Targeting Artificial Tumor Stromal Targets for Molecular Imaging of Tumor Vascular Hypoxia.

Developed and tested for many years, a variety of tumor hypoxia detection methods have been inconsistent in their ability to predict treatment outcomes or monitor treatment efficacy, limiting their present prognostic capability. These variable results might stem from the fact that these approaches are based on inherently wide-ranging global tumor oxygenation levels based on uncertain influences of necrotic regions present in most solid tumors. Here, we have developed a novel non-invasive and specific method for tumor vessel hypoxia detection, as hypoxemia (vascular hypoxia) has been implicated as a key driver of malignant progression, therapy resistance and metastasis. This method is based on high-frequency ultrasound imaging of α-pimonidazole targeted-microbubbles to the exogenously administered hypoxia marker pimonidazole. The degree of tumor vessel hypoxia was assessed in three mouse models of mammary gland carcinoma (4T1, SCK and MMTV-Wnt-1) and amassed up to 20% of the tumor vasculature. In the 4T1 mammary gland carcinoma model, the signal strength of α-pimonidazole targeted-microbubbles was on average 8-fold fold higher in tumors of pimonidazole-injected mice than in non-pimonidazole injected tumor bearing mice or non-targeted microbubbles in pimonidazole-injected tumor bearing mice. Overall, this provides proof of principle for generating and targeting artificial antigens able to be 'created' on-demand under tumor specific microenvironmental conditions, providing translational diagnostic, therapeutic and treatment planning potential in cancer and other hypoxia-associated diseases or conditions.

Evaluation and immunohistochemical qualification of carbogen-induced ΔR2 as a noninvasive imaging biomarker of improved tumor oxygenation.

PURPOSE: To evaluate and histologically qualify carbogen-induced ΔR2 as a noninvasive magnetic resonance imaging biomarker of improved tumor oxygenation using a double 2-nitroimidazole hypoxia marker approach. METHODS AND MATERIALS: Multigradient echo images were acquired from mice bearing GH3 prolactinomas, preadministered with the hypoxia marker CCI-103F, to quantify tumor R2 during air breathing. With the mouse remaining positioned within the magnet bore, the gas supply was switched to carbogen (95% O2, 5% CO2), during which a second hypoxia marker, pimonidazole, was administered via an intraperitoneal line, and an additional set of identical multigradient echo images acquired to quantify any changes in tumor R2. Hypoxic fraction was quantified histologically using immunofluorescence detection of CCI-103F and pimonidazole adduct formation from the same whole tumor section. Carbogen-induced changes in tumor pO2 were further validated using the Oxylite fiberoptic probe. RESULTS: Carbogen challenge significantly reduced mean tumor R2 from 116 ± 13 s(-1) to 97 ± 9 s(-1) (P<.05). This was associated with a significantly lower pimonidazole adduct area (2.3 ± 1%), compared with CCI-103F (6.3 ± 2%) (P<.05). A significant correlation was observed between ΔR2 and Δhypoxic fraction (r=0.55, P<.01). Mean tumor pO2 during carbogen breathing significantly increased from 6.3 ± 2.2 mm Hg to 36.0 ± 7.5 mm Hg (P<.01). CONCLUSIONS: The combined use of intrinsic susceptibility magnetic resonance imaging with a double hypoxia marker approach corroborates carbogen-induced ΔR2 as a noninvasive imaging biomarker of increased tumor oxygenation.

Hypoxia regulation of phosphokinases and the prognostic value of pAKT in breast cancer.

