mcgill radiobiology tutorial session: assays for … · assays for measuring radiation survival...
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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
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Overview
Assays for radiosensitization
• In vitro – Radiation clonogenic survival assays
• In vivo – Tumor growth delay
– TCD50
– Ex vivo clonogenic survival
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Joiner M & van der Kogel A (eds). Basic Clinical Radiobiology. Edward Arnold 2009.
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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
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Clonogenic Survival Assay
Day 1:
Seed cells
Day 2 :
radiation treatment
~ Day 10:
stain and count
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Munshi et al. Methods in Molecular Medicine vol 110.
Plotting clonogenic assay
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Example of clonogenic assay to assess differential survival
Joiner M & van der Kogel A (eds). Basic Clinical Radiobiology. Edward Arnold 2009.
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In vivo assays
Hallahan and Weinberg. Cell 2000
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In vivo assays
–Tumor growth delay
–TCD50
– Ex vivo clonogenic survival
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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
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Tumor growth delay
Hall E. Radiobiology for the Radiologist. Tumor regresses in volume then regrows after IR
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Tumor growth delay
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TCD50
Joiner M & van der Kogel A (eds). Basic Clinical Radiobiology. Edward Arnold 2009.
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Ex vivo clonogenic assay
Irradiation in situ
Excise tumor and obtain single cell
suspension
Setup clonogenic assay
Hall E. Radiobiology for the Radiologist.
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Summary
• Radiation clonogenic survival assays
– Adv: quick, cheap, measures clonogenic survival
– Dis: cannot replicate tumor microenvironment (fluctuating hypoxia, IFP, stromal cells, immune system)
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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
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Summary
• Ex vivo clonogenic survival
– Adv: measures clonogenic survival, quicker than other two assays
– Dis: does not account for ongoing effects of tumor microenvironment