Hypoxyprobe -1 Plus Kit for the Detection of Tissue Hypoxia

Transcription

1 Hypoxyprobe -1 Plus Kit for the Detection of Tissue Hypoxia 100 mg Pimonidazole 500 ul Hypoxyprobe -1 Mab1 FITC-Labeled 500 ul Secondary anti-fitc Mab Labeled with HRP Cat. No. HP2-100 FOR RESEARCH USE ONLY Not for use in diagnostic procedures USA & Canada Phone: +1(800) Fax: +1 (951) Europe +44 (0) Australia Germany ISO Registered Worldwide custserv@chemicon.com techserv@chemicon.com

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3 Introduction Oxygen gradients exist in normal and tumor tissue. These gradients affect gene expression and are important in normal and pathological conditions. Until recently it was difficult to measure these gradients at the cellular level; however, with the development of 2-nitroimidazole hypoxia markers, it is now possible to accurately measure oxygen gradient at the cellular level. There are a number of ways that hypoxia markers can be detected. The immunohistochemical technique is particularly attractive because oxygen gradients can be visualized and compared with underlying hierarchical structures in tissues and with gene expression on a cell-by-cell basis. This insert describes the Hypoxyprobe system and its application to studies of tissue hypoxia under both normal and pathological conditions. The hypoxia marker that has received most attention in the Hypoxyprobe system of markers is Hypoxyprobe -1, which is also known as pimonidazole hydrochloride. Hypoxyprobe -1 (pimonidazole hydrochloride) The kit contains reagents for an improved two-step immunohistochemical procedure for detecting hypoxia in rodent tissues using a primary fluorescein (FITC)-conjugated mouse monoclonal antibody (Mab) directed against pimonidazole protein adducts and a secondary mouse anti-fitc Mab conjugated to horseradish peroxidase (HRP). These reagents produce slides that are virtually free of the non-specific background staining that is frequently observed when rodent antibodies are utilized to detect antigens in rodent tissues (see Ref 50: J. Histochem Cytochem 52: , 2004) 1

4 Background In 1976, Varghese et al. reported that 14 C-labelled misonidazole formed adducts in hypoxic cells in vitro and in vivo (1). It was subsequently found that adducts form with thiol groups in proteins, peptides and amino acids in a way that all atoms of the ring and side-chain of the 2-nitroimidazole are retained (2-5). Hypoxia (po 2 < 10 mmhg) is required for binding but binding is not dependent on the presence of specialized redox enzymes such as P450 nitroreductases. Furthermore, wide variations in NADH and NADPH levels do not change the oxygen dependence of binding (6, 7). Chapman et al. showed that the oxygen dependence of binding was fortuitously close to that for radiation resistance and suggested that misonidazole binding might be used as a hypoxia marker in solid tumors (8). The clinical feasibility of the hypoxia marker idea was demonstrated by means of autoradiographic analyses of 3 H-misonidazole binding in a variety of human tumors (9). While the 3 H-misonidazole approach had limited clinical utility, it spurred the development of a variety of non-invasive assays for tissue hypoxia based on 2- nitroimidazoles. These included single photon electron capture tomography, positron emission tomography, nuclear medicine analysis and magnetic resonance spectroscopy of suitably labeled 2-nitroimidazole analogues (for review see ref. (10) ). During 19 F MRS investigations of tumor hypoxia with the hexafluorinated 2- nitroimidazole, CCI-103F, it became clear that an histological assessment of hypoxia would be a useful complement to non-invasive assays (11-13). This led to the invention of the immunochemical hypoxia marker technique based on monoclonal and polyclonal antibodies raised against protein adducts of reductively activated 2-nitroimidazoles (14, 15). Preclinical testing of immunochemical reagents in spontaneous canine tumors showed that immunochemical hypoxia markers would be useful in their own right (16-21). In addition to providing a quantitative measure of hypoxia, immunohistochemical markers provide insights into microregional relationships between hypoxia and factors such as necrosis, proliferation, differentiation, apoptosis, and oxygen regulated protein expression. A variety of immunochemical hypoxia markers have now been used in clinical (22-27) and preclinical studies (16, 28-33) of such relationships. An example of the unique value of the immunohistochemical marker approach is the observation that neither metallothionein nor vascular endothelial growth factor are expressed in the majority of hypoxic cells in human squamous cell carcinomas (24, 25) even though in vitro studies would have predicted otherwise (34, 35). With respect to quantifying hypoxia by the immunochemical technique, image analysis (24-27) or flow cytometric analysis (36, 37) appear most promising. Preclinical studies of sampling error showed that stratification of patients is 2

