CSULA-COH Cancer Collaborative Cancer Cell Culturing and Cytotoxicity Assays for Anticancer Screening at City of Hope CSULA-COH Cancer Collaborative Presented by: Anthony Martinez

Research: Response and Resistance of cancer cells to therapeutic agents Failure of cancer treatment One main cause: drug resistance by tumor Breast cancer is the most common cancer in women (incurable if metastatic) Her2 is overexpressed in 25-30% of breast cancer and helps the cancer resist conventional treatments (Azambuja, 2007)

Hypothesis Some antibiotics are also anti-cancer drugs Examples Doxyrubicin Clioquinol Immune defense in infectious agents and tumor defense display some similarities Antimicrobial lipids produced naturally in body fluids might exert some anticancer activity

Collaborative Design Dr. Kane’s Lab Dr. Porter’s Lab Discover drugs that overcome resistance in cancer Dr. Porter’s Lab Characterize host-derived lipids with antimicrobial activity Molecular Express, Inc. Develop liposomal formulations of antimicrobial lipids University of Wisconsin

Cell Maintenance and Technique Cell growth and passage Tissue culture medium Antibiotics Trypsin, serum, L-glutamine Cryopreservation and storage Cell Counting Hemocytometer Coulter counter Safety and technique An example of removing Media from a T25 flask Masters, J., Stacey, G. (2007). Changing medium and passaging cell lines. Nature Protocols 2: 2276-2284.

Measuring Cytotoxicity Clonogenic Assay (colony forming assay) Sulforhodamine B (SRB Assay)

Colony Forming Assay Treatment is applied to a sample of cells. The cells are plated in a tissue culture vessel and allowed to grow. The colonies produced are fixed, stained, and counted Cell survival – surviving fraction vs. drug concentration

Before Treatment After Treatment Too many to count Franken N., Rodermond H., Stap J, Haveman J, van Bree C. (2006). Clonogenic assay of cells in vitro. Nature Protocols 1: 2315-2319.

The sulforhodamine B (SRB) Assay SRB binds to protein components of cell SRB is bright-pink aminoxanthene dye and binds basic amino groups – binding is stoichiometric to cell mass Adaptable to 96-well, high throughput screening Dose Response Analysis Vichai, V., Kirtikara, K. (2006). Sulforhadamine B colorimetric assay for cytotoxicity screening. Nature Protocols 1: 1112-1116.

Herceptin Study Herceptin (trastuzumab) – monoclonal antibody Used to treat breast cancers over-expressing Her2/neu receptor Disrupt cell signalling pathway – PI-3-kinase/Akt signal transduction pathway

First Results Cells/ml 6 well plate 2 ml/plate Cells/ml A No Herceptin B Cells/ml No Herceptin .01 M 1 M Figure 1: Effect of anti-HER2 monoclonal antibody herceptin on SK-BR-3 cell proliferation. Cell number was determined on the third, seventh and tenth day using the hemocytometer. (A) 3000 cells/ml per well were seeded on day 0. (B) 10,000 cells/ml per well were seeded. Each count was done in duplicate with the error bars representing +/- SD.

Next Steps Complete training at City of Hope Implement various screening protocols with antimicrobial lipid based liposome preparations privided by Molecular Express, Inc. at CSULA

Acknowledgements Beckman Research Institute of City of Hope and Graduate School of Biological Sciences Dr. Susan Kane Dr. Steven Novak Dr. Tom Lebon Dr. Long Gu Cecile Donahue Students and Staff California State University, Los Angeles Faculty and Staff Dr. Edith Porter Dr. Jamil Momand Dr. Sandra Sharp Dr. Nancy McQueen Annie Mirsoian Ronnie Cheng Dr. Gary Fujii Dr. William Ernst Joan-En Chang Funding NIH grants: 1P20CA118783-01A1 and 1P20CA118775-01A1

References Freshney, R. (2005). Culture of Animal Cells: A Manual of Basic Technique, 5th ed. Hoboken, NJ, John Wiley & Sons. Kane, S. (2006). Cancer therapies targeted to the epidermal growth factor receptor and its family members. Expert Opinion on Therapeutic Patents 2: 147-164. Langdon, S. (2004). Cancer Cell Culture: Methods and Protocols, Totowa, NJ, Humana Press. Masters, J., Stacey, G. (2007). Changing medium and passaging cell lines. Nature Protocols 2: 2276-2284. Vichai, V., Kirtikara, K. (2006). Sulforhadamine B colorimetric assay for cytotoxicity screening. Nature Protocols 1: 1112-1116. Franken N., Rodermond H., Stap J, Haveman J, van Bree C. (2006). Clonogenic assay of cells in vitro. Nature Protocols 1: 2315-2319. Azambuja E., Durbecq V., Rosa D.D., Colozza M., Larsimont D., Piccart-Gebhart M., Cardoso F. (2008). HER-2 overexpression/amplification and its interaction with taxane-based therapy in breast cancer. Annals of Oncology 19: 223-232.