Tumor hypoxia results in poor treatment response and is an indicator of poor outcome in cancer patients. TRIB3 is a hypoxia-upregulated protein involved in the ability of breast cancer cells to survive in hypoxic conditions. It is also involved in the prognosis of cancer patients, possibly by affecting several kinase-signaling pathways. We set out to establish which kinase-signaling pathways are regulated by hypoxia and whether these kinases are relevant for breast cancer prognosis. Using a phosphokinase antibody array comparing cells cultured under hypoxic conditions with those cultured during normoxia, we found that the phosphorylation status of ERK1/2, AKT, p70 S6 kinase, Lck and STAT3 was altered in both MCF7 and MDA-MB-231 breast cancer cells. Using Western blotting, we found that phosphorylated AKT (pAKT) increased in hypoxic conditions. Knockdown of TRIB3 attenuated this effect of hypoxia on AKT activation. Both pAKT and TRIB3 were expressed in pimonidazole-positive, hypoxic areas of human breast cancer tumors. In breast cancer patients significantly lower 5-year disease-free survival was observed for the pAKT-positive compared to the pAKT-negative group (64.6% vs 86.1%, p=0.03). In conclusion, the phosphorylation status of AKT is increased in hypoxic conditions and TRIB3 knockdown attenuates this response. Furthermore, pAKT expression denotes a worse prognosis in breast cancer patients. The hypoxia-related activation of AKT could explain the resistance to various treatments including chemotherapy and radiotherapy.

Tribbles homolog 3 denotes a poor prognosis in breast cancer and is involved in hypoxia response.

INTRODUCTION: Hypoxia in solid tumors is associated with treatment resistance, resulting in poor prognosis. Tribbles homolog 3 (TRIB3) is induced during hypoxia and is involved in multiple cellular pathways involved in cell survival. Here, we investigated the role of TRIB3 in breast cancer. METHODS: TRIB3 mRNA expression was measured in breast tumor tissue from 247 patients and correlated with clinicopathological parameters and clinical outcome. Furthermore, we studied TRIB3 expression regulation in cell lines, xenografts tissues and human breast cancer material using Reverse transcriptase, quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical staining. Finally, the effect of small interfering RNA (siRNA) mediated TRIB3 knockdown on hypoxia tolerance was assessed. RESULTS: Breast cancer patients with low, intermediate or high TRIB3 expression exhibited a mean disease free survival (DFS) of 80 (95% confidence interval [CI] = 74 to 86), 74 (CI = 67 to 81), and 63 (CI = 55 to 71) months respectively (P = .002, Mantel-Cox log-rank). The prognostic value of TRIB3 was limited to those patients that had received radiotherapy as part of their primary treatment (n = 179, P = .005) and remained statistically significant after correction for other clinicopathological parameters (DFS, Hazard Ratio = 1.90, CI = 1.17 to 3.08, P = .009). In breast cell lines TRIB3 expression was induced by hypoxia, nutrient starvation, and endoplasmic reticulum stress in an hypoxia inducible factor 1 (HIF-1) independent manner. TRIB3 induction after hypoxia did not increase with decreasing oxygen levels. In breast tumor xenografts and human breast cancer tissues TRIB3 co-localized with the hypoxic cell marker pimonidazole. The induction of TRIB3 by hypoxia was shown to be regulated via the PERK/ATF4/CHOP pathway of the unfolded protein response and knockdown of TRIB3 resulted in a dose-dependent increase in hypoxia sensitivity. CONCLUSIONS: TRIB3 is independently associated with poor prognosis of breast cancer patients, possibly through its association with tumor cell hypoxia.

Tuberculous granulomas are hypoxic in guinea pigs, rabbits, and nonhuman primates.

Understanding the physical characteristics of the local microenvironment in which Mycobacterium tuberculosis resides is an important goal that may allow the targeting of metabolic processes to shorten drug regimens. Pimonidazole hydrochloride (Hypoxyprobe) is an imaging agent that is bioreductively activated only under hypoxic conditions in mammalian tissue. We employed this probe to evaluate the oxygen tension in tuberculous granulomas in four animal models of disease: mouse, guinea pig, rabbit, and nonhuman primate. Following infusion of pimonidazole into animals with established infections, lung tissues from the guinea pig, rabbit, and nonhuman primate showed discrete areas of pimonidazole adduct formation surrounding necrotic and caseous regions of pulmonary granulomas by immunohistochemical staining. This labeling could be substantially reduced by housing the animal under an atmosphere of 95% O(2). Direct measurement of tissue oxygen partial pressure by surgical insertion of a fiber optic oxygen probe into granulomas in the lungs of living infected rabbits demonstrated that even small (3-mm) pulmonary lesions were severely hypoxic (1.6 +/- 0.7 mm Hg). Finally, metronidazole, which has potent bactericidal activity in vitro only under low-oxygen culture conditions, was highly effective at reducing total-lung bacterial burdens in infected rabbits. Thus, three independent lines of evidence support the hypothesis that hypoxic microenvironments are an important feature of some lesions in these animal models of tuberculosis.

