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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 treatmentOne 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 DesignDr. Porter’s Lab
Characterize host-derived lipids with
antimicrobial activity
University of Wisconsin
Molecular Express, Inc.
Develop liposomal formulations of antimicrobial
lipids
Dr. Kane’s LabDiscover drugs that overcome
resistance in cancer
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
Franken N., Rodermond H., Stap J, Haveman J, van Bree C. (2006). Clonogenic assay of cells in vitro. Nature Protocols 1: 2315-2319.
Before Treatment
After Treatment
Too many to count
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
A
3K cells/ml per well
0
200,000
400,000
600,000
800,000
1,000,000
1,200,000
1,400,000
1,600,000
0 3 7 10
Day
Cell
s/m
l
No Herceptin
.01 mM
1 mM
Herceptin applied day 1
No Herceptin.01 M1 MC
ells
/ml
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.
B 10K cells/ml per well
0
200,000
400,000
600,000
800,000
1,000,000
1,200,000
1,400,000
1,600,000
0 3 7 10
Day
Cells/m
l
No Herceptin
.01 mM
1 mM
Herceptin applied day 1
Cel
ls/m
l
No Herceptin.01 M1 M
First Results
6 well plate
2 ml/plate
Next Steps
Complete training at City of HopeImplement various screening protocols with antimicrobial lipid based liposome preparations privided by Molecular Express, Inc. at CSULA
Acknowledgements
Funding NIH grants: 1P20CA118783-01A1 and 1P20CA118775-01A1
California State University, Los Angeles Faculty and Staff
Dr. Edith PorterDr. Jamil MomandDr. Sandra SharpDr. Nancy McQueenAnnie MirsoianRonnie Cheng
Beckman Research Institute of City of Hope and Graduate School of Biological Sciences
Dr. Susan KaneDr. Steven NovakDr. Tom LebonDr. Long GuCecile DonahueStudents and Staff
Dr. Gary FujiiDr. William ErnstJoan-En Chang
ReferencesFreshney, 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.