5 feasible with the immunohistochemical approach if 3-4 biopsies are obtained from geographically separate regions of each tumor. Precision can be increased by increasing the number of sections analyzed per biopsy from 1 to 3 but analysis of multiple biopsies is the most important factor. Interestingly, the accuracy of the immunochemical analysis increases as the amount of hypoxia decreases (19,21). Protein adducts of reductively-activated pimonidazole are effective immunogens for the production of both polyclonal and monoclonal antibodies. The antibodies have been used for immunoperoxidase analysis of formalin-fixed, paraffinembedded sections (6, 16, 23-26, 32, 40) ; for immunofluorescence analysis of frozen fixed sections (27, 41-43) ; and, for flow cytometry with directly labeled or secondary fluorescent antibodies (36).The antibodies have also been used in enzyme linked immunosorbent assays (6, 16, 40). One final attractive feature of pimonidazole is the fact that pimonidazole adducts in vivo are long-lived (16). This provides flexibility in the timing of biopsy taking which is an advantage in a clinical setting. In summary Hypoxyprobe -1 and associated antibodies form a very attractive basis on which to develop a low tech, low cost kit for measuring normal and tumor tissue hypoxia. Application The rationale for developing pimonidazole hydrochloride (Hypoxyprobe -1) as a hypoxia marker for experimental and clinical use was based on its chemical stability, water solubility, wide tissue distribution and, in the case of clinical studies, the fact that human toxicity data were available from earlier radiosensitizer trials. This facilitated early clinical application and Hypoxyprobe kits have now been used in many experimental studies and clinical trials worldwide. Solid Hypoxyprobe -1 is very stable being unchanged after storage for 2 years at room temperature in subdued light. Saline solutions of Hypoxyprobe -1 used for clinical studies (34 millimolar in 0.9% saline, ph 3.9 ± 0.1) are extremely stable being unchanged after 1.5 years at 4 o C in subdued light as determined by high performance liquid chromatography and ultraviolet spectroscopy. In addition to chemical stability, Hypoxyprobe -1 has high water solubility (400 millimolar; 116 grams per 1000 ml) that facilitates intravenous marker infusion and produces a short plasma half-life of 5.1 ± 0.8 hours. In spite of the water solubility of its hydrochloride salt (pka 8.7), pimonidazole itself has an octanol-water partition coefficient of 8.5 (38) and diffuses readily into tumors and normal tissues including brain (39). Consistent with a large, 155 liter volume of distribution, pimonidazole concentrates approximately 3 fold above plasma levels in tumors and normal tissues (39) thereby increasing the sensitivity of hypoxia marking. At the Hypoxyprobe -1 dose of 0.5 g/m 2 used 3

6 in hypoxia marking, pimonidazole causes neither central nervous system toxicity nor sensation (e.g., flushing) (39)..Central nervous system toxicity was of particular interest because this was the dose limiting toxicity for Hypoxyprobe -1 at the higher, multiple doses used in radiosensitizer trials. In addition to the absence of central nervous system effects, the overall procedure from Hypoxyprobe -1 infusion to tumor biopsy is well-tolerated in both inpatient and outpatient settings. Currently, rodent (mouse and rat) animal models are frequently used to study hypoxia in normal and tumor tissues, including human-to-rodent xenotransplantation experiments. Because the Hypoxyprobe -1 Mab1, which recognizes pimonidazole adducts in hypoxic tissue proteins, is of mouse origin, the presence of normal mouse or highly homologous rat immunoglobulins makes it difficult for the secondary antibody reagent to selectively bind to Mab1. Therefore, even when special blocking procedures are employed, the immunostaining of rodent tissues with Mab1 may result in a strong non-specific background staining that can obscure the specific binding pattern of this primary antibody. The Hypoxyprobe -1 Plus Kit for the Detection of Tissue Hypoxia was developed to address this obstacle and ensure fast (two-step) and accurate detection of hypoxia gradients in mouse and rat animal models. In this detection system, the primary monoclonal antibody Mab1 is directly labeled with FITC, while the secondary Mab (directed against FITC) is labeled with HRP. Since the secondary Mab does not cross-react with antigen epitopes present in rodent tissues, no non-specific background staining is generated using this set of reagents. For Research Use Only; Not for use in diagnostic procedures 4

