McGill Radiobiology tutorial session:

Assays for measuring radiation survival

following irradiation

Stanley Liu, MD, PhD Depts. Of Medical Biophysics and Radiation Oncology, University of Toronto

Sunnybrook Odette Cancer Centre, Sunnybrook Health Sciences Centre

Overview

Assays for radiosensitization

• In vitro – Radiation clonogenic survival assays

• In vivo – Tumor growth delay

– TCD50

– Ex vivo clonogenic survival

Joiner M & van der Kogel A (eds). Basic Clinical Radiobiology. Edward Arnold 2009.

Cell death after IR

Mitosis

DNA

Damage

Response Apoptosis

Senescence Autophagy

Necrosis

Mitotic Catastrophe

Clonogenic

Survival

Adapted from Wouters B (2015). Molecular Basis of Cell Death. Clin and Exp Rad

Bio Course U of T; and Minafra & Bravatà. Trans Cancer Res. 3(1): 2014

Clonogenic Survival Assay

Day 1:

Seed cells

Day 2 :

radiation treatment

~ Day 10:

stain and count

Munshi et al. Methods in Molecular Medicine vol 110.

Plotting clonogenic assay

Example of clonogenic assay to assess differential survival

Joiner M & van der Kogel A (eds). Basic Clinical Radiobiology. Edward Arnold 2009.

In vivo assays

Hallahan and Weinberg. Cell 2000

In vivo assays

–Tumor growth delay

–TCD50

– Ex vivo clonogenic survival

Tumor growth delay

Inject tumor cells SC into flank of

nude mouse

Irradiate tumor

measure tumor volumes

Tumor will typically regress in

volume then regrow

Tumor growth delay

Hall E. Radiobiology for the Radiologist. Tumor regresses in volume then regrows after IR

Tumor growth delay

TCD50

Joiner M & van der Kogel A (eds). Basic Clinical Radiobiology. Edward Arnold 2009.

Ex vivo clonogenic assay

Irradiation in situ

Excise tumor and obtain single cell

suspension

Setup clonogenic assay

Hall E. Radiobiology for the Radiologist.

Summary

• Radiation clonogenic survival assays

– Adv: quick, cheap, measures clonogenic survival

– Dis: cannot replicate tumor microenvironment (fluctuating hypoxia, IFP, stromal cells, immune system)

Summary

• Tumor growth delay

– Adv: cheaper and easier than TCD50

– Dis: doesn’t measure clonogenic survival (kill of clonogenic and non-clonogenic cells)

• TCD50

– Adv: measures clonogenic survival

– Dis: very expensive, time consuming

Summary

• Ex vivo clonogenic survival

– Adv: measures clonogenic survival, quicker than other two assays

– Dis: does not account for ongoing effects of tumor microenvironment