized prostate cancer before radiotherapy. Measurements were made along two tracks through regions of suspected tumor in the prostate, and core needle biopsies were then obtained from the same regions. ResultsThe median pO2 ranged from 0.2 to 57.3 mm Hg, and the grand median pO2 was 4.5 mm Hg. The percentage of oxygen readings <5 mm Hg (HP5) ranged from 0% to 100% (median 60%). The track 1 oxygen readings were greater than those from track 2. Statistically significant heterogeneity was found in the individual oxygen readings: the between- and within-tumor components accounted for 32% and 68% of the total variability, respectively. However, the between-tumor variability in HP5 significantly exceeded the within-tumor variability (61% vs. 39%). No association was found between oxygen values and clinical factors, including age, T stage, Gleason score, prostate-specific antigen level, hemoglobin concentration, or prior hormonal treatment. No difference was noted in the oxygenation between regions of tumor and normal prostate tissue, as determined from the core biopsies. ConclusionLocalized prostate cancer is characterized by marked hypoxia and significant heterogeneity in oxygenation, similar to other human tumors. The normal prostate may contain regions of low oxygen concentration. HP5, as determined in this study, should adequately discriminate among patients with prostate cancer and allow the independent prognostic significance of oxygenation to be evaluated once the study matures.

Influence of body temperature on the BOLD effect in murine SCC tumors.

Changes in the blood oxygen level dependent (BOLD) enhancements in tumors (squamous cell carcinoma, (SCCVII)) implanted in mice maintained at core temperatures of 30 degrees C or 37 degrees C were measured using MRI and compared to tumor oxygen levels obtained using an oxygen-sensitive Eppendorf electrode. Tumors were implanted in a hindleg of the mice intramuscularly. Tumor-bearing mice were imaged by BOLD MRI, while first breathing air and then carbogen (95% O2, 5% CO2) for 15-min intervals at a core temperature of 30 degrees C. After an equilibration period, the identical regimen was conducted with the same animal maintained at 37 degrees C. This procedure was repeated with additional mice starting at 37 degrees C followed by imaging at 30 degrees C. Likewise, oxygen electrode measurements of the tumor were determined at core temperatures of 30 degrees C and 37 degrees C. The Eppendorf measurements showed that tumors in animals maintained at 30 degrees C were significantly more hypoxic than at 37 degrees C. MRI studies demonstrated stronger BOLD enhancement at 30 degrees C than at 37 degrees C, suggesting significant changes in hypoxia and/or blood flow in tumors at these temperatures. The findings of the study stress the importance of maintaining normal core temperature when assessing tumor oxygen status using functional imaging modalities or oxygen-sensitive electrodes.

Vascular targeting effects of ZD6126 in a C3H mouse mammary carcinoma and the enhancement of radiation response

Purpose The aim of this study was to investigate the pathophysiologic effects induced by the novel vascular targeting agent ZD6126 in a C3H mouse mammary carcinoma and to evaluate the agent's ability to inhibit tumor growth either when given alone or in combination with radiation. Methods and materials A C3H mammary carcinoma grown in the right rear foot of female CDF1 mice was treated when at 200 mm 3 in size. ZD6126 was dissolved in saline and injected intraperitoneally. Blood perfusion was measured using the RbCl extraction procedure, tumor oxygen (pO 2) status was assessed with the Eppendorf electrode, and tumor necrosis was estimated from histologic sections. Radiation (240-kV X-rays) was locally administered to tumors of restrained nonanesthetized mice, and response was assessed using a tumor growth assay. Results ZD6126 induced a significant dose- and time-dependent decrease in tumor perfusion, reaching a maximal 70% reduction around 3 h after injecting 150–300 mg/kg. However, full recovery was seen within 6 h. A 200 mg/kg dose significantly decreased tumor oxygenation status at 3 h (median pO 2 decreased from 7 to 3 mm Hg; % pO 2 values ≤2.5 mm Hg increased from 30% to 55%) and by 24 h had significantly increased necrotic fraction from 14.5% to 25.2%. This ZD6126 dose resulted in a small, yet significant, 1.4 days inhibition of tumor growth when given alone. It also enhanced the tumor response to radiation, giving rise to a significant 1.3-fold increase in the slope of the radiation dose–response curve. Of the normal tissues, only muscle (at 3 h) and spleen (at 6 h) showed any significant reduction in perfusion after injecting 200 mg/kg, but these transient decreases were only 32% and 49%, respectively. Conclusions Our preclinical studies clearly demonstrate a tumor-specific reduction in blood perfusion by ZD6126. Although these changes were transient, they were sufficient to increase tumor necrosis, inhibit tumor growth, and enhance radiation response.

