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.