7 Kit Components 1. Hypoxyprobe-1: (Part No.: 90203) One vial containing 100 mg of Pimonidazole Hydrochloride 2. Hypoxyprobe-1 Mab1: (Part No.: 90531) One vial containing 500 µl of purified Mab1 (clone ) conjugated with FITC. 3. Anti-FITC secondary Mab: (Part No.: 90532) One vial containing 500 µl of purified Mab against FITC (clone 5D6.2) conjugated with HRP. Storage The FITC-labeled Hypoxyprobe 1-Mab1 and the HRP-labeled Secondary Anti- FITC monoclonal antibody can be stored at 2-8 C until the expiration date indicated on the label. Keep dark. Hypoxyprobe -1 (Pimonidazole Hydrochloride) has great chemical stability in solid form and in aqueous solutions and requires no stabilizer. Solid Hypoxyprobe -1 has been stored for two years at room temperature in subdued light without detectable degradation as assessed by UV and HPLC analyses. Hypoxyprobe -1 solutions in 0.9% saline have been stored at a concentration of 10 g/liter (34 millimolar) at 4 C in subdued light for 1.5 years without detectable degradation (UV and HPLC analyses). When exposed to laboratory light, Hypoxyprobe -1 slowly turns yellow. Assay Instructions 1. Although doses of Hypoxyprobe -1 up to 400 mg/kg have been used without measurable toxicity in mice (36), a dose of 60 mg/kg body weight is routinely used in studies of tissue hypoxia in rodents. The high water solubility of Hypoxyprobe -1 permits small volume injections to be made which is convenient for studies with small animals. Intravenous injection or intraperitoneal injection can be used. The plasma half-life of Hypoxyprobe -1 is 0.5 hours in C3H/He mice. Hypoxyprobe -1 is distributed to all tissues in the body including brain but binds only to cells that have oxygen concentrations less than 14 micromolar, which is equivalent to a po 2 of 10 mm Hg at 37 o C. Tumors and normal liver, kidney and skin have cells at, or below, this po 2. For dogs, whole body doses of 0.28 gm/m 2 are recommended (16). The plasma half-life for Hypoxyprobe - 1 in dogs is 1.5 ± 1.0 hours. 5

8 In addition to animal studies, Hypoxyprobe kits can be used for cells in tissue culture (6, 44). Typically, cell suspensions are incubated under hypoxia for 1 to 2 hours in the presence of 100 to 200 micromolar Hypoxyprobe - 1. The cells are harvested by cytospin, fixed and immunostained with an antibody for pimonidazole adducts. Sufficient concentrations of pimonidazole adducts are formed on the surface of cells to elicit a response to complement or activated cytotoxic lymphocytes (44). Hypoxyprobe -1 Plus Kit has enough hypoxia marker to administer to 67 (25 gram) mice or 6-7 (250 gram) rats. 2. The primary antibody reagent is a conjugate consisting of Hypoxyprobe - 1 MAb1 (mouse IgG 1 ) covalently bound to FITC and is intended to detect protein adducts of Hypoxyprobe -1 in hypoxic cells. The conjugate is provided as a purified preparation at a concentration of 1 mg/ml. Fifteen to ninety minutes after the injection of Hypoxyprobe -1, tumor or normal tissue is excised or biopsied. The tissue of interest is then studied as frozen sections, formalin-fixed paraffin-embedded sections, or as disaggregated tissues in flow cytometry assays. In the case of formalin-fixed, paraffinembedded tissue sections, 100 µl of a 1:50 dilution of the conjugate is added to each tissue section. Hypoxyprobe -1 Plus Kit has enough primary conjugate to analyze 250 tissue sections at a 1:50 dilution. Optimal working dilutions must be determined by the end user. 3. The secondary antibody reagent is a conjugate consisting of a mouse anti- FITC covalently bound to HRP and is intended to detect Mab1-FITC stained structures in target slides. The conjugate is provided as a purified preparation at a concentration of 1 mg/ml. In the case of formalin-fixed, paraffin-embedded tissue sections, 100 µl of a 1:50 dilution of the conjugate is added to each tissue section. We recommend utilizing standard colorimetric systems used in visualizing HRP-stained tissue sections in routine IHC protocols. Hypoxyprobe -1 Plus Kit has enough secondary conjugate to analyze 250 tissue sections at a 1:50 dilution. Optimal working dilutions must be determined by the end user. 6

