Radiobiology

1. CellSurvivalCurves
Dr.Avilash Banerjee
PGY1,Department of Radiation Oncology
Yashoda Superspeciality Hospital

2. What is a Cell Survival Curve ?
• A cell survival curve is the relationship
between the fraction of cells retaining their
reproductive integrity and the absorbed
dose of radiation.
• The surviving fraction is depicted on a
logarithmic scale, and is plotted on the y-
axis
against
• Dose on the x-axis.

3. Cell Death
• Following irradiation,cells may be intact and may be able to produce
proteins,synthesize new DNA and even go through few more cell divisions.
• However if it lost the capability to reproduce indefinitely it is considered dead.
• LAW OF BERGONI’E & TRIBONDEAU : 1906
• Quickly dividing tumor cells are generally more sensitive than the majority of
body cells
• Radiation Damage and Cell maturity are inversely related

4. Clonogenic Cell: A Cell that is able to produce indefinitely,and forms a huge
colony from a single cell
Non proliferative/ Differentiated cell
• Eg.Nerve, Muscle etc.
• Loss of function occurs at 100 Gy
Proliferative/ Undifferentiated cell
• Eg.Hematopoetic Stem cell, Epithelial
cells
• Loss of reproductive capacity occurs
at 2 Gy

5. Cell Death Mechanisms
MITOTIC APOPTOTIC
Main mechanism of damage mediated by Radiation
Cell dies at the time of division.
Programmed Cell Death.Mostly in embryonal tissues.
DIRECT EFFECT INDIRECT EFFECT
Radiation interacts directly with
critical targets in cell.Direct DNA
strand breaks.
Radiation interacts with other molecules ( mainly
water ) within the cells to produce ,
which through diffusion in the cells damage critical
organ within cell.
Radiation induced Cell Damage

6. DNA Damage by Radiation CONCEPT OF CELL SURVIVAL
• Cell when retains its capacity to divide
after radiation:Cell Survival.
• when they can not divide anymore it is
called Cell Death.

7. Concept of cell survival curve:
LINEAR LOG SEMI LOG
Equal Interval of
same number.
Easier.
Eg, Will be used
here for DOSE
of radiation
Exponential
Interval.
Eg, Will be used
for cell survival
as Radiation has
an exponential
effect on the
number of cell
killing
One axis linear
One axis LOG
Cell Survival
Curve will be
having linear X
axis( Dose of
Radiation) and Y
axis ( Cell
Survival)

8. • Plating Efficiency
PE=
NUMER OF COLONIES COUNTED/NUMBER OF
COLONIES SEEDED x 100
For a 100 cells seeded into the dish, the number of
colonies counted may be expected to be in the
range of 50 to 90.
Ideally, it should be 100, but it seldom is for various
reasons, including suboptimal growth medium,
errors and uncertainties in counting the cell
suspension, and the trauma of trypsinization and
handling.
In Vitro Cell Survival Curve

9. How to get intrinsic Cell Survival Curve?
STEPS
1.Specimen of tumor chopped into pieces.
2.Trypsin is added.(Dissolution of cell membranes)
3.Cell Suspension seeed in n number of dishes
4. 2 week Incubation .
5.Exposed to increased frequency of radiation dose.
6.Surving Fraction is Calculated.

10. Surviving Fraction
• Surviving fraction is the ratio of colonies produced to cells plated
with Constant Radiation Exposure, with a correction necessary for
plating efficiency (PE).
• A cell survival curve is plot of log of surviving fraction of cells
(logarithmic y-axis) against the single radiation dose (linear x-axis) .
• All the curves are plotted on semi-log plots

11. Characteristics of Cell Survival Curves
• Slope of the cell survival curve:
Slope ∞ Radiosensitivity
Steeper is the slope more radio
sensitive the cell is.
The diagram clearly shows first curve
producing the same effect at much
lower dose.
7 Gy 17 Gy

12. Shape of the Cell survival curve
• Lower Organism • Mammalian
SHOULDER EFFECT
SURVING
FRACTION
RADIATION DOSE
SURVIVING
FRACTION
RADIATION DOSE

13. • Each increment in dose will decrease the survival fraction by
same proportion,not by number.
• This is called Exponential Relationship.
• Survival is exponential function of dose.
• So Survival Fraction can never become Zero.

14. Cell Survival Curve in Low
LET and High LET
radiation
HIGH LET:
High linear energy transfer eg. Alpha
Particles,more dense energy transfer,Can
kill cell as single hit.
curve is straight line from the begining.
Low LET
Low energy transfer,eg XRays Less dense
Curve starts straight at low dose then bends at high
dose and finally straightens at very high dose.

15. Random Nature of Cell killing by Radiation
• Nature of cell killing is random.
• So increasing the dose of radiation does not equate to similar effect in cell kill.
• Ionisations produced by radiation are distributed randomly,some cells get more some none.
• For example,If 100 cells are there in a medium and exposed to 2 Gy radiation some will get 2,some 1,some 0.
• So always the surviving fraction will be in exponential relation.

16. Concept of D10
D 10 is the dose of radiation that
causes 1 log reduction of survival
fraction
or
reduce the survival fraction to 10 %.

17. Mean Lethal Dose D0
• D0 is the dose required to reduce the survival fraction to 37%= e ^-1
• 2 D0 is the dose that causes reduction of survival fraction to two exponential
reduction,ie. 0.37 x 0.37
SF = e^-D/Do
TOTAL DOSE OF RADIATION
MEAN LETHAL DOSE
• MLD for E.Coli: 100 Gy
• MLD for Viruses : 1500 Gy
• Generally for mammalian cells :1-2 Gy

18. Relation between D0 and D10
D 10 = D0 x 2.3 D 0
SURVIVAL FRACTION
RADIATION DOSE
0.1
0.