Pimonidazole labelling and response to fractionated irradiation of five human squamous cell carcinoma (hSCC) lines in nude mice: the need for a multivariate approach in biomarker studies.

OBJECTIVE: To investigate the influence on local control after fractionated radiotherapy of hypoxia measured in unirradiated tumours using the hypoxic marker Pimonidazole, using multivariate approaches. MATERIAL AND METHODS: Five human squamous cell carcinoma lines (FaDu, UT-SCC-15, UT-SCC-14, XF354, and UT-SCC-5) were transplanted subcutaneously into the right hind-leg of NMRI nude mice. Histological material was collected from 60 unirradiated tumours after injection of Pimonidazole. The relative hypoxic area within the viable tumour area (Pimonidazole hypoxic fraction, pHF) was determined in seven serial 10 microm cross-sections per tumour by fluorescence microscopy and computerized image analysis. Local tumour control was evaluated in a total of 399 irradiated tumours at 120 days after 30 fractions given within 6 weeks with total doses between 30 and 115 Gy. RESULTS: Tumour lines showed pronounced heterogeneity in both pHF and TCD50. Mean pHF values varied between 5% and 37%, TCD50 values between 47 and 130 Gy. A Cox Proportional Hazards model of time to recurrence with two covariates, dose and pHF, yielded significant contributions of both parameters on local control (p < 0.005) but violated the proportional hazards assumption, suggesting that other factors also influence tumour control. Introduction of histological grade as an example of a confounding factor into the model improved the fit significantly. Local control rates decreased with increasing pHF and this effect was more pronounced at higher doses. CONCLUSIONS: This study confirms that tumour hypoxia measured using Pimonidazole in untreated tumours is a significant determinant of local control after fractionated irradiation. The data support the use of multivariate approaches for the evaluation of a single prognostic biomarker such as Pimonidazole, and more generally, suggest that they are required to establish accurate prognostic factors for tumour response.

The prognostic value of pimonidazole and tumour pO2 in human cervix carcinomas after radiation therapy: a prospective international multi-center study.

BACKGROUND AND PURPOSE: Hypoxia adversely affects treatment outcome in human uterine cervical cancer. Here, we present the results of a prospective international multi-centre study evaluating the prognostic value of pre-treatment tumour oxygen partial pressure (pO(2)) and the hypoxia marker pimonidazole (pimo). MATERIALS AND METHODS: One hundred and twenty-seven patients with primary cervix cancer were entered. Pre-treatment tumour pO(2) measurements were obtained, and reported by the median tumour pO(2), the fraction of pO(2) values

A comparison of oral and intravenous pimonidazole in canine tumors using intravenous CCI-103F as a control hypoxia marker.

PURPOSE: Pimonidazole HCl is widely used in immunohistochemical analyses of hypoxia in normal and malignant tissues. The present study investigates oral administration as a means of minimizing invasiveness. METHODS AND MATERIALS: Twelve dogs with confirmed malignancy received 0.5 g/m2 of pimonidazole HCl: 6 by mouth and 6 by i.v. infusion. All dogs received i.v. CCI-103F as a control. Plasma levels of pimonidazole, pimonidazole N-oxide, and CCI-103F were measured. Tumor biopsies were formalin fixed, paraffin embedded, sectioned, immunostained, and analyzed for pimonidazole and CCI-103F binding. pH dependence for pimonidazole and CCI-103F binding was studied in vitro. RESULTS: Pimonidazole and CCI-103F binding in carcinomas and sarcomas was strongly correlated for both oral and i.v. pimonidazole HCl (r2=0.97). On average, the extent of pimonidazole binding exceeded that for CCI-103F by a factor of approximately 1.2, with the factor ranging from 1.0 to 1.65. Binding of both markers was pH dependent, but pimonidazole binding was greater at all values of pH. CONCLUSIONS: Oral pimonidazole HCl is effective as a hypoxia marker in spontaneously arising canine tumors. Selective cellular uptake and concomitant higher levels of binding in regions of hypoxia at the high end of pH gradients might account for the greater extent of pimonidazole binding.