9 Peroxidase Immunohistochemistry Technique Recommended procedure for immunostaining Hypoxyprobe-1 adducts in formalin-fixed, paraffin-embedded tissues from a variety of animal species including mice and rats. Other tissue fixation methods will require optimization of the suggested protocol. Improved immunohistochemical method for detecting hypoxia gradients in mouse tissues and tumors. Samoszuk MK, Walter J, Mechetner E. J. Histochem. Cytochem. 52(6): , Step Procedure Time, min. Temp. Reagents Notes Heat Fix tissue slides Deparaffinize tissue Deparaffinize tissue Deparaffinize tissue Clear slides of Xylene Clear slides of Xylene Begin hydrating tissue Continue to hydrate o C None 1 3 RT Xylene 2 3 RT Xylene 3 RT Xylene 1 RT 100% Ethanol 1 RT 100% Ethanol 1 RT 70% Aqueous ethanol 1 RT 30% Aqueous ethanol 9 Hydrate tissue 1 RT Deionized water 10 Hydrate tissue 1 RT Deionized water 11 Transfer slides to rinse buffer 2 RT 1x TBS + Tween

10 Step Procedure Time, min. Temp. Reagents Notes 12 Quench tissue peroxidase 5 RT 3% H 2 O 2 in distilled water 4 13 Antigen retrieval 20 Steamer 10mM Citrate, ph Transfer slides to rinse buffer Block non-specific binding 2 RT 1x TBS + Tween RT Blocking reagent 7 16 Incubate in Primary conjugate 30 RT Hypoxyprobe-1 MAb1 conjugated with FITC (1:50) 8 17 Wash in rinse buffer 2 RT 1x TBS + Tween Incubate in Secondary conjugate 30 RT Anti-FITC Mab conjugated with HRP (1:50) 9 19 Wash in rinse buffer 2 RT 1x TBS + Tween Peroxidase chromogen 10 RT DAB Stop DAB reaction 1 RT Distilled water 22 Wash in rinse buffer 1 RT 1x TBS + Tween Counterstaining 1 RT Hematoxylin Wash in rinse buffer 1 RT 1x TBS + Tween Dehydration & Clearing See notes RT 100% alcohol and Xylene Coverslip N/a RT Permount 13 8

11 Technical Notes 1. This step may be performed in microwave oven on high for 2 minutes. If the sections are still wet, the water will turn to steam and separate the tissue from slides. Caution, glass can get very hot. 2. Per xylene incubation use clean xylene. Clear-Rite 3 is a non-toxic alternative to xylene and is available from Richard-Allan Scientific, Kalamazoo, MI (Cat. No. 6901). If using this alternative, increase the incubation periods accordingly x TBS is Tris buffered saline, 10 mm (Chemicon Cat. No ). All rinses were done with 1x TBS working solution. 4. Make sure to rinse off the 3% H 2 O 2 since it can interfere with results. Store brand hydrogen peroxide may be utilized. 5. Antigen retrieval solution is 10x Citrate Buffer (Chemicon Cat. No ) used at a 1x working solution. 6. HIER method is at the end users discretion. 7. Blocking reagent (Chemicon Cat. No ). 1% BSA or 1% normal mouse serum in TBS may also be used. 8. The primary antibody reagent is a conjugate between Hypoxyprobe -1 MAb1 (mouse IgG 1 ) and FITC. The conjugate is provided as a purified preparation at a concentration of 1 mg/ml. Dilute the primary conjugate solution 1:50 in Antibody Diluent (Chemicon Cat. No ). Typically, 100 µl of diluted primary conjugate solution is applied to each tissue section. 9. The secondary antibody reagent is a conjugate between a monoclonal antibody against FITC and HRP provided as a purified preparation at a concentration of 1 mg/ml. Dilute the secondary antibody solution 1:50 in Antibody Diluent (Chemicon Cat. No ). Typically, 100 µl of diluted secondary conjugate is added to each tissue section. 10. Liquid 3,3'-diaminobenzidine reagent (DAB). DAB Chromogen-A (Chemicon Cat. No ) and DAB Chromogen-B (Chemicon Cat. No ) mix at 1:25 ratio respectively. 11. Hematoxylin counter staining reagent (Chemicon Cat. No ). 12. Use clean and fresh solutions to incubate slides for 1 minute in 4 changes of 100% alcohol and then to three changes of clean xylene. 13. Permount solution is available from Fisher Scientific (Cat. No. SP15-500). 9

