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Identification of Prognostic
Markers and Selective Targeting Agents
of the Tumour Microenvironment
David Hedley
Our lab investigates protein expression in hypoxic regions of
cervical and pancreatic tumours, and in patient biopsy samples
taken as part of the Hypoxia Program. The main long term goal is
better understanding of the molecular processes involved in
tumour hypoxia, and the development of new forms of cancer
treatment that selectively target these mechanisms.
We use the drug EF5 as a hypoxia marker. We also study
the vasculature in tumors using antibodies to the endothelial
marker CD31. Tumour cells are grown both in cell culture and as
xenografts in SCID mice. Drug effects are evaluated by measuring
tumor growth delay in xenografts and alterations in protein
expression in the tumour cells.
We study protein expression using flow cytometry, Western
blots and wide-field fluorescence microscopy. Microscopy is
usually done by “tiling” the image, using one of our two
epifluorescence microscopes equipped with CCD cameras and
motorized stages. In this way an entire tumour section several
millimeters in diameter can be imaged at up to 200X
magnification. Up to 4 antibodies can be visualized at one time,
thus allowing us to study the colocalization of up to 4 proteins
simultaneously. Because the motorized stages are very accurate,
the images of the 4 different antibodies are exactly aligned,
allowing accurate colocalization measurements to be performed.
Our lab has recently acquired a new fluorescence imaging system
called the TISSUEscope®, made by Biomedical Photometrics Inc.
of Waterloo, Ontario. Although the resolution of this system is
only 2 microns, it is capable of imaging a section up to 20mm x
20mm in size ten times faster than tiling. It can visualize up
to 3 different antibodies at once. We also have a transmitted
light microscope equipped with a camera and motorized stage for
tiling immunochemistry slides.
We are interested in computerized image analysis of
immunofluorescence images. The amount of expression of each
protein and the degree of colocalization of the different
proteins is assessed. The expression of a protein as a function
of distance from either blood vessels or the tumor border is
sometimes calculated as well. This can be useful in evaluating
drug penetration into a tumour. Image analysis is done using
either MCID or software developed in our lab by Andrew Morrison,
based on earlier programs written by Dr. Vojo Vukovic, a former
postdoc in our lab. When this program is finished development,
we hope to license it commercially. It allows rapid analysis of
images and does plots of the results. The program runs on
Windows and Macintosh computers, and it has the potential to run
on various UNIX workstations as well.
A SiHa xenograft
immunofluorescently labeled for EF5 (red), Hif-1a (green) and
CAIX (blue). The close-up is of the region in the white
box. The image was tiled using one of the epifluorescence
microscopes (20X objective lens).
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