Immunofluorescence intensity was visually inspected and EF5-positive labeling was represented by intensities above the 75th percentile, as described previously ( 34). Measurements of fluorescence intensity in viable tumor areas were done using Image-Pro Plus 6.1.0 (Media Cybernetics). Fluorescence of debris and other artifacts was omitted. ![]() H&E images were reviewed to generate masks of viable tumor areas for individual images of immunofluorescence using image analysis tools developed in-house on the basis of IDL 6.3 programming language. Immunohistochemical and immunofluorescent staining The percentages of EF5- and BrdUrd-positive cells were analyzed using WinList 6.0 (Verity). Single-parameter DNA content histograms were analyzed using ModFit LT 3.0 (Verity). Samples were analyzed using an LSRII flow cytometer (BD Biosciences). Anti-vimentin and anti-cytokeratin 19 (both from Abcam) were also used for staining. Cameron Koch, University of Pennsylvania), anti-BrdUrd (PRBQD-Alexa 488 Phoenix Flow Systems), and 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI). For dual EF5 and BrdUrd labeling, cells were acid denatured, neutralized, and then stained with anti-EF5 monoclonal antibody (Cy5 conjugates, ELK3-51, obtained from Dr. Aliquots of single-cell suspensions were then fixed either in 80% ethanol to allow DNA denaturation for anti-BrdUrd labeling or in 4% formaldehyde for 10 minutes to optimize intracellular protein staining. Single-cell suspensions from tumors were prepared by an enzymatic technique (Collagenase XI, Protease, and DNase I cocktail) for flow cytometry as described previously ( 32, 33). The results suggest that hypoxia is a major adverse prognostic factor in pancreatic cancer patients and support the introduction of techniques to measure hypoxia directly in patients and the development of treatment protocols to target hypoxia. mRNA expression analysis showed increased expression of genes involved in cell survival and proliferation in the hypoxic models. Hypoxia was highly correlated with rapid tumor growth, increased BrdUrd uptake, and with spontaneous metastasis formation. EF5 labeling, tumor growth rates, and metastatic patterns were highly consistent within replicates, indicating a significant transmissible (genetic or epigenetic) component. ![]() The 16 primary xenografts showed a wide range in their growth rates and metastatic potential, reminiscent of the spectrum of behavior seen in the clinic. Orthotopic implants closely resembled the histology of the original surgical samples. Bromodeoxyuridine (BrdUrd) uptake, microvessel density, cleaved caspase-3, and the differentiation markers E-cadherin, cytokeratin 19, and vimentin were analyzed in relation to hypoxia. ![]() Hypoxic cells were labeled using the 2-nitroimidazole probe EF5 and stained for immunofluorescence microscopy of tissue sections or as cell suspensions for flow cytometry. Early passage xenografts were established from 16 patients undergoing surgery for pancreatic cancer and maintained in the pancreas of immune-deprived mice. To address this, we examined the associations between hypoxia and biological aggression in a series of patient-derived xenografts grown orthotopically. Although hypoxia has been reported in pancreatic cancer patients, there is little direct evidence that this contributes to their overall poor prognosis. Hypoxia in solid tumors is associated with treatment resistance and increased metastatic potential.
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