12 Frequently Asked Questions Q: What is the best solvent for Hypoxyprobe -1? A: Hypoxyprobe -1 is the hydrochloride salt of a weak base and, as such, is very soluble in aqueous solutions including neutral buffered saline (116 mg/ml or 400 millimolar). Q. What dose of Hypoxyprobe -1 should be used for hypoxia marking experiments? A: For small animals of uniform size such as laboratory rats and mice, a dose of Hypoxprobe -1 of 60 mg/kg body weight is recommended. Doses ranging from 30 mg/kg (47) to 400 mg/kg (36) have been used in mice and rats without toxicity or altered oxygen levels due to blood flow effects. Nevertheless, significant effects on blood flow at doses above 100 mg/kg of Hypoxyprobe -1 have been observed in tumors implanted in the hind legs of mice. Caution must be taken, therefore, when doses > 100 mg/kg are used. For larger animals with non-uniform body size, the dose is calculated on the basis of surface area. For humans, the recommended dose is 0.5 gm/m2 (23) while for dogs a dose of 0.28 gm/m2 has been used (16). Q. Does Hypoxyprobe -1 penetrate hypoxic brain and brain tumor tissue? A: Although Hypoxyprobe -1 is water soluble, its corresponding free base has an octanol water coefficient of 8.5 and, as a result, the marker freely penetrates into both brain and brain tumor tissue (48, 49). 10

13 Q. Is Hypoxyprobe -1 the best probe for detecting hypoxia in vivo? A: Although other markers are available for detecting hypoxia in vivo, Hypoxyprobe -1 has some real advantages. Foremost is its high solubility in aqueous solution (400 millimolar; 116 mg/ml of saline), which allows the marker to be administered to rodents as small volume (= 0.1 ml) injections of saline either intraperitoneally or intravenously. Other markers such as the hexafluorinated CCI-103F have aqueous solubilities of 10 millimolar or less. Although Hypoxyprobe -1, the hydrochloride salt of pimonidazole, is very water soluble, pimonidazole itself has an octanol-water partition coefficient of 8.5 and penetrates all tissues including brain. Another advantage is that pimonidazole binding can be detected by immunofluorescence in frozen fixed tissue sections; by immunoperoxidase in formalin-fixed paraffin-embedded tissue sections; by ELISA or by flow cytometry. Q. Can the monoclonal antibody to Hypoxyprobe -1 adducts be used on mouse or rat tissues? A: Yes. The described procedure results in a clean IHC staining that is free of non-specific background caused by mouse or rat immunoglobulins impregnating the surrounding tissue (50). 11

