ahmed group lecture 6 cell and tissue survival assays lecture 6
TRANSCRIPT
Ahmed GroupAhmed GroupLecture 6Lecture 6
Cell and Tissue Survival Assays
Lecture 6
Ahmed GroupAhmed GroupLecture 6Lecture 6
• In vitro clonogenic assays• Calculation of plating efficiency and surviving fraction• In vivo clonogenic assays• Bone marrow stem cell assays, jejunal crypt stem cell assay, skin clones, kidney tubules• Functional endpoints
Ahmed GroupAhmed GroupLecture 6Lecture 6
• In-vitro clonogenic assays• Calculation of plating efficiency and surviving fraction• In-vivo clonogenic assays• Bone marrow stem cell assays, jejunal crypt stem cell assay, skin clones, kidney tubules• Functional endpoints
Ahmed GroupAhmed GroupLecture 6Lecture 6
1. Division Delay - (Dose = 0.1 to 10 Gy)2. Interphase Death - Apoptosis3. Reproductive Failure - Loss of clonogenicity
Fate of irradiated cells
Ahmed GroupAhmed GroupLecture 6Lecture 6
Measurement of Killing
A. In-vitro
1. Plating Efficiency2. Survival
B. In-vivo
1. Xenografts2. In-situ tumors3. Survival
Ahmed GroupAhmed GroupLecture 6Lecture 6
In-vitro clonogenic assays
• Clonogenic assays – techniques in which the endpointobserved depends directly on the reproductive integrityof individual cells.
• These systems are directly analogous to cell survivalin vitro.
Ahmed GroupAhmed GroupLecture 6Lecture 6
Tissue (tumor) sample
Chop it up + trypsin
Single cell suspension
Culture dish with medium at 370 C
1 – 2 Weeks
Colonies
In-vitro clonogenic assay
Ahmed GroupAhmed GroupLecture 6Lecture 6
In-vitro clonogenic assay
Ahmed GroupAhmed GroupLecture 6Lecture 6
• In-vitro clonogenic assays• Calculation of plating efficiency and surviving fraction• In-vivo clonogenic assays• Bone marrow stem cell assays, jejunal crypt stem cell assay, skin clones, kidney tubules• Functional endpoints
Ahmed GroupAhmed GroupLecture 6Lecture 6
Reproductive DeathLoss of proliferation ability of cell exposed to radiation, as assessed by colony forming assay.
Ahmed GroupAhmed GroupLecture 6Lecture 6
Ahmed GroupAhmed GroupLecture 6Lecture 6
• The influence of cellular multiplicity (number of cells per potential colony-forming unit) on the determination of radiation sensitivity can be evaluated for a range of multiplicity distributions.
• Cell surviving fraction can be calculated using no multiplicity correction, an average multiplicity correction or the fractional distribution of multiplicities of the control and irradiated population.
• Multiplicity corrections are required when the number of cells per potential colony-forming unit is greater than 1.00 either immediately after plating or at the time of irradiation.
• Both the control and irradiated populations must be corrected for multiplicity. • Multiplicity errors are most pronounced in the low-dose range, e.g. in the survival range
with 2 Gy. • The error introduced by using an average vs fractional distribution of multiplicities
increases with the multiplicity dispersion. • Seemingly small errors due to uncorrected multiplicity effects lead to markedly different
predicted isoeffect doses when amplified through multiple (e.g. 30) fractions.
MULTIPLICITY
Ahmed GroupAhmed GroupLecture 6Lecture 6
Survival Curves for Mammalian cells
First in-vitro survival curve was reported in 1956
Ahmed GroupAhmed GroupLecture 6Lecture 6
Ahmed GroupAhmed GroupLecture 6Lecture 6
Ahmed GroupAhmed GroupLecture 6Lecture 6
Surviving fraction for cells irradiated to 6 Gy
C3H cells C3H cells V-79 cells(plateau phase) (Exponential) (plateau phase)Treatment
Controls (0.37) (0.34) (0.59)0.3 Gy x 20 fractions 0.30 0.24 0.281 Gy x 6 fractions 0.36 0.33 0.342 Gy x 3 fractions 0.52 0.55 0.653 Gy x 2 fractions 0.11 0.20 0.146 Gy x 1 fractions 0.06 0.10 0.08
Smith et al, IJROBP1999
Ahmed GroupAhmed GroupLecture 6Lecture 6
Parameters of survival curves
PE: Plating efficiency. Percentage of cells able to form colonies
Dq: The quasi-threshold dose for a given population that often measures the width of the shoulder
D0: The dose that reduces the surviving fraction to e-1 (= 0.37) on the exponential portion of the curve or the dose that produces 37% survival.
n: Extrapolation number. This value is obtained by extrapolating the exponential portion of the curve to the abscissa.
Ahmed GroupAhmed GroupLecture 6Lecture 6
Radiation sensitivity profiles for cells of human origin
Ahmed GroupAhmed GroupLecture 6Lecture 6
• In-vitro clonogenic assays• Calculation of plating efficiency and surviving fraction• In-vivo clonogenic assays• Bone marrow stem cell assays, jejunal crypt stem cell assay, skin clones, kidney tubules• Functional endpoints
Ahmed GroupAhmed GroupLecture 6Lecture 6
In-vivo clonogenic assays
- The techniques developed by Withers and his colleagues are based on the observation of a clone of cells regenerating in situ in irradiated tissue. In situ re-growth techniques include the skin, crypt cells in the jejunum or colon, testes stem cells, and kidney tubules
- Systems in which cell survival is assessed by transplantation into another site include bone-marrow stem cells, thyroid cells, and mammary cells
The various types of normal tissue assay systems are described in following slides.