Hypoxic cell turnover in different solid tumor lines.

PURPOSE: Most solid tumors contain hypoxic cells, and the amount of tumor hypoxia has been shown to have a negative impact on the outcome of radiotherapy. The efficacy of combined modality treatments depends both on the sequence and timing of the treatments. Hypoxic cell turnover in tumors may be important for optimal scheduling of combined modality treatments, especially when hypoxic cell targeting is involved. METHODS AND MATERIALS: Previously we have shown that a double bioreductive hypoxic marker assay could be used to detect changes of tumor hypoxia in relation to the tumor vasculature after carbogen and hydralazine treatments. This assay was used in the current study to establish the turnover rate of hypoxic cells in three different tumor models. The first hypoxic marker, pimonidazole, was administered at variable times before tumor harvest, and the second hypoxic marker, CCI-103F, was injected at a fixed time before harvest. Hypoxic cell turnover was defined as loss of pimonidazole (first marker) relative to CCI-103F (second marker). RESULTS: The half-life of hypoxic cell turnover was 17 h in the murine C38 colon carcinoma line, 23 h and 49 h in the human xenograft lines MEC82 and SCCNij3, respectively. Within 24 h, loss of pimonidazole-stained areas in C38 and MEC82 occurred concurrent with the appearance of pimonidazole positive cell debris in necrotic regions. In C38 and MEC82, most of the hypoxic cells had disappeared after 48 h, whereas in SCCNij3, viable cells that had been labeled with pimonidazole were still observed after 5 days. CONCLUSIONS: The present study demonstrates that the double hypoxia marker assay can be used to study changes in both the proportion of hypoxic tumor cells and their lifespan at the same time. The present study shows that large differences in hypoxic cell turnover rates may exist among tumor lines, with half-lives ranging from 17-49 h.

Erythropoietin and erythropoietin receptor expression in head and neck cancer: relationship to tumor hypoxia.

PURPOSE: Erythropoietin, an oxygen-regulated glycoprotein hormone, is a hematopoietic cytokine that stimulates erythropoiesis by binding to its cellular receptor [erythropoietin receptor (EPOR)]. The recombinant form of human erythropoietin is used to prevent or treat anemia in cancer patients. However, in a recent randomized, placebo-controlled trial involving patients receiving curative radiotherapy for squamous cell carcinoma of the head and neck, erythropoietin treatment was associated with poorer locoregional progression-free survival. The purpose of our study was to determine whether EPOR and its ligand erythropoietin are expressed in primary head and neck cancer. We also investigated the hypothesis that erythropoietin expression in malignant cells may be associated with the presence of tumor hypoxia, an important factor involved in resistance to radiation treatment, tumor aggressiveness, and poor prognosis. EXPERIMENTAL DESIGN: Twenty-one patients received an i.v. infusion of the hypoxia marker pimonidazole hydrochloride before multiple tumor biopsies. Contiguous sections from 74 biopsies were analyzed by immunohistochemistry for EPOR and erythropoietin expression and pimonidazole binding. RESULTS: EPOR expression was present in tumor cells in 97% of the biopsies. Coexpression of erythropoietin was observed in 90% of biopsies. Erythropoietin and pimonidazole adduct staining did not always colocalize within tumors, but there was a significant positive correlation between levels of microregional erythropoietin expression and pimonidazole binding. CONCLUSIONS: The coexpression of erythropoietin and EPOR in tumor cells suggests that erythropoietin may potentially function as an autocrine or paracrine factor in head and neck cancer. The expression of the hypoxia-inducible protein erythropoietin in tumor cells correlates with levels of tumor hypoxia.

