Document Type

Thesis - University Access Only

Award Date

2004

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Alan Young

Abstract

Metastasis is a major complicating factor of most cancers. In breast cancer, metastasis primarily occurs through the axillary lymph node, mimicking the recirculation of lymphocytes. The mechanisms that allow metastatic cells to exit the lymph node in the efferent lymph remain unclear. The novel immunosuppressant FTY720 was shown to sequester lymphocytes in secondary lymphoid organs (Yanagawa et al, 1998; Chiba et al, 1998) and has also been shown to reduce tumor size and prevent metastasis in a mouse model of breast cancer at low doses (Azuma et al, 2002). Because of its ability to halt the exit of lymphocytes from lymph nodes and prevent metastasis, we studied FTY720' s ability to prevent metastatic human breast cancer cells, MDA-MB-231 cell line, from exiting a single lymph node m an ovine model.

Apoptosis assays were done on both ovine lymphocytes and MDA-MB-231 cells to determine the appropriate concentration ofFTY720 to use; In both lymphocytes and MDA-MB-231 cells, the percent of cells undergoing necrosis or apoptosis was great when concentrations of 15 µM or higher FTY720 were used. At concentrations of FTY720 at or below 10 µM, the percent of cells undergoing apoptosis and necrosis was relatively stable and the majority of cells were alive. Based on this, we chose to use 10 µM FTY720 treatments for the subsequent studies.

To determine if FTY720 was able to sequester peripheral blood lymphocytes in an ovine model, we undertook whole blood labeling experiments. When FTY720 was injected near the draining area of lymph nodes, the percent of labeled peripheral blood lymphocytes recovered from that lymph node was significantly increased compared to saline controls. This confirmed that an ovine model worked similar to other animal models used previously. To determine if FTY720 prevented the exit oflymphocytes and MDA-MB-231 cells from lymph nodes, we performed half and full perfusions. Catheters were surgically inserted in the afferent popliteal lymphatic vessel to allow both treated and untreated cells to be perfused into the lymph node. In the half-perfusion experiments, the lymph nodes were harvested the following day and single cell suspensions were made to compare the amount of treated and untreated lymphocytes or MDA-MB-231 cells. In both lymphocyte and MDA-MB-231 studies, there was no significant difference in the percent recovery of treated versus non-treated cells. In full perfusion models, the efferent popliteal lymphatic vessel was also cannulated, allowing for the collection of efferent lymph over the course of the experiment. The efferent lymph was used to analyze the percent of treated and untreated cells that had exited the lymph node. Surprisingly, the percent of treated cells was always higher than that of the untreated cells. While not significant, in each animal this occurred, possibly indicating a trend.

Last, we looked at FTY720's ability to alter gene expression in the MDA-MB-231 cell line. RNA was extracted from treated and untreated cells, reverse transcribed into cDNA, and the DNA was amplified by PCR using different degenerate primers. V This cDNA was then run on an agarose gel to determine if FTY720 changed the expression of any genes. It appeared that FTY720 up-regulated the expression of at least one gene.

Library of Congress Subject Headings

Breast -- Cancer.
Metastasis -- Prevention.
Immune response -- Regulation.
Lymph nodes.
Lymphocytes.
Cell migration.

Publisher

South Dakota State University

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Rights Statement

In Copyright