Philip Maximilian Scherer and Dr. Kim L. O’Neill, Microbiology and Molecular Biology
In 2005, the National Cancer Society estimated that about 211,240 new cases of invasive breast cancer in the United States will be diagnosed. Overall, an estimated 1,372,910 new cases of cancer and 570,280 cancer deaths in the United States are predicted for 2005. The National Institutes of Health estimated that the overall costs for cancer in 2004 were $189.9 billion. Cancer is thus a growing health concern. While past research has investigated genes in order to find a cure to cancer, current research now emphasizes a study of the microenvironment surrounding the cancer. The purpose of this research was to study the interaction between cancer cells and the immune system. This project addressed the following question: Do breast cancer cells elicit higher levels of blood vessel formation on their own, or do they require mononuclear leukocytes (MNLs) to induce significantly higher levels of angiogenesis?
Tumor angiogenesis is the proliferation of a network of blood vessels that penetrates into cancerous growths in order to remove waste products and to supply nutrients and oxygen. Angiogenesis is important for tumor growth, expansion, and migration. Tumors can initiate angiogenesis by releasing chemical signals that stimulate the surrounding normal host tissue.
Complex inter-relationships between cancer cells and the immune system result in progressive tumor development. The cascade of cytokines in the tumor microenvironment determines the pro- or anti-cancer activity of immune cells. The chick chorioallantoic membrane (CAM) assay is a model system for studying these interactions and examining blood vessel formation. The CAM assay permits the investigation of angiogenic interactions in vivo. I hypothesized that breast cancer cells would induce greater levels of angiogenesis when stimulated by MNLs.
MDA-MB-231 and MDA-MB-435 cells were cultured in RPMI 1640 medium supplemented with 10% bovine calf serum (BCS). MCF-7 cells were cultured in MEM with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 1 mM sodium pyruvate, and supplemented with 10% BCS. All three breast cancer cell lines were incubated at 37°C and 5% CO2. Cell viability was maintained above 95% as determined by the trypan blue exclusion method. MNLs were isolated from human blood using an LSM® Leukocyte Separation Media protocol.
Fertilized chicken eggs were incubated at 100°F and 60% humidity. On day 7, a small flashlight was used to locate the air sac and prominent blood vessels in the eggs. A Dremel® drill was used to make a shallow hole at the blunt end of the egg and another perpendicular hole in the center of the egg. Mild suction at the blunt end hole displaced the air sac and separated the CAM from the shell. The CAM was exposed by removing a small portion of the shell over the air sac. The breast cancer cells and MNLs were injected in various combinations at a concentration of 500,000 cells/20µl. The window was sealed with Parafilm®. The eggs were allowed to incubate for 3 additional days at 37°C. On day 10, the window in the egg shell was enlarged, completely exposing the chorioallantoic membrane. Photos of the CAM were taken using a digital camera, and angiogenesis was quantified using a CAM analysis computer program.
I quantified blood vessel formation based on vessel length and density. Normal levels of angiogenesis on untreated embryos served as negative controls, while embryos treated with MCF-7 cells, MDA-MB-231 cells, MDA-MB-435 cells, and MNLs alone served as positive controls. Embryos were treated with combinations of each cell line with MNLs. MCF-7 cells alone elicited an increase of 32% in vessel length and 14% in vessel density when compared with untreated embryos. MDA-MB-231 cells alone elicited an increase of 86% in length and 78% in density. MDA-MB-435 cells alone elicited an increase of 69% in length and 84% in density, and MNLs alone elicited an increase of 18% in length and 10% in density. MCF-7 cells with leukocytes elicited an increase of 16% in length and 18% in density compared with the corresponding positive controls, indicating an increase in angiogenic activity in the normally less aggressive MCF-7 cells as a result of MNL interaction. Experiments using MDA-MB-231 cells with leukocytes showed an increase of 2% in length and 1% in density, indicating that MNL interaction did not affect the already angiogenically aggressive MDA-MB-231 cells. However, experiments using MDA-MB-435 cells with leukocytes elicited a decrease of 35% in length and 43% in density, possibly due to increased metastatic aggression as a result of MNL interaction.
MCF-7 breast cancer cells and MNL interactions caused significant increases in angiogenic activity on the chick chorioallantoic membrane. This is possibly due to stimulation of MNLs by MCF-7 cells to secrete immunosuppressive cytokines or proangiogenic factors that alter normal immune system functions, resulting in increased angiogenesis. MNL interactions with MDA-MB-231 cells did not significantly alter levels of angiogenesis, suggesting that the MDA-MB-231 cell-induced level of angiogenesis in the chick egg was already at the maximum angiogenic level for a newly forming chick embryo. MNL interactions with MDA-MB-435 cells showed a dramatic decrease in angiogenesis. This decrease could be the result of a cytokine cascade that increased metastasis instead of angiogenesis in the metastatically aggressive MDA-MB-435 cells. Contrary to my original hypothesis, MNLs interacting with MCF-7, MDA-MB-231, and MDA-MB-435 breast cancer cells result in differential profiles of angiogenic aggressiveness that differ relative to the specific characteristics of each cell line. Thus, MNLs are major contributors to cancer survival as they induce amplified levels of angiogenesis and aggressiveness.
This project focused solely on the angiogenic activity of MNL and breast cancer interactions. More research needs to be conducted to identify the physiological conditions and chemical messenger cascades in the tumor microenvironment that influence MNL infiltration and the resultant angiogenic or metastatic response. Further research involving angiogenesis cytokine ELIZA kits or DNA microarrays is necessary to elucidate the complex interactions of pro- and anti-angiogenic factors and the signaling pathways involved in MNL and breast cancer crosstalk.