Hypoxia and differentiation in squamous cell carcinomas of the uterine cervix: pimonidazole and involucrin.

PURPOSE: Pimonidazole binding (hypoxia) and involucrin expression (differentiation) overlap extensively in squamous cell carcinomas. This study asks whether involucrin might serve as an endogenous marker for tumor hypoxia. A second question is whether differentiation affects hypoxia-inducible metallothionein (MT) expression in normal human epithelia and squamous cell carcinomas as it does in rodent epithelia. EXPERIMENTAL DESIGN: Thirty-four patients with squamous cell carcinoma of the uterine cervix were infused with pimonidazole hydrochloride solution. The next day, multiple biopsies were formalin-fixed, paraffin-embedded and sectioned at 4 micro m. Qualitative and quantitative analyses for involucrin expression, pimonidazole binding, and human MT-IIa mRNA expression were performed. RESULTS: No overall correlation between the extent of involucrin expression and pimonidazole binding was observed. The lack of correlation was because of heterogeneous patterns of immunostaining for involucrin generally related to tumor grade. Colocalized immunostaining for involucrin and pimonidazole binding was observed in intermediate grade tumors but not in well-differentiated or poorly differentiated tumors. Human MT-IIa mRNA and MT protein were expressed in basal lamina of normal human epithelia and in the proliferative rims of tumor nests. CONCLUSIONS: Colocalization of immunostaining for involucrin and pimonidazole binding is consistent with oxygen regulation, but the lack of involucrin expression in hypoxic regions of poorly differentiated tumors indicates that its transcriptional status with respect to hypoxia induction is altered by cell differentiation. The localization of MT message and protein in the outer rims of most tumor nests indicates that the transcriptional status of metallothionein is also altered by differentiation.

Chronic intragastric alcohol exposure causes hypoxia and oxidative stress in the rat pancreas.

The effect of chronic enteral ethanol on pancreatic hypoxia was investigated using the hypoxia marker, pimonidazole. Male Wistar rats were fed an ethanol-containing diet for 3 weeks using an enteral model shown to cause pancreatic damage; pimonidazole (120 mg/kg i.v.) was injected 1h before sacrifice. Pimonidazole and 4-hydroxynonenal (an index of lipid peroxidation) adducts were detected immunochemically. Breathing air with low oxygen content (8% O(2)) for 1h increased pimonidazole adduct accumulation approximately 2-fold in pancreata of nai;ve rats, confirming that this technique will detect increases in hypoxia in pancreata. Pancreata of rats fed ethanol began to show signs of damage after 3 weeks. Ethanol feeding also significantly increased pimonidazole adducts in pancreas approximately 2-fold (1 or 3 weeks of ethanol produced similar values). Concomitant with increasing hypoxia in the pancreas, alcohol also caused a significant increase in 4-hydroxynonenal adducts, indicative of increased oxidative stress. These results indicate that chronic ethanol causes hypoxia at the cellular level in the pancreas in vivo; further, the data support the hypothesis that hypoxia is involved in mechanisms of chronic alcoholic pancreatitis.

Measurements of hypoxia using pimonidazole and polarographic oxygen-sensitive electrodes in human cervix carcinomas.

BACKGROUND AND PURPOSE: The measurement of tumour oxygenation using Eppendorf oxygen-sensitive needle electrodes can provide prognostic information but the method is limited to accessible tumours that are suitable for electrode insertion. In this paper the aim was to study the relationship between such physiological measurements of tumour hypoxia and the labelling of tumours with the hypoxia-specific marker pimonidazole. MATERIALS AND METHODS: Assessment of tumour oxygen partial pressure (pO(2)) using an Eppendorf pO(2) histograph and immunohistochemical pimonidazole labelling was carried out in 86 patients with primary cervix carcinomas. Pimonidazole was given as a single injection (0.5 g/m(2) i.v.) and 10-24 h later pO(2) measurements were made and biopsies taken. Tumour oxygenation status was evaluated as the median tumour pO(2) and the fraction of pO(2) values

GLUT-1 and CAIX as intrinsic markers of hypoxia in carcinoma of the cervix: relationship to pimonidazole binding.

