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 pO(2) 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 pO(2). 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% pO(2)) rather than severe (defined as approximately 0.1% pO(2)). 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.     

EF5 binding and clinical outcome in human soft tissue sarcomas

PURPOSE: To study the 2-nitroimidazole agent EF5 as a surrogate for measuring hypoxia in a series of patients with soft tissue sarcomas, and to determine whether hypoxia measured with this technique was associated with patient outcome. METHODS AND MATERIALS: Patients with soft tissue sarcomas of the head and neck, extremity, trunk, or retroperitoneum for whom surgical excision was the initial treatment of choice, were given 21 mg/kg EF5 24-48 hours before surgery. Biopsy specimens were stained for EF5 binding with fluorescence-labeled monoclonal antibodies, and the images were analyzed quantitatively. Endpoints included the relationship between EF5 binding, clinically important prognostic factors, and patient outcome. RESULTS: Two patients with recurrent and 14 patients with de novo sarcomas were studied. There were seven low-grade, one intermediate-grade, and eight high-grade tumors. No relationship was found between EF5 binding and patient age, sex, hemoglobin level, or tumor size. In de novo tumors, the presence of mitoses and histologic grade were positively correlated with hypoxia. High-grade and -stage de novo tumors had higher levels of EF5 binding compared with low-grade and -stage tumors. Patients with de novo tumors containing moderate to severe hypoxia (> or = 20% EF5 binding), high grade, or > or = 7% mitoses were more likely to develop metastases. CONCLUSIONS: Further studies in a larger cohort of patients are necessary to determine whether hypoxia, as measured by EF5 binding, is an independent prognostic factor for outcome in high-grade sarcomas. Such data should be useful to identify high-risk patients for clinical trials to determine whether early chemotherapy will influence the occurrence of metastasis.     

Hypoxia and necrosis in rat 9L glioma and Morris 7777 hepatoma tumors: comparative measurements using EF5 binding and the Eppendorf needle electrode

PURPOSE: The purpose of this study was to assess the presence of tumor hypoxia using two independent techniques: binding of the 2-nitroimidazole EF5 and Eppendorf needle electrode measurements. The distribution of tumor hypoxia was assessed with respect to tumor necrosis in corresponding histological studies. METHODS AND MATERIALS: Each of several rats bearing a subcutaneous 9L glioma or Morris 7777 hepatoma tumor was given EF5 i.v. to a final, whole-body concentration of 100 microM. About 2.5 h later, each rat was anesthetized, and needle electrode measurements were made in the tumor along 1-5 tracks (30-200 individual measurements). At 3 h post-EF5 injection, the tumor was excised and frozen. Frozen sections were analyzed for the presence and distribution of binding of EF5 and necrosis using immunohistochemical techniques followed by staining with hematoxylin and eosin (H&E). The histochemical analysis and electrode readings in similar regions of the tumor were compared. RESULTS: Electrode measurements were taken at 0.4-mm intervals along one-dimensional tracks, whereas EF5 binding measurements from tissue sections contained two-dimensional information at high spatial resolution ( approximately 2.5 micro). The EF5 measurements showed greater spatial heterogeneity than did the electrode measurements. In tumor regions with minimal necrosis, needle tracks with relatively high pO(2) readings were usually found to contain relatively low EF5 binding, and vice versa. Because EF5 binding is inversely related to tissue pO(2), this result was expected. The expected inverse correlation of the two techniques was most disparate in necrotic tumor regions (confirmed by H&E staining), where needle electrode measurements showed low to zero pO(2) values, but little or no EF5 binding was found. CONCLUSION: The two methods compared in this study operate in fundamentally different ways and provide substantially different information. EF5 binding provided detailed spatial information on the distribution of hypoxia in viable tumor tissue. There was no EF5 binding in necrotic tumor tissue because cells in such tissue were unable to metabolize the drug. In contrast, output from the needle electrode method appeared to represent a "track-average" tissue pO(2) and did not distinguish between extreme hypoxia and either macroscopic or microscopic necrosis. At the present time, the importance of tumor necrosis in determining treatment response is unknown. However, our data suggest that the Eppendorf needle electrode technique will overestimate the presence of hypoxia. Both techniques are potentially limited by sampling errors in tumors with heterogeneous distributions of hypoxia.     

