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Immune Response and Splenomegaly in B16 Melanoma Dominika A Jurkovic Advisor: Dr. Spilatro April 2, 2003 Overview of Presentation Background Information Hypothesis Procedures Data Conclusions Discussion Future Experiments Background Information What is melanoma? B16 Melanoma Splenomegaly www.melanoma.com Hypothesis Splenomegaly in mice implanted with B16 melanoma is an immunological response to the developing tumor Spleen Size in B16 Melanoma Purpose is to examine Spleen enlargement in response to a developing tumor Procedure Establish Tumor Line Implantation Allow to grow Excise tissue Weigh tissues Preserve tissues Appearance of B16 Melanoma in Mice Spleen Size Over Time 0.18 0.16 0.14 Spleen Size (g) 0.12 0.1 0.08 0.06 0.04 0.02 0 0 3 6 9 Day of Experiment 12 15 18 Spleen Enlargement Size Comparison of Day 0 and Day 18 Spleen Spleen Weight Compared to Tumor Size 0.2 0.18 0.16 Average Spleen Size (g) 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 0 0.5 1 Average Tum or Size (g) 1.5 2 Procedure for Cell-Free Homogenate Homogenize B16 Tissue Centrifuge Filter Sterilization Inject 8µg Protein Inject PBS Buffer Spleen Enlargement in Control Groups 250% 225% 200% Increase in Size 175% 150% 125% 100% 75% 50% 25% 0% PBS Buffer Cell-free Extract Antigen B16 Melanoma Summary Spleen enlarges as the tumor grows PBS Control negative Cell free extract less effect indicating response to soluble tumor antigens Histological Changes in the Spleen Examine histology of spleen in normal, melanoma implanted, and cell-free extract injected mice Procedures Fixation Embedding Staining Microtome Nikon camera Anatomy and Function of the Spleen Primary Follicles Secondary Follicle Sears, D. 1997 Gude, W. 1982 Structural Changes Day 0 Day 18 Histology Results Normal B16 Melanoma Cell-Free Extract Average Diameter of Follicles 279.21um +/- .509* 249.49 +/- .394* 311.11 +/- .564 Follices per mm2 61.55/mm2 +/- 9.71#^ 55.02/mm2 +/- 5.03# 38.2/mm2 +/- 8.79^ Total Follicles of Cross-section 72.66 +/- 17.01* 126.93 +/- 20.49* 74 +/- 12.16 *, #, ^ =Significant Difference Summary Structural changes indicate a humoral response is occurring in the B16 Melanoma Humoral response is seen in Cell-free extract injected mice, but not to the same degree Overall Conclusions Splenomegaly is an immune response to B16 Melanoma Histology of spleen indicates a humoral immune response to B16 melanoma suggesting production of B16 specific antigens Discussion The immune system can recognize cancer cells as antigens, or foreign invaders Researchers have identified that some cancer cells do carry antigens on their surfaces. By training the immune system to attack these antigens, or "build up an immunity," doctors may be able to halt the disease Future Experiments Confirm type of immune response Identify specific antigen associated with the melanoma tissue Examine splenomegaly in other mice strains implanted with B16 melanoma Acknowledgements Dr. Spilatro-Advisor Dr. Brown and the capstone class Dr. Kramer Questions?