The presence of hypoxia in tumours results in the overexpression of certain genes, which are controlled via the transcription factor HIF-1. Hypoxic cells are known to be radioresistant and chemoresistant, thus, a reliable surrogate marker of hypoxia is desirable to ensure that treatment may be rationally applied. Recently, the HIF-1-regulated proteins Glut-1 and CAIX were validated as intrinsic markers of hypoxia by comparison with pO(2) measured using oxygen electrodes. We compare the expression of Glut-1 and CAIX with the binding of the bioreductive drug hypoxia marker pimonidazole. Pimonidazole was administered to 42 patients with advanced carcinoma of the cervix, 16 hr before biopsy. Sections of single or multiple biopsies were then immunostained for Glut-1 and CAIX, and the area of staining scored by eye, using a "field-by-field" semi-quantitative averaging system. Using 1 biopsy only, Glut-1 (r = 0.54, p = <0.001) correlated with the level of pimonidazole binding, and Glut-1 and CAIX expression also correlated significantly (r = 0.40, p = <0.009). Thus, our study has shown that HIF-1 regulated genes have potential for future use as predictors of the malignant changes mediated by hypoxia, and warrant further investigation as indicators of response to cancer therapy.

Synergy between vascular targeting agents and antibody-directed therapy.

PURPOSE: Tumor heterogeneity necessitates the use of combined therapies. We have shown that combining antibody-directed therapy with antivascular agents converts a subcurative to a curative treatment. The purpose of this study was to investigate, by radioluminographic and microscopic techniques, the regional effects of the two complementary therapies. METHODS AND MATERIALS: Nude mice bearing colorectal tumors were injected with 125I-labeled anti-carcinoembryonic antigen antibody, and images were obtained for antibody distribution and modeling studies using radioluminography. For therapy studies, the mice were given radioimmunotherapy alone (131I-A5B7 anti-carcinoembryonic antigen antibody), the antivascular agent combretastatin A-4 3-0-phosphate (200 mg/kg), or both. Extra mice were used to study the regional tumor effects of these therapies over time: relevant histochemical procedures were performed on tissue sections to obtain composite digital microscopic images of apoptosis, blood vessels, perfusion, hypoxia, and morphology. RESULTS: Antibody distribution, modeling, and immunohistochemistry showed how radioimmunotherapy (7.4 MBq/40 microg antibody) effectively treated the outer, well-oxygenated tumor region only. Combretastatin A-4 3-0-phosphate treated the more hypoxic center, and in doing so altered the relationship between tumor parameters. CONCLUSION: The combined complementary therapies produced cures by destroying tumor regions with different pathophysiologies. Relating these regional therapeutic effects to the relevant tumor parameters microscopically allows optimization of therapy and improved translation to clinical trials.

HIF-1A, pimonidazole, and iododeoxyuridine to estimate hypoxia and perfusion in human head-and-neck tumors.

