W. Timothy Jenkins

Hypoxia is important in the biology and aggression of human glial brain tumors.

We investigated whether increasing levels of tissue hypoxia, measured by the binding of EF5 [2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] or by Eppendorf needle electrodes, were associated with tumor aggressiveness in patients with previously untreated glial brain tumors. We hypothesized that more extensive and severe hypoxia would be present in tumor cells from patients bearing more clinically aggressive tumors. Hypoxia was measured with the 2-nitroimidazole imaging agent EF5 in 18 patients with supratentorial glial neoplasms. In 12 patients, needle electrode measurements were made intraoperatively. Time to recurrence was used as an indicator of tumor aggression and was analyzed as a function of EF5 binding, electrode values and recursive partitioning analysis (RPA) classification. On the basis of EF5 binding, WHO grade 2 tumors were characterized by modest cellular hypoxia (pO2s ≈ 10%) and grade 3 tumors by modest-to-moderate hypoxia (pO2s ≈ 10%- 2.5%). Severe hypoxia (≈0.1% oxygen) was present in 5 of 12 grade 4 tumors. A correlation between more rapid tumor recurrence and hypoxia was demonstrated with EF5 binding, but this relationship was not predicted by Eppendorf measurements.

Comparative Measurements of Hypoxia in Human Brain Tumors Using Needle Electrodes and EF5 Binding

Hypoxia is known to be an important prognostic marker in many human cancers. We report the use of two oxygen measurement techniques in human brain tumors and compare these data with semiquantitative histological end points. Oxygenation was measured using the Eppendorf needle electrode and/or EF5 binding in 28 brain tumors. These data were compared with necrosis, mitosis, and endothelial proliferation. In some tumors, absolute EF5 binding was converted to tissue pO2 based on in vitro calibrations. Eppendorf electrode readings could not be used to identify WHO grade 1/2 versus WHO grade 3/4 tumors, they could not differentiate grade 3 versus grade 4 glial-derived neoplasms, nor did they correlate with necrosis or endothelial proliferation scores. EF5 binding increased as the tumor grade increased and was significantly associated with necrosis and endothelial proliferation. There was no statistically significant correlation between the two hypoxia detection techniques, although both methods indicated similar absolute ranges of tissue pO2. There was substantial inter- and intratumoral heterogeneity of EF5 binding in WHO grade 4 glial neoplasms. The majority of cells in glial-derived tumor had levels of hypoxia that were mild to moderate (defined herein as 10% to 0.5% pO2) rather than severe (defined as approximately 0.1% pO2). Immunohistochemical detection of EF5 binding tracks histological parameters in adult brain tumors, with increased binding associated with increasing necrosis and endothelial proliferation. The proportion of moderately to severely hypoxic cells is relatively low, even in the high-grade tumors. Human brain tumors are dominated by oxic to moderately hypoxic cells.

Early detection of radiation therapy response in non-Hodgkin's lymphoma xenografts by in vivo1H magnetic resonance spectroscopy and imaging

The purpose of the study was to investigate the capability of 1H MRS and MRI methods for detecting early response to radiation therapy in non‐Hodgkins lymphoma (NHL). Studies were performed on the WSU‐DLCL2 xenograft model in nude mice of human diffuse large B‐cell lymphoma, the most common form of NHL. Radiation treatment was applied as a single 15 Gy dose to the tumor. Tumor lactate, lipids, total choline, T2 and apparent diffusion coefficients (ADC) were measured before treatment and at 24 h and 72 h after radiation. A Hadamard‐encoded slice‐selective multiple quantum coherence spectroscopy sequence was used for detecting lactate (Lac) while a stimulated echo acquisition mode sequence was used for detection of total choline (tCho) and lipids. T2‐ and diffusion‐weighted imaging sequences were used for measuring T2 and ADC. Within 24 h after radiation, significant changes were observed in the normalized integrated resonance intensities of Lac and the methylenes of lipids. Lac/H2O decreased by 38 ± 15% (p = 0.03), and lipid (1.3 ppm, CH2)/H2O increased by 57 ± 14% (p = 0.01). At 72 h after radiation, tCho/H2O decreased by 45 ± 14% (p = 0.01), and lipid (2.8 ppm, polyunsaturated fatty acid)/H2O increased by 970 ± 36% (p = 0.001). ADC increased by 14 ± 2% (p = 0.003), and T2 did not change significantly. Tumor growth delay and regression were observed thereafter. This study enabled comparison of the relative sensitivities of various 1H MRS and MRI indices to radiation and suggests that 1H MRS/MRI measurements detect early responses to radiation that precede tumor volume changes. Copyright

In vivo profiling of hypoxic gene expression in gliomas using the hypoxia marker EF5 and laser-capture microdissection

