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Expression of cathepsins and their inhibitors in U87<br />
spheroids embedded in collagen matrices:<br />
characterisation of migrating versus non-migrating cells<br />
Boris Gole, María Beatriz Durán Alonso, Simon Caserman and<br />
Tamara T. Lah<br />
Department of Genetic Toxicology and Cancer Biology, National Institute of Biology,<br />
Ve~na pot 111, 1000 Ljubljana, Slovenia<br />
In malignant tumours, cell populations are present that differ in their migratory and<br />
invasive behaviour. Characterisation of invasive tumour cells has revealed that they<br />
also differ from non-invasive tumour cells in other crucial biological properties such<br />
as proliferation ability and resistance/susceptibility to apoptosis. We are interested<br />
in the role of cathepsins and their inhibitors in the biology of tumour cells. In vitro<br />
and in vivo data point at these proteases as important players in tumour progression.<br />
Cathepsins B, L and D have so far been the cathepsins most extensively studied in<br />
various stages in tumourigenesis and malignant progression of cancer and in vivo<br />
studies show an increase of their expression levels in tumours, especially at the<br />
invading front. We are currently characterising the expression patterns of Cathepsin<br />
B and Cathepsin L and their inhibitors in invasive versus non-invasive U87 cells. To<br />
do this we are preparing U87 spheroids using the hanging-drop method and then<br />
placing these spheroids into collagen matrices, which is one of the models available<br />
to study invasion of tumour cells in vitro. After various lengths of time in collagen<br />
the migrating cells are separated from the non-migrating fraction and possible<br />
differences in protease and inhibitor expression are sought-for, regarding both the<br />
type of cells (i.e. migrating vs. non-migrating) and the length of time the cells<br />
82p11<br />
have been kept in collagen for. Studies at the mRNA level indicate changes in the<br />
expression levels of some of these genes when looking at the two U87 phenotypes.<br />
Analyses of the activity levels of these proteases complement the mRNA data. Using<br />
confocal microscopy we observe intracellular degradation of a quenched fluorescent<br />
substrate, DQ-collagen type IV, in agreement with previour reports.
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