Detection of hypoxia in human squamous cell carcinoma by EF5 binding

Localization and quantitation of 2-nitroimidazole drug binding in low pO2 tumors is a technique that can allow the assessment of hypoxia as a predictive assay. EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] is such a drug, and it has been shown to be predictive of radiation response in rodent tumors. Using fluorescence immunohistochemical techniques, we provide data on the presence, distribution, and levels of EF5 binding as a surrogate for hypoxia in human head and neck and uterine cervix squamous cell cancers (SCCs). Six patients with SCC were studied. Four patients had head and neck tumors, and two had uterine cervix cancers. The incubation of fresh tissue cubes in EF3 under hypoxic conditions ("reference binding") demonstrated that all tumors were capable of binding drug, and that this binding varied by a factor of 2.9-fold (174.5-516.1) on an absolute fluorescence scale. In the five patients treated at the lowest drug doses (9 mg/kg), in situ binding was quantitatable. For all six patients, the maximum rate of in situ binding varied by a factor of 6.7 between the lowest and highest binding tumor (24.8-160.3) on an absolute fluorescence scale. In tumors with high binding regions, intratumoral heterogeneity was large, extending from minimal fluorescence (<1%) up to 88.6% of reference binding. In tumors with minimal binding, there was little intratumoral heterogeneity. These studies demonstrate substantial heterogeneity of in situ binding between and within individual squamous cell tumors.     

Hypoxic fractions in xenografted human colon tumors

We investigated the percentage of radiobiologically hypoxic cells within 11 different xenografted human colon tumors using an in vivo-in vitro excision assay technique. Tumors were excised at average volumes of 750 mm3, and it was found that hypoxic fractions varied from less than 1% (clone D) to over 80% (HCT-8). The geometric mean hypoxic percentage was 10.4% (95% confidence interval, 4.9 to 22.1%). Comparison of the percentage of hypoxia results from the xenografted human colon tumors to published data from xenografted melanomas suggests that transplanted colorectal tumors as a class contain significantly less hypoxia than do the melanomas.     

Multiparameter analysis of vasculature, perfusion and proliferation in human tumour xenografts.

A method is presented in this report for concurrent analysis of vascular architecture, blood perfusion and proliferation characteristics in whole-tumour cross-sections of human larynx carcinoma and glioblastoma xenografts. Tumours were implanted subcutaneously in nude mice. After i.v. injection with Hoechst 33342 and bromodeoxyuridine (BrdUrd) as perfusion and proliferation markers, animals were killed. An antiendothelial antibody (9F1) was used to delineate vascular structures. Cross-sections were analysed by a multistep immune staining and a computer-controlled microscope scanning method. Each tumour section was stained and scanned four times (Hoechst, 9F1, BrdUrd and Fast Blue for all nuclei). When these images were combined, vasculature, perfusion and proliferation parameters were analysed. The labelling index (LI) was defined as the ratio of the BrdUrd-labelled area to the total nuclear area. The LI based on manual counting and the LI calculated by flow cytometry (FCM) were in good agreement with the LI based on surface analysis. LI decreased at increasing distance from its nearest vessel. In the vicinity of perfused vessels, the LI was 30-70% higher than near non-perfused vessels. This method shows that both vasculature/perfusion and proliferation characteristics can be measured in the same whole-tumour section in a semiautomatic way. This could be applied in clinical practice to identify combined human tumour characteristics that predict for a favourable response to treatment modifications.     

Hypoxic fractions of human tumors xenografted into mice: a review

Transplanted tumors in rats and mice have been used extensively in radiobiology studies examining the biological basis and therapeutic implications of hypoxia in solid tumors, and in studies evaluating new agents and regimens which may circumvent the radioprotective effects of hypoxic cells. This use of rodent tumors assumes that data obtained using rapidly-growing transplanted tumors arising in inbred rodents can be extrapolated meaningfully to the treatment of primary and/or metastatic lesions in heterozygous humans. The studies reported here examine one facet of this critical assumption by comparing the hypoxic fractions of transplanted rodent tumors with those of human tumor cell lines xenografted into immune deficient mice. No significant differences were found between the hypoxic fractions of the xenografts and those of mouse tumors. This finding eliminates several possible bases for predicting that the oxygenation of human tumors might be systematically different from that of the rodent tumors often used in preclinical radiobiology studies, and supports the hypothesis that human tumors contain hypoxic cells which may be critical in determining their response to therapy.     