Hypoxia is a key determinant of tumor aggressiveness, yet little is known regarding hypoxic global gene regulation in vivo. We used the hypoxia marker EF5 coupled with laser-capture microdissection to isolate RNA from viable hypoxic and normoxic regions of 9L experimental gliomas. Through microarray analysis, we identified several mRNAs (including the HIF targets Vegf, Glut-1, and Hsp27) with increased levels under hypoxia compared with normoxia both in vitro and in vivo. However, we also found striking differences between the global in vitro and in vivo hypoxic mRNA profiles. Intriguingly, the mRNA levels of a substantial number of immunomodulatory and DNA repair proteins including CXCL9, CD3D, and RAD51 were found to be downregulated in hypoxic areas in vivo, consistent with a protumorigenic role of hypoxia in solid tumors. Immunohistochemical staining verified increased HSP27 and decreased RAD51 protein levels in hypoxic versus normoxic tumor regions. Moreover, CD8(+) T cells, which are recruited to tumors upon stimulation by CXCL9 and CXCL10, were largely excluded from viable hypoxic areas in vivo. This is the first study to analyze the influence of hypoxia on mRNA levels in vivo and can be readily adapted to obtain a comprehensive picture of hypoxic regulation of gene expression and its influence on biological functions in solid tumors.

Imaging and Analytical Methods as Applied to the Evaluation of Vasculature and Hypoxia in Human Brain Tumors

Abstract Evans, S. M., Jenkins, K. W., Jenkins, W. T., Dilling, T., Judy, K. D., Schrlau, A., Judkins, A., Hahn, S. M. and Koch, C. J. Imaging and Analytical Methods as Applied to the Evaluation of Vasculature and Hypoxia in Human Brain Tumors. Radiat. Res. 170, 677–690 (2008). Tissue hypoxia results from the interaction of cellular respiration, vascular oxygen carrying capacity, and vessel distribution. We studied the relationship between tumor vasculature and regions of low pO2 using quantitative analysis of binding of the 2-nitroimidazole EF5 given to patients intravenously (21 mg/kg) approximately 24 h preceding surgery. We describe new computer algorithms for determining EF5 binding as a function of radial distance from individual blood vessels and converting this value to tissue pO2. Tissues from six human brain tumors were assessed. In a hemangiopericytoma, a WHO Grade 2 and WHO Grade 3 glial brain tumor, all tissue pO2 values calculated by EF5 binding were >20 mmHg (described as “physiologically oxygenated”). In these three tumors, EF5 binding gradients (measured as a function of distance from each observed vessel) were low, with small positive and negative values averaging close to zero. Much lower tissue oxygen levels were found, including near some vessels, in glioblastomas. Gradients of EF5 binding away from vessels were larger in glioblastomas than in the low-grade tumors, but positive and negative values again averaged to near zero. Based on these preliminary data, we hypothesize a new paradigm for tumor blood flow in human brain tumors whereby in-flowing and out-flowing blood patterns may have contrasting effects on average tissue EF5 (and by inference, oxygen) gradients. Our studies also imply that neither distance to the nearest blood vessel nor distance from each observed blood vessel provide reliable estimates of tissue pO2.

The efficiency of DNA strand-break repair in two fibrosarcoma tumors and in normal tissues of mice irradiated in vivo with X rays.

We have used alkaline elution to study the repair of X-ray-induced DNA strand breaks in vivo in two fibrosarcoma tumors and in several normal mouse tissues after whole-body irradiation of mice with 10-12.5 Gy of X rays. Both tumors were found to repair damage significantly faster and to a greater extent than any of the normal tissues, so that by 2 hr after irradiation the level of damage in both tumors was indistinguishable from unirradiated control values. Of the normal tissues studied, liver repaired the fastest. The kinetics for the other normal tissues were essentially the same, showing an appreciable level (7-16%) of unrepaired lesions still evident after 2 hr. Even as late as 12 hr there was a significant amount of residual damage in some tissues, with testes and spleen showing the greatest level (ca. 15%). The repair kinetics for each tissue were not appropriately described by a sum of two exponentials. In contrast, previously reported data for many homogeneous mammalian cell systems in vitro and for some tissues in vivo have shown biphasic repair kinetics. This difference may be related to heterogeneity of both cell type and environment within the tissue populations used in the investigation. The faster repair of DNA strand breaks by tumor cells relative to cells from normal tissues was not readily explainable in terms of such radiobiological parameters as overall tissue oxygenation or sulfhydryl content. Rather, it appears that the degree of differentiation of the cells within the tissue population may be a major determinant of repair proficiency. Based on a model incorporating a competition between repair and fixation of sublethal lesions, these data are consistent with the idea that tumor cells may have a repair, and hence survival, advantage over normal cells in response to ionizing radiation.

