1. Ahmed Group Lecture 6 Cell and Tissue Survival Assays Lecture 6

2. Ahmed Group Lecture 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

3. Ahmed Group Lecture 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

4. Ahmed Group Lecture 6 1.Division Delay-(Dose = 0.1 to 10 Gy) 2.Interphase Death-Apoptosis 3.Reproductive Failure-Loss of clonogenicity Fate of irradiated cells

5. Ahmed Group Lecture 6 Measurement of Killing A.In-vitro 1.Plating Efficiency 2.Survival B.In-vivo 1.Xenografts 2.In-situ tumors 3.Survival

6. Ahmed Group Lecture 6 In-vitro clonogenic assays Clonogenic assays – techniques in which the endpoint observed depends directly on the reproductive integrity of individual cells. These systems are directly analogous to cell survival in vitro.

7. Ahmed Group Lecture 6 Tissue (tumor) sample Chop it up + trypsin Single cell suspension Culture dish with medium at 37 0 C 1 – 2 Weeks Colonies In-vitro clonogenic assay

8. Ahmed Group Lecture 6 In-vitro clonogenic assay

9. Ahmed Group Lecture 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

10. Ahmed Group Lecture 6 Reproductive Death Loss of proliferation ability of cell exposed to radiation, as assessed by colony forming assay.

11. Ahmed Group Lecture 6

12. Ahmed Group Lecture 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

13. Ahmed Group Lecture 6 Survival Curves for Mammalian cells First in-vitro survival curve was reported in 1956

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15. Ahmed Group Lecture 6

16. Ahmed Group Lecture 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 0.28 1 Gy x 6 fractions 0.36 0.33 0.34 2 Gy x 3 fractions 0.52 0.55 0.65 3 Gy x 2 fractions 0.11 0.20 0.14 6 Gy x 1 fractions 0.06 0.10 0.08 Smith et al, IJROBP1999

17. Ahmed Group Lecture 6 Parameters of survival curves PE:Plating efficiency. Percentage of cells able to form colonies D q :The quasi-threshold dose for a given population that often measures the width of the shoulder D 0 :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.

18. Ahmed Group Lecture 6 Radiation sensitivity profiles for cells of human origin

19. Ahmed Group Lecture 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

20. Ahmed Group Lecture 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.

21. Ahmed Group Lecture 6 Clones re-growing in-situ Skin colonies

22. Ahmed Group Lecture 6 Clones re-growing in-situ Skin colonies

23. Ahmed Group Lecture 6 Clones re-growing in-situ Skin colonies

24. Ahmed Group Lecture 6 Clones re-growing in-situ Crypt cells of the mouse jejunum

25. Ahmed Group Lecture 6 Crypt cells of the mouse jejunum Clones re-growing in-situ

26. Ahmed Group Lecture 6 Crypt cells of the mouse jejunum Clones re-growing in-situ

27. Ahmed Group Lecture 6 Crypt cells of the mouse jejunum Clones re-growing in-situ

28. Ahmed Group Lecture 6 Testes stem cells Clones re-growing in-situ

29. Ahmed Group Lecture 6 Testes stem cells Clones re-growing in-situ

30. Ahmed Group Lecture 6 Testes stem cells Clones re-growing in-situ

31. Ahmed Group Lecture 6 Kidney tubules Clones re-growing in-situ

32. Ahmed Group Lecture 6 Kidney tubules Clones re-growing in-situ

33. Ahmed Group Lecture 6 Cells transplanted to another site Bone-marrow stem cells

34. Ahmed Group Lecture 6 Cells transplanted to another site Bone-marrow stem cells Photograph of a spleen showing the colonies to be counted

35. Ahmed Group Lecture 6 Cells transplanted to another site Bone-marrow stem cells

36. Ahmed Group Lecture 6 Cells transplanted to another site Mammary cells

37. Ahmed Group Lecture 6 Cells transplanted to another site Thyroid cells

38. Ahmed Group Lecture 6 Summary of dose-response curves for all of the clonogenic assays in normal tissues Note the substantial range of radiosensitivities with shoulder width being the principal variable

39. Ahmed Group Lecture 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

40. Ahmed Group Lecture 6 Functional endpoints Dose-response relationships Pig skin

41. Ahmed Group Lecture 6 Pig skin Functional endpoints Dose-response relationships

42. Ahmed Group Lecture 6 Functional endpoints Dose-response relationships Pig skin

43. Ahmed Group Lecture 6 Rodent skin Functional endpoints Dose-response relationships

44. Ahmed Group Lecture 6 Functional endpoints Dose-response relationships Rodent skin

45. Ahmed Group Lecture 6 Functional endpoints Early and late response of the lung based on breathing rate. Breathing frequency increases progressively with dose after a treshold of about 11 Gy

46. Ahmed Group Lecture 6 Functional endpoints Spinal 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.

47. Ahmed Group Lecture 6 Functional endpoints Spinal cord myelopathy Fractionation and protraction. The effect of a large number of very small fractions is shown.

48. Ahmed Group Lecture 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 the length of the cord irradiated in the rat.

49. Ahmed Group Lecture 6 Functional endpoints Dose-response relationships The 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.

50. Ahmed Group Lecture 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