PURPOSE: Tumor hypoxia measured by microelectrodes has been shown to indicate poor patient outcome. Here we investigated four potentially more widely applicable immunohistochemical parameters of tumor oxygenation and perfusion in human head-and-neck tumors. METHODS: Twenty patients with squamous cell carcinomas of the head and neck treated with primary surgery were injected with pimonidazole and IdUrd the evening before operation. Consecutive paraffin-embedded sections were stained for blood vessels, pimonidazole, IdUrd, and HIF-1alpha. IdUrd labeling and Ki-67 labeling around individual blood vessels were scored. The spatial relationship between HIF-1alpha and pimonidazole was studied, as well as the distribution of both markers as a function of distance from the nearest blood vessel. RESULTS: Measurement of all four parameters (diffusion-limited fraction, pimonidazole fraction, HIF-1alpha fraction, IdUrd-negative vessels) was feasible, and a significant difference between tumors was found for all parameters. IdUrd-labeled cells were absent around some vessels, indicating lack of perfusion, because these regions were positive for Ki-67. There was a positive correlation between diffusion-limited fraction and pimonidazole area for all images from all tumors, although no correlation for mean values per tumor. Colocalization of pimonidazole and HIF-1alpha was low (0.02%-25%). Most expression profiles showed a more homogenous distribution for HIF-1alpha than pimonidazole. There was no significant correlation between the pimonidazole and HIF-1alpha fractions in the 10 tumors studied. CONCLUSIONS: Simultaneous immunohistochemical measurements related to hypoxia and perfusion are feasible (and easily applicable) in resected human tumors. The different geographic distributions of HIF-1alpha and pimonidazole indicate that HIF-1alpha might not be suitable as a marker for chronic hypoxia. Each parameter will be correlated with outcome in a larger ongoing study on head-and-neck tumors treated with surgery with or without postoperative radiotherapy.

Orally administered pimonidazole to label hypoxic tumor cells.

Pimonidazole, a "hypoxia marker" normally delivered i.v. or i.p., was instead administered in the drinking water of tumor-bearing mice. As pimonidazole exposure was increased from 3-96 h ad libitum, both the fraction of hypoxic tumor cells and the relative number of pimonidazole adducts in those cells increased. Furthermore, the sustained ingestion of pimonidazole revealed a larger hypoxic fraction than did a single injection of an alternative hypoxia marker, CCI-103F. The "additional" hypoxia seen with longer-term oral administration apparently reflects the inclusion of transiently hypoxic tumor cells. Thus, in addition to its convenience and versatility when compared with hypoxia marker injection, oral administration of pimonidazole appears to permit identification of all of the physiologically and therapeutically relevant hypoxic tumor cells.

Pimonidazole binding and tumor vascularity predict for treatment outcome in head and neck cancer.

Hypoxia is associated with tumor aggressiveness and is an important cause of resistance to radiation treatment. Assays of tumor hypoxia could provide selection tools for hypoxia-modifying treatments. This study correlated the exogenous 2-nitroimidazole hypoxia marker 1-[(2-hydroxy-3-piperidinyl)propyl]-2-nitroimidazole hydrochloride (pimonidazole) with the endogenous hypoxia-related marker carbonic anhydrase 9 (CA9) and with vascular parameters using immunohistochemical techniques and a computerized image analysis system. Tumor biopsies were obtained from patients with head and neck carcinomas that were potential candidates for a Phase II trial with accelerated radiotherapy combined with carbogen and nicotinamide (ARCON). If, after completion of the diagnostic workup, the eligibility criteria were met and informed consent was obtained, patients were treated with ARCON. Those patients that were not eligible or refused ARCON were treated with radiotherapy, surgery, or a combined modality. Forty-three biopsies were analyzed, and the results were related with treatment outcome. The distribution patterns of pimonidazole and CA9 were similar, although the CA9 signal was generally observed already at shorter distances from blood vessels. There was a weak but significant correlation between the relative tumor areas positive for pimonidazole binding and areas with CA9 expression. Locoregional tumor control was significantly lower for patients who had hypoxic tumors or tumors with low vascular density. The 2-year control rates were 48 versus 87% for tumors with high and low pimonidazole binding levels (stratified by median, P = 0.01) and 48 and 88% for tumors with low and high vascular density (stratified by median, P = 0.01). These associations disappeared in the subgroup of patients treated with ARCON. There was no relationship between the level of CA9 expression and treatment outcome. It is concluded that pimonidazole binding and vascular density can predict treatment outcome in head and neck cancer and may be useful as selection tools for hypoxia-modifying treatments. Pimonidazole and CA9 demonstrate concordant staining patterns, but the latter is a less specific marker for hypoxia.