Improved Methods to Generate Spheroid Cultures from Tumor Cells, Tumor Cells & Fibroblasts or Tumor-Fragments: Microenvironment, Microvesicles and MiRNA

Diagnostic and prognostic indicators are key components to achieve the goal of personalized cancer therapy. Two distinct approaches to this goal include predicting response by genetic analysis and direct testing of possible therapies using cultures derived from biopsy specimens. Optimally, the latter method requires a rapid assessment, but growing xenograft tumors or developing patient-derived cell lines can involve a great deal of time and expense. Furthermore, tumor cells have much different responses when grown in 2D versus 3D tissue environments. Using a modification of existing methods, we show that it is possible to make tumor-fragment (TF) spheroids in only 2–3 days. TF spheroids appear to closely model characteristics of the original tumor and may be used to assess critical therapy-modulating features of the microenvironment such as hypoxia. A similar method allows the reproducible development of spheroids from mixed tumor cells and fibroblasts (mixed-cell spheroids). Prior literature reports have shown highly variable development and properties of mixed-cell spheroids and this has hampered the detailed study of how individual tumor-cell components interact. In this study, we illustrate this approach and describe similarities and differences using two tumor models (U87 glioma and SQ20B squamous-cell carcinoma) with supporting data from additional cell lines. We show that U87 and SQ20B spheroids predict a key microenvironmental factor in tumors (hypoxia) and that SQ20B cells and spheroids generate similar numbers of microvesicles. We also present pilot data for miRNA expression under conditions of cells, tumors, and TF spheroids.

Co-Localization of Hypoxia and Apoptosis in Irradiated and Untreated HCT116 Human Colon Carcinoma Xenografts

HCT116 human colon carcinoma xenografts were grown in nude mice. Frozen sections of control and irradiated tumors were stained and analysed for the distribution and extent of hypoxia and apoptosis. Tissue oxygen partial pressure was measured by immunohistochemical staining of hypoxia-dependent metabolites of the 2-nitroimidazole EF5. Apoptosis was assessed using a commercial kit which stains damaged DNA. Although the apoptosis stain was unlikely to exclude other forms of cell death (necrosis, pyknosis) all staining was found to associate with regions of near anoxia.

Abstract 5004: In vivo profiling of hypoxic mRNA and miRNA expression in gliomas and head and neck tumors

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Hypoxia plays a key role in tumor aggressiveness and radiation resistance, yet little is known regarding hypoxic global gene regulation in vivo. Although the regulation of mRNA and miRNA expression by hypoxia has been investigated in isolation in vitro, it has not yet been analyzed directly in tumor samples. We have used the hypoxia marker EF5 coupled with laser capture microdissection (LCM) to isolate RNA from viable hypoxic and normoxic regions of 9L experimental rat gliomas and from human head and neck (H&N) tumor samples. This was followed by microarray analysis of mRNA expression and comparison of this signature with that obtained from treating 9L cells with hypoxia in vitro. Through this, we have identified several mRNAs (including the HIF targets Vegf, Glut-1 and Hsp27) with increased levels under hypoxia compared to normoxia both in vitro and in vivo. We also found striking differences between the global in vitro and in vivo hypoxic mRNA profiles. An intriguing finding was the hypoxic-downregulation of a number of immunomodulatory and DNA repair proteins including CXCL9, CD3D and RAD51 in vivo, consistent with a pro-tumorigenic role of hypoxia in solid tumors. Immunohistochemical staining verified increased HSP27 and decreased RAD51 protein levels in hypoxic vs. normoxic tumor regions. Moreover, CD8+ T cells which are recruited to tumors upon stimulation by CXCL9 and CXCL10, were largely excluded from viable hypoxic areas in vivo. Global microRNA expression changes have been reported to occur in response to hypoxia in vitro. Hence, our second objective was to identify the cluster of miRNAs differentially expressed in hypoxic vs. normoxic regions of the human tumors using TaqMan® Array MicroRNA Cards. We employed the same technique used with the 9L tumors to analyze miRNA expression patterns in hypoxic vs. normoxic samples from H&N cancer patients who have been administered EF5 prior to surgical removal of the tumor. Consistent with published in vitro studies, miR-210 emerged as a major miRNA robustly induced in the hypoxic regions. We also confirmed miR-210 induction by an individual real time-PCR assay and found it to be upregulated > 2-fold in all hypoxic RNA samples. In addition, we have also found several miRNAs whose levels were upregulated and downregulated in hypoxic vs. normoxic areas. This is the first study to analyze the influence of hypoxia on mRNA and miRNA levels in vivo and can be readily adapted to obtain a comprehensive picture of hypoxic regulation of gene expression and its influence on biological functions in solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5004. doi:10.1158/1538-7445.AM2011-5004