Clinical significance of macrophage heterogeneity in human malignant tumors

The fact that various immune cells, including macrophages, can be found in tumor tissue has long been known. With the recent introduction of the novel concept of macrophage differentiation into a classically activated phenotype (M1) and an alternatively activated phenotype (M2), the role of tumor‐associated macrophages (TAMs) is gradually beginning to be elucidated. Specifically, in human malignant tumors, TAMs that have differentiated into M2 macrophages act as “protumoral macrophages” and contribute to the progression of disease. Based on recent basic and preclinical research, TAMs that have differentiated into protumoral or M2 macrophages are believed to be intimately involved in the angiogenesis, immunosuppression, and activation of tumor cells. In this paper, we specifically discuss both the role of TAMs in human malignant tumors and the cell–cell interactions between TAMs and tumor cells.     

Direct relationship between radiobiological hypoxia in tumors and monoclonal antibody detection of EF5 cellular adducts

While the potential importance of hypoxia in limiting the sensitivity of tumor cells to ionizing radiation has long been appreciated, methods for accurately quantifying the number of radiation-resistant hypoxic cells within tumors have been lacking. We have used the pentafluorinated derivative [2-(2-nitro-IH-imidazol-l-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide] of etanidazole (EF5), which binds selectively to hypoxic cells. The adducts formed between EF5 and cellular proteins in the hypoxic cells were detected using the specific monoclonal antibody (MAb), ELK3-51 conjugated to the flurochrome Cy3, and the number of hypoxic cells was quantified by flow cytometry. To verify the validity of this technique for the detection of hypoxic cells, mice bearing KHT sarcomas were treated with various agents to alter tumor oxygenation and hence vary the fraction of radiobiologically hypoxic tumor cells. The percentage of EF5 binding cells was then compared directly with the clonogenic survival of the tumor cells following radiation treatment under the various pretreatment conditions. The results showed that allowing the mice to breathe carbogen (5% CO₂/95% O₂) prior to irradiation reduced clonogenic cell survival approx. 6-fold and led to an absence of cells binding high levels of EF5. In contrast, pretreating the tumor-bearing animals with either hydralazine, which decreased tumor blood flow, or phenylhydrazine hydrochloride, which made the mice anemic, increased tumor cell survival following irradiation 2- to 4-fold, indicative of an increase in the fraction of hypoxic tumor cells. EF5 measurements made under identical conditions illustrated a shift in the cells in the tumor to high EF5 binding. Our results demonstrate that flow cytometric measurement by fluorescent MAb binding to EF5 adducts may relate directly to radiobiological hypoxia in KHT tumors measured by conventional methods. © 1996 Wiley-Liss, Inc.     

In vivo colocalization of 2-nitroimidazole EF5 fluorescence intensity and electron paramagnetic resonance oximetry in mouse tumors.

BACKGROUND AND PURPOSE: The primary objective of this study was to establish in vivo the relationship between 2-2-nitro-1H-imidazol-1yl-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5) adduct formation and intratumoral oxygen concentrations measured by electron paramagnetic resonance (EPR) in a tumor model mimicking a clinical situation. The secondary objective was an attempt to calibrate in situ the immunofluorescence (IF) signal with EPR oximetry. MATERIALS AND METHODS: IM syngeneic fibrosarcoma (NFSA) bearing C3H mice were used. Three days after injection of a paramagnetic charcoal into the tumor, the mice were anesthetized, injected with the hypoxic marker EF5, and monitored every 20 min for 3 h with a low-frequency EPR spectrometer. Animals were allowed to breath either under 21 or 100% O(2). Tumors were then harvested, frozen, cut into sections including the charcoal and processed for EF5 adducts detection using monoclonal antibodies. Slices were viewed with a fluorescence microscope and 190x140 micrometer areas surrounding the charcoal were digitized and analyzed with the NIH-Image and Adobe Photoshop software. The fluorescence intensity (FI) was measured in the whole pictures and in strips of 10 micrometer around the charcoal. RESULTS: EF5 binding increased with decreasing pO(2), most substantially at pO(2) below 5 mm Hg. Baseline (ambient air) pO(2) reached 3.2+/-2.1 mm Hg in NFSA tumors. It increased to 9.8+/-3.2 mm Hg under 100% O(2). A statistically significant correlation was observed on an individual tumor basis between the FI in the first 10 micrometer strip around the charcoal and the pO(2) determined by EPR oximetry (Wilcoxon signed rank test: P<0.001). CONCLUSIONS: The present study confirms the intrinsic relationship between EF5 adduct binding and intratumoral pO(2) in an in vivo environment under biologically-relevant pO(2) values of less than 10 mm Hg.     