14 References 1. Varghese, A. J., Gulyas, S., and Mohindra, J. K. (1976). Hypoxia-dependent reduction of 1-(2-nitro-1-imidazolyl)-3-methoxy-2- propanol by Chinese hamster ovary cells and KHT tumor cells in vitro and in vivo, Cancer Res. 36: Chacon, E., Morrow, C. J., Leon, A. A., Born, J. L., and Smith, B. R. (1988). Regioselective formation of a misonidazole-glutathione conjugate as a function of ph during chemical reduction., Biochem. Pharmacol. 37: Raleigh, J. A., Franko, A. J., Koch, C. J., and Born, J. L. (1985). Binding of misonidazole to hypoxic cells in monolayer and spheroid culture: evidence that a side-chain label is bound as efficiently as a ring label. Br. J. Cancer. 51: Raleigh, J. A. and Koch, C. J. (1990). Importance of thiols in the reductive binding of 2-nitroimidazoles to macromolecules. Biochem Pharmacol. 40: Varghese, A. J. (1983). Glutathione conjugates of misonidazole. Biochem Biophys Res Commun. 112: Arteel, G. E., Thurman, R. G., Yates, J. M., and Raleigh, J. A. (1995). Evidence that hypoxia markers detect oxygen gradients in liver: pimonidazole and retrograde perfusion of rat liver. Br J Cancer. 72: Arteel, G. E., Thurman, R. G., and Raleigh, J. A. (1988). Reductive metabolism of the hypoxia marker pimonidazole is regulated by oxygen tension independent of the pyridine nucleotide redox state. Eur. J. Biochem. 253: Chapman, J. D., Franko, A. J., and Sharplin, J. A (1981). marker for hypoxic cells in tumours with potential clinical applicability. Br. J. Cancer. 43: Urtasun, R. C., Koch, C. J., Franko, A. J., Raleigh, J. A., and Chapman, J. D. (1986). A novel technique for measuring human tissue po2 at the cellular level. Br. J. Cancer. 54: Raleigh, J. A., Dewhirst, M. W., and Thrall, D. E. Measuring tumor hypoxia., Sem. Radiat. Oncol. 6:37-45, Jin, G. Y., Li, S. J., Moulder, J. E., and Raleigh, J. A. Dynamic measurements of hexafluoromisonidazole (CCI-103F) retention in mouse tumours by 1H/19F magnetic resonance spectroscopy, Int. J. Radiat. Biol. 58: , Raleigh, J. A., Franko, A. J., Treiber, E. O., Lunt, J. A., and Allen, P. S. Covalent binding of a fluorinated 2-nitroimidazole to EMT-6 tumors in BALB/C mice: Detection by F-19 nuclear magnetic resonance at 2.35T, Int. J. Radiat. Oncol. Biol. Phys. 12: ,

15 13. Raleigh, J., Franko, A., Kelly, D., Trimble, L., and Allen, P. (1991). Development of an in vivo 19F magnetic resonance method for measuring oxygen deficiency in tumors. Magn. Res. Med. 22: Raleigh, J. A., Miller, G. G., Franko, A. J., Koch, C. J., Fuciarelli, A. F., and Kelly, D. A. (1987). Fluorescence immunohistochemical detection of hypoxic cells in spheroids and tumours. Br J Cancer 56: Raleigh, J. A., Miller, G. G., Franko, A. J., and Chapman, J. D. Immunochemical detection of hypoxia in normal and tumor tissue. USA patent 5,086,068, February, Azuma, C., Raleigh, J. A., and Thrall, D. E. (1997). Longevity of pimonidazole adducts in spontaneous canine tumors as an estimate of hypoxic cell lifetime. Radiat. Res. 148: Cline, J. M., Thrall, D. E., Page, R. L., Franko, A. J., and Raleigh, J. A. (1990). Immunohistochemical detection of a hypoxia marker in spontaneous canine tumours. Br. J. Cancer 62: Cline, J. M., Thrall, D. E., Rosner, G. L., and Raleigh, J. A. (1994). Distribution of the hypoxia marker CCI-103F in canine tumors. Int J Radiat Oncol Biol Phys. 28: Cline, J., Rosner, G., Raleigh, J., and Thrall, D. (1997). Quantification of CCI- 103F labeling heterogeneity in canine solid tumors. Int. J. Radiat. Oncol. Biol. Phys. 37: Thrall, D. E., McEntee, M. C., Cline, J. M., and Raleigh, J. A. (1994). ELISA quantification of CCI-103F binding in canine tumors prior to and during irradiation. Int. J. Radiat. Oncol. Biol. Phys. 28: Thrall, D. E., Rosner, G. L., Azuma, C., McEntee, M. C., and Raleigh, J. A. (1997). Hypoxia marker labeling in tumor biopsies: quantification of labeling variation and criteria for biopsy sectioning. Radiother Oncol. 44: Evans, S. M., Hahn, S., Pook, D. R., Jenkins, W. T., Chalian, A. A., Zhang, P., Stevens, C., Weber, R., Weinstein, G., Benjamin, I., Mirza, N., Morgan, M., Rubin, S., McKenna, W. G., Lord, E. M., and Koch, C. J. (2000). Detection of hypoxia in human squamous cell carcinoma by EF5 binding. Cancer Res. 60: Kennedy, A. S., Raleigh, J. A., Perez, G. M., Calkins, D. P., Thrall, D. E., Novotny, D. B., and Varia, M. A. (1997). Proliferation and hypoxia in human squamous cell carcinoma of the cervix: first report of combined immunohistochemical assays. Int J Radiat Oncol Biol Phys. 37:

16 24. Raleigh, J. A., Calkins-Adams, D. P., Rinker, L. H., Ballenger, C. A., Weissler, M. C., Fowler, W. C., Jr., Novotny, D. B., and Varia, M. A. (1998). Hypoxia and vascular endothelial growth factor expression in human squamous cell carcinomas using pimonidazole as a hypoxia marker. Cancer Res. 58: Raleigh, J., Chou, S.-C., Calkins-Adams, D., Ballenger, C., Rinker, L., Novotny, D., and Varia, M. (2000). A clinical study of hypoxia and metallothionein protein expression in squamous cell carcinomas. Cl. Cancer Res. 6: Varia, M. A., Calkins-Adams, D. P., Rinker, L. H., Kennedy, A. S., Novotny, D. B., Fowler, W. C., Jr., and Raleigh, J. A. (1998). Pimonidazole : a novel hypoxia marker for complementary study of tumor hypoxia and cell proliferation in cervical carcinoma. Gynecol. Oncol. 71: Wijffels, K., Kaanders, J., Rijken, P., Bussink, J., Van den Hoogen, F., Marres, H., de Wilde, P., Raleigh, J., and Van der Kogel, A. (2000). Vascular architecture and hypoxic profiles in human head and neck squamous cell carcinomas. Br. J. Cancer. 83: Carmeliet, P., Dor, Y., Herbert, J. M., Fukumura, D., Brusselmans, K., Dewerchin, M., Neeman, M., Bono, F., Abramovitch, R., Maxwell, P., Koch, C. J., Ratcliffe, P., Moons, L., Jain, R. K., Collen, D., and Keshet, E. (1998). Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature 394: Hodgkiss, R. J. (1998). Use of 2-nitroimidazoles as bioreductive markers for tumour hypoxia. Anticancer Drug Des. 13: Koch, C. J., Chasan, J. E., Jenkins, W. T., Chan, C. Y., Laughlin, K. M., and Evans, S. M. (1998). Co-localization of hypoxia and apoptosis in irradiated and untreated HCT116 human colon carcinoma xenografts. Adv Exp Med Biol. 454: Raleigh, J. A., Zeman, E. M., Calkins, D. P., McEntee, M. C., and Thrall, D. E. (1995). Distribution of hypoxia and proliferation associated markers in spontaneous canine tumors. Acta Oncol. 34: Raleigh, J. A., Chou, S.-C., Tables, L., Suchindran, S., Varia, M. A., and Horsman, M. R. (1998). Relationship of hypoxia to metallothionein expression in murine tumors. Int. J. Radiat. Oncol. Biol. Phys. 42: Waleh, N. S., Brody, M. D., Knapp, M. A., Mendonca, H. L., Lord, E. M., Koch, C. J., Laderoute, K. R., and Sutherland, R. M. (1995). Mapping of the vascular endothelial growth factor-producing hypoxic cells in multicellular tumor spheroids using a hypoxia-specific marker. Cancer Res. 55:

