Direct visualization of metastases and apoptosis in the lung : a model for tumor progression in the lung / Christopher W. Wong.

Wong, Christopher W.
xi, 77 p. : color ill. ; 29 cm.
Medical subjects:
Dissertations, Academic.
Local subjects:
Penn dissertations -- Pathology.
Pathology -- Penn dissertations.
Metastatic inefficiency refers to the large number of circulating tumor cells required to form one metastasis. The limiting step in metastatic inefficiency could lie at any step in the metastatic cascade. These steps include the inability of tumor cells to attach at a distant site, survive in a foreign environment, or initiate growth. Furthermore, the mechanisms resulting in the death of circulating tumor cells is not known.
Research has been hampered by the inability to directly visualize cells during metastasis. Now, tumor cells labeled by expression of green fluorescent protein (GFP) can be visualized in isolated lung preparations using fluorescent microscopy. This makes it possible to directly visualize the sequence of events in lung metastases. We observed that tumor cells, regardless of their metastatic potential, were able to attach to the lung endothelium. A large number of tumor cells that initially attached in the lung were cleared from the lungs by 24 h. We observed that tumor cells preferentially attached in the pre- or post-capillary arterioles, and the cells that survived proliferated radially outward along and within the capillaries. We did not observe cell dormancy, or extravasation of cells prior to proliferation.
Using TUNEL staining, we determined that many tumor cells undergo apoptosis. To confirm that apoptosis of attached tumor cells occurred, we developed an assay that allowed direct visualization of apoptosis in vivo. Bad, a pro-apoptotic member of the Bcl-2 family translocates from the cytoplasm to the mitochondria during apoptosis. When a Bad-GFP fusion protein is over-expressed in healthy tumor cells, a diffuse fluorescence is observed. However during apoptosis, translocation of Bad-GFP to the mitochondria results in a "speckled" phenotype.
Using these methods, we observed that non-metastatic cells were more susceptible to apoptosis than metastatic cells. To further verify the importance of apoptosis in regulating metastasis, we inhibited apoptosis with Bcl-2 and found increased formation of lung metastases in experimental metastasis assays.
Based on these observations, a model for tumor progression is proposed in this thesis. Evidence is presented to support the conclusions that (a) the mechanism leading to tumor cell death in the lung is apoptosis; (b) differential levels of apoptosis correlates with differences in metastatic potential; and (c) apoptosis is an important component of metastatic inefficiency.
Supervisor: Ruth J. Muschel.
Thesis (Ph.D. in Pathology) -- University of Pennsylvania, 2001.
Includes bibliographical references.
Local notes:
University Microfilms order no.: 3003704.
Muschel, Ruth J., advisor.
University of Pennsylvania.
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