Functional significance of erythropoietin receptor expression in breast cancer.

Erythropoietin (EPO) is the principal hematopoietic cytokine that regulates mammalian erythropoiesis by binding to its transmembrane receptor EpoR. Recent experimental evidence suggests that the biologic effects of EPO are not limited to the regulation of erythropoiesis. In studies focusing on nonhematopoietic effects of EpoR signaling, we found high levels of EpoR protein expression in human breast cancer cells. The purpose of the present study was to evaluate clinical breast cancer specimens for EPO and EpoR expression, characterize the relationship between EPO expression and tumor hypoxia in biopsies prelabeled with hypoxia marker pimonidazole, analyze breast cancer cell lines for EpoR expression, and study the functional significance of EpoR expression in breast cancer cells in vivo. Immunohistochemical analysis for EPO, EpoR expression, and pimonidazole adducts was performed on 26 tumor biopsies with contiguous sections from 10 patients with breast cancer. High levels of EpoR expression were found in cancer cells in 90% of tumors. EPO expression was found in 60% of tumors and EPO and EpoR colocalization in tumor cells was present in many cases. The expression pattern of EPO with respect to tumor hypoxia was variable, without consistent colocalization of EPO and hypoxia in tumor cells. Human and rat breast cancer tissue culture cells express EpoR mRNA and protein. To study the in vivo function of EpoR expression in breast cancer cells, we used rat syngeneic R3230Ac mammary adenocarcinoma cells in a tumor Z-chamber model (dual porous plexiglass chambers containing fibrin gel, cancer cells, and a putative anti-tumor compound implanted into the subcutaneous tissue of rats). Local, one-time administration of a neutralizing anti-EPO antibody, soluble EPO receptor, or an inhibitor of Jak2, a cytoplasmic tyrosine kinase essential for EPO-mediated mitogenesis, resulted in a delay in tumor growth with 45% reduction in maximal tumor depth in tumor Z-chambers in a dose-dependent manner. These studies demonstrate the expression of functional receptors for EPO in breast cancer cells.

Tumor-specific regulation of angiogenic growth factors and their receptors during recovery from cytotoxic therapy.

After cytotoxic treatment, up-regulation of vascular growth factors and their receptors may be crucial for tumor relapse or progression. To determine how cytotoxic therapy (radioimmunotherapy) alters expression of angiogenic growth factors and receptors, athymic mice bearing LoVo, GW-39, HT-29, or Calu3 human tumor xenografts were treated with one dose (240 microCi) of (131)I-MN-14 anti-CEA IgG or (295 microCi) (131)I-RS-7-3G11 anti-EGP-1 IgG. Tumors removed at 1-week intervals up to week 6 were probed by immunohistochemistry (n = 3-11 samples) for vascular endothelial growth factor (VEGF), placental growth factor (PlGF), flk-1 and flt-1, angiopoietin-1 and -2, and Tie-1 and -2. Tumor extracts were also assayed for VEGF by immunoblot and for PlGF by comparative reverse transcription-PCR. During weeks 2-5 after radioimmunotherapy, significantly up-regulated tumor cell VEGF was only detected in HT-29 (immunohistochemistry; 2-fold; week 4; P < 0.05). The increased VEGF of HT-29 was paralleled by a 2-fold (week 4) rise in VEGF receptor flk-1 on vessels as well as increased ang-2/Tie-2. HT-29, GW-39, and Calu-3 increased tumor cell expression of PlGF by week 2 (HT-29 and Calu-3, P < 0.05). In Calu-3, PlGF mRNA increased 8-fold at week 5. PlGF was detected in hypoxic areas at week 2. Vascular expression of orphan receptor Tie-1 increased in all four of the tumors by week 2 (LoVo, GW-39, and Calu-3, P < 0.05). Regulation of angiogenic factors and angiogenesis after tumoricidal therapy may be tumor-specific. Antiangiogenic therapy may require a tumor-specific combination of inhibitors for PlGF, the angiopoietins, or their receptors, in addition to VEGF/flk-1.