Ultrasound guided pO 2 measurement of breast cancer reoxygenation after neoadjuvant chemotherapy and hyperthermia treatment

The objective of this study was to determine whether neoadjuvant chemotherapy in combination with hyperthermia (HT) would improve oxygenation in locally advanced breast tumours. The study describes a new optimized ultrasound guided technique of pO 2 measurement using Eppendorf polarographic oxygen probes in 18 stage IIB-III breast cancer patients. Prior to treatment, tumour hypoxia (median pO 2 < 10 mmHg) was present in 11/18 patients (average median pO 2 = 3.2 mmHg). Seven patients had well oxygenated tumours (median pO 2 of 48.3 mmHg). Eight patients with hypoxic tumours prior to treatment had a significant improvement ( p = 0.0008) in tumour pO 2 after treatment (pO 2 increased to 19.2 mmHg). In three patients, tumours remained hypoxic (average median pO 2 = 4.5 mmHg). The advantages of the ultrasound guided pO 2 probe are in the accuracy of the Eppendorf electrode placement in tumour tissue, the ability to monitor electrode movement through the tumour tissue during the measurement and the ability to avoid electrode placement near or in large blood vessels by using colour Doppler imaging. The results of this preliminary study suggest that the combination of neoadjuvant chemotherapy and hyperthermia improves oxygenation in locally advanced breast tumours that are initially hypoxic.

Intratumoral Sickling in a Patient with Cervix Cancer and Sickle Trait: Effect on Blood Flow and Oxygenation

Background. Sickle trait affects approximately 8% of the black population in the United States and up to 40% of individuals in some parts of tropical Africa, but rarely causes clinically significant illness. This report provides the first conclusive evidence that erythrocytes in patients with sickle trait may sickle in the microvasculature of solid tumors, leading to impaired perfusion and hypoxia.Case. A black woman who was sickle trait positive presented with stage IIIB squamous cell carcinoma of the cervix. A biopsy showed extensive intravascular sickling of erythrocytes. An aspirate of blood obtained directly from the tumor also showed numerous sickled cells. A peripheral blood smear was normal. Direct measurement of oxygen tension using the Eppendorf electrode revealed the tumor to be markedly hypoxic, with 93% of a total of 142 individual oxygen reading <5 mm Hg.Conclusions. Erythrocytes in patients with sickle trait may sickle in the microvasculature of solid tumors and contribute to reduced blood flow and the development of hypoxia. Hypoxia is a strong independent prognostic factor in patients with cervix cancer, and further study is needed to evaluate the impact of intratumoral sickling on long-term outcome.

A polarographic electrode study of tumor oxygenation in localized prostate cancer

A polarographic electrode study of tumor oxygenation in localized prostate cancer

Ultrasound-guided pO2 measurement in breast cancer patients before and after hyperthermia treatment

The significance of tumor hypoxia extends beyond conventional radiation resistance. It has been found that tumor hypoxia affects drug resistance, angiognesis, cytokine production, cell cycle control, apoptosis and development of distant metastases. Recently, it has been reported that hyperthermia improves tumor oxygenation in both canine as well as human soft tissue sarcoma. This study describes a new optimized technique for pO2 measurement in breast cancer patients using ultrasound-guided placement of Eppendorf polarographic oxygen probes. Locally advanced breast cancer patients, participating in a phaseI/II study of neoadjuvant liposomal doxorubicin/paclitaxel/hyperthermia treatment, were the subjects of this study. Tumor oxygenation was measured before and 24 h after hyperthermia treatment.Advantages of the ultrasound-guided pO2 probe placement are the following: accuracy with visualization and verification of the Eppendorf electrode placement in tumor tissue; monitoring of the electrode movement through the tumor tissue during the measurement; ability to avoid electrode placement near or in large blood vessels by using color Doppler imaging; and spatial reproducibility of the second measurement. Despite progress in the technology that can be used to measure tumor hypoxia, accurate and verifiable placement of the oxygen probes in tumor tissue is of tremendous importance. Ultrasound-guided pO2 probe placement should become standard technique to improve accuracy and reliability in the assessment of tumor oxygenation for disease sites in which it is appropriate. Supported by a grant from the NIH CA42745.