Transcript Cancer Cell Culturing and Cytotoxicity Assays for Anticancer

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. Porter’s Lab
Characterize hostderived lipids with
antimicrobial activity
Dr. Kane’s Lab
Discover drugs that overcome
resistance in cancer
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 overexpressing Her2/neu receptor
 Disrupt cell signalling pathway –

 PI-3-kinase/Akt
signal transduction pathway
First Results
3K cells/ml per well
1,600,000
A
1,400,000
Cells/ml
Cells/ml
1,200,000
1,000,000
No
NoHerceptin
Herceptin
.01
.01mM
M
800,000
11mM
M
600,000
400,000
200,000
0
0
3
7
10
6 well plate
Day
Herceptin applied day 1
10K cells/ml per well
B
1,600,000
2 ml/plate
1,400,000
Cells/ml
Cells/ml
1,200,000
1,000,000
No
NoHerceptin
Herceptin
.01
mM
.01 M
800,000
11mM
M
600,000
400,000
200,000
0
0
Herceptin applied day 1
3
7
10
Day
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
California State University, Los Angeles
Faculty and Staff
Beckman Research Institute of City of Hope
and Graduate School of Biological Sciences
Dr. Edith Porter
Dr. Jamil Momand
Dr. Sandra Sharp
Dr. Nancy McQueen
Annie Mirsoian
Ronnie Cheng
Dr. Susan Kane
Dr. Steven Novak
Dr. Tom Lebon
Dr. Long Gu
Cecile Donahue
Students and Staff
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 taxanebased therapy in breast cancer. Annals of Oncology 19: 223-232.