Patterns of proliferation related to vasculature in human head-and-neck carcinomas before and after transplantation in nude mice

Purpose: The predictive potential of tumor cell kinetic parameters may be improved when they are studied in relation to other microenvironmental parameters. The purpose of this investigation was to quantitatively categorize human tumor samples according to proliferation patterns. Second, it was examined whether these characteristics are retained after xenotransplantation.

Methods and Materials: Fifty tumor samples from head-and-neck cancer patients were immunohistochemically stained for Ki-67 and vessels. Also, parts of the samples were transplanted into nude mice. Tumors were categorized according to previously described patterns of proliferation. Vascular and proliferation patterns were analyzed using an image processing system.

Results: The 50 tumors were categorized into four patterns of proliferation by visual assessment: marginal (6), intermediate (10), random (21), and mixed (12). One tumor could not be classified. These patterns were quantified by calculating the Ki-67 labeling index in distinct zones at increasing distance from vessels yielding good discrimination and significant differences between patterns. The probability of growth after xenotransplantation was significantly higher for tumors with a labeling index and vascular density above the median value compared to tumors with both parameters below the median (82% vs. 35%). Fifty percent of the tumors retained their proliferation patterns after xenotransplantation.

Conclusion: The categorization by proliferation pattern previously described by others was reproduced quantitatively and spatially related to the vascular network using a computerized image processing system. The combination of quantitative and architectural information of multiple microenvironmental parameters adds a new dimension to the study of treatment resistance mechanisms. Tumor models representative of the various patterns can be used to further investigate the relevance of these architectural patterns.     

Arabinofuranosyl-5-azacytosine: activity against human tumors in athymic mice

Summary Arabinofuranosyl-5-azacytosine (Ara-AC), a new compound structurally related to arabinofuranosyl-cytosine (Ara-C) and 5-azacytidine (5-AC), has demonstrated significant therapeutic activity against a wide spectrum of murine tumors and three human tumor xenografts in the NCI tumor panel. Studies on the activity of Ara-AC in these and other human tumor xenograft models were undertaken to define its potential antihuman-tumor profile more completely. Ara-AC demonstrated marked antitumor activity against human tumor xenografts, including leukemias and solid tumors that do not respond to Ara-C or 5-AC. An important finding was the demonstration that Ara-AC was as effective by the oral route as when given intraperitoneally. Furthermore, the compound demonstrated synergism when combined with cisplatin in the treatment of refractory solid tumors and also induced monocyte-type differentiation of promyelocytic leukemia (HL-60) cells in vitro. Ara-AC is a promising new compound that may have utility in the treatment of human cancer beyond that anticipated for a cytotoxic nucleoside.     

Cures and partial regression of murine and human tumors by recombinant human tumor necrosis factor

We tested the effect of recombinant human tumor necrosis factor (TNF) on the growth of the murine methylcholanthrene induced fibrosarcoma and the human ovarian carcinoma (NIH:OVCAR-3) in mice. The mice received multiple doses (25-250 micrograms/kg) of TNF starting 7-10 days after s.c. transplantation of tumors when they were easily palpable. TNF was administered i.v. every other day for a total of 6 injections per mouse, or i.p. daily for 7 days. Complete tumor regression was observed in the methylcholanthrene induced tumor bearing mice in 90% of the mice treated with TNF (100 micrograms/kg), 67% treated with TNF (50 micrograms/kg), and 34% treated with TNF (25 micrograms/kg). Tumors which did not completely regress were growth retarded during the course of TNF treatment. All mice given the highest TNF dose are still alive and tumor free (currently over 400 days), whereas the median survival of control mice was 28-39 days. Partial regression was observed in 100% of mice bearing the ovarian carcinoma treated i.p. with 250 micrograms/kg. Injections of TNF i.v. resulted in higher percentage of cures than i.p. injections at similar dose levels. These results suggest that tumor necrosis factor represents a likely potent drug against solid tumors and that the method of administration is critical in optimizing its use in cancer.