Ahmed GroupAhmed GroupLecture 6Lecture 6
Clones re-growing in-situ
Skin colonies
Ahmed GroupAhmed GroupLecture 6Lecture 6
Clones re-growing in-situ
Skin colonies
Ahmed GroupAhmed GroupLecture 6Lecture 6
Clones re-growing in-situ
Skin colonies
Ahmed GroupAhmed GroupLecture 6Lecture 6
Clones re-growing in-situ
Crypt cells of the mouse jejunum
Ahmed GroupAhmed GroupLecture 6Lecture 6
Crypt cells of the mouse jejunum
Clones re-growing in-situ
Ahmed GroupAhmed GroupLecture 6Lecture 6
Crypt cells of the mouse jejunum
Clones re-growing in-situ
Ahmed GroupAhmed GroupLecture 6Lecture 6
Crypt cells of the mouse jejunum
Clones re-growing in-situ
Ahmed GroupAhmed GroupLecture 6Lecture 6
Testes stem cells
Clones re-growing in-situ
Ahmed GroupAhmed GroupLecture 6Lecture 6
Testes stem cellsClones re-growing in-situ
Ahmed GroupAhmed GroupLecture 6Lecture 6
Testes stem cells
Clones re-growing in-situ
Ahmed GroupAhmed GroupLecture 6Lecture 6
Kidney tubules
Clones re-growing in-situ
Ahmed GroupAhmed GroupLecture 6Lecture 6
Kidney tubules
Clones re-growing in-situ
Ahmed GroupAhmed GroupLecture 6Lecture 6
Cells transplanted to another site
Bone-marrow stem cells
Ahmed GroupAhmed GroupLecture 6Lecture 6
Cells transplanted to another site
Bone-marrow stem cells
Photograph of a spleen showing the colonies to be counted
Ahmed GroupAhmed GroupLecture 6Lecture 6
Cells transplanted to another site
Bone-marrow stem cells
Ahmed GroupAhmed GroupLecture 6Lecture 6
Cells transplanted to another site
Mammary cells
Ahmed GroupAhmed GroupLecture 6Lecture 6
Cells transplanted to another site
Thyroid cells
Ahmed GroupAhmed GroupLecture 6Lecture 6
Summary of dose-response curves for all of the clonogenic assays in normal tissues
Note the substantial range of radiosensitivitieswith shoulder width beingthe principal variable
Ahmed GroupAhmed GroupLecture 6Lecture 6
• In-vitro clonogenic assays• Calculation of plating efficiency and surviving fraction• In-vivo clonogenic assays• Bone marrow stem cell assays, jejunal crypt stem cell assay, skin clones, kidney tubules• Functional endpoints
Ahmed GroupAhmed GroupLecture 6Lecture 6
Functional endpointsDose-response relationships
Pig skin
Ahmed GroupAhmed GroupLecture 6Lecture 6
Pig skin
Functional endpointsDose-response relationships
Ahmed GroupAhmed GroupLecture 6Lecture 6
Functional endpointsDose-response relationships
Pig skin
Ahmed GroupAhmed GroupLecture 6Lecture 6
Rodent skin
Functional endpointsDose-response relationships
Ahmed GroupAhmed GroupLecture 6Lecture 6
Functional endpointsDose-response relationshipsRodent skin
Ahmed GroupAhmed GroupLecture 6Lecture 6
Functional endpointsEarly and late response of the lung based on breathing rate.
Breathing frequency increases progressivelywith dose after a treshold of about 11 Gy
Ahmed GroupAhmed GroupLecture 6Lecture 6
Functional endpointsSpinal cord myelopathy
A dose-response relationship can be determined for late damage caused by local irradiation of the spinal cords of rats. After latent periods of 4 to 12 months, symptoms of myelopathy develop: palpable muscle atrophy, followed by impaired use of hind legs
Fractionation and protraction. Dose per fraction is very important, with the dose to produce paralysis increasing dramatically with number of fractions.
Ahmed GroupAhmed GroupLecture 6Lecture 6
Functional endpointsSpinal cord myelopathy
Fractionation and protraction. The effect of a large number of very small fractions is shown.
Ahmed GroupAhmed GroupLecture 6Lecture 6
Functional endpoints Spinal cord myelopathy
Volume effects. The total volume of irradiated tissue has an influence on the development of tissue injury. Shown is the relation between tolerance dose and thelength of the cord irradiated in the rat.
Ahmed GroupAhmed GroupLecture 6Lecture 6
Functional endpointsDose-response relationshipsThe parameters of the dose-response curve for any normal tissue system for which a
functional endpoint can be observed may be inferred by performing multifractionexperiment and estimating the α/β ratio. Because α/β is the dose at which cell killing bylinear and by quadratic components are equal, the implication is that dose-response relationships for late-responding tissues are “curvier” than for early-responding tissues.This is important in the discussion of fractionation in radiotherapy.
Ahmed GroupAhmed GroupLecture 6Lecture 6
Summary- In vitro clonogenic assays – techniques in which the endpoint observed
depends directly on the reproductive integrity of individual cells.
- In vivo clonogenic endpoints include systems in which cells re-grow in-situ and some in which cells are transplanted to another site.
- Dose-response curves can be obtained as a result of clonogenic assays.
- The radiosensitivity of cells from normal tissues varies widely. The width of the shoulder of the curve is the principal variable.
- Dose-response curves for functional endpoints, distinct from cell survival, can be obtained for:
- The shape of the dose-response relationship for functional endpoints, obtained from multifraction experiments, is more pertinent to radiotherapy than clonogenic assays
- The ratio α/β may be inferred from multifraction experiments in systems scoring nonclonogenic endpoints
•pig skin and rodent skin by measuring skin reactions•early and late response of the lung by measuring breathing rate•spinal cord by observing myelopathy