17 34. Leith, J. T. and Michelson, S. (1995). Secretion rates and levels of vascular endothelial growth factor in clone A or HCT-8 human colon tumour cells as a function of oxygen concentration. Cell Prolif. 28: Murphy, B. J., Andrews, G. K., Bittel, D., Discher, D. J., McCue, J., Green, C. J., Yanovsky, M., Giaccia, A., Sutherland, R. M., Laderoute, K. R., and Webster, K. A. (1999). Activation of metallothionein gene expression by hypoxia involves metal response elements and MTF-1. Cancer Res. 59: Durand, R. E. and Raleigh, J. A. (1998). Identification of nonproliferating but viable hypoxic cells in vivo. Cancer Res. 58: Webster, L., Hodgkiss, R. J., and Wilson, G. D. (1995). Simultaneous triple staining for hypoxia, proliferation, and DNA content in murine tumours. Cytometry 21: Smithen, C., Clarke, E., Dale, J., Jacobs, R., Wardman, P., Watts, M., and Woodcock, M. Novel (nitro-1-imidazoyl)-alkanolamines as potential radiosensitizers with improved therapeutic properties. In: L. Brady (ed.) Radiation Sensitizers. Their Use in the Clinical Management of Cancer., pp New York: Masson, Saunders, M. I., Anderson, P. J., Bennett, M. H., Dische, S., Minchinton, A. I., and Stratford, M. R. L. (1984). The clinical testing of Ro pharmacokinetics, toxicology, tissue and tumor concentrations. Int. J. Radiat. Oncol. Biol. Phys. 10: Arteel, G. E., Iimuro, Y., Yin, M., Raleigh, J. A., and Thurman, R. G. (1997). Chronic enteral ethanol treatment causes hypoxia in rat liver tissue in vivo. Hepatology 25: Bussink, J., Kaanders, J. H., Rijken, P. F., Raleigh, J. A., and Van Der Kogel, A. J. (2000). Changes in blood perfusion and hypoxia after irradiation of a human squamous cell carcinoma xenograft tumor line. Radiat. Res. 153: Rijken, P., Bernsen, H., Peters, J., Hodgkiss, R., Raleigh, J., and van der Kogel, A. (2000). Spatial relationship between hypoxia and the (perfused) vascular network in human glioma xenografts: a quantitative multi-parameter analysis. Int. J. Radiat. Oncol. Biol. Phys. 48: Bernsen, H. J., Rijken, P. F., Peters, H., Raleigh, J. A., Jeuken, J. W., Wesseling, P., and van der Kogel, A. J. (2000). Hypoxia in a human intracerebral glioma model. J Neurosurg. 93: Chou, S. C., Flood, P. M., and Raleigh, J. A. (1996). Marking hypoxic cells for complement and cytotoxic T lymphocyte- mediated lysis: using pimonidazole. Br J Cancer Suppl. 27:S213-S

18 45. Ljungkvist,ASE, Bussink, J, Raleigh, JA, Denekemp, J and van der Kogel, AJ. (2000). Changes in tumor hypoxia measured with a double hypoxic marker technique. Int. J. Radiat. Oncol. Biol. Phys. 48: Wykoff, CC, Beasley, NJ, Watson, PH, Turner, KJ, Pastorek, J, Sibtain, A, Wilson, GD, Turley, H, Talks, KL, Maxwell, PH, Pugh, CW, Ratcliffe, PJ and Harris, AL. (2000). Hypoxia-inducible expression of tumor-associated carbonic anhydrases. Cancer Res. 60: Rofstad, EK and Maseide, K. (1999). Radiobiological and immunohistochemical assessment of hypoxia in human melanoma xenografts: acute and chronic hypoxia in individual tumours. Int. J. Radiat. Biol. 75: Newman, HFV, Bleehen, NM, Ward, R and Workman, P. (1988). Hypoxic cell radiosensitizers in the treatment of high grade gliomas: A new direction using combined Ro (pimonidazole) and SR 2508 (etanidazole). Int. J. Radiat. Oncol. Biol. Phys. 15: Roberts, JT, Bleehen, NM, Walton, MI, and Workman, P. (1986). A clinical phase I toxicity study of Ro : plasma, urine, tumour and normal brain pharmacokinetics. Br. J. Radiology 59: Samoszuk MK, Walter J, and Mechetner E. (2004). Improved immunohistochemical method for detecting hypoxia gradients in mouse tissues and tumors. J Histochem Cytochem. 52: Warranty These products are warranted to perform as described in their labeling and in CHEMICON literature when used in accordance with their instructions. THERE ARE NO WARRANTIES, WHICH EXTEND BEYOND THIS EXPRESSED WARRANTY AND CHEMICON DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE. CHEMICON s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be, at the option of CHEMICON, to repair or replace the products. In no event shall CHEMICON be liable for any proximate, incidental or consequential damages in connection with the products. 2004: CHEMICON International, Inc. - By CHEMICON International, Inc. All rights reserved. No part of these works may be reproduced in any form without permissions in writing. 16

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20 Cat. No. HP2-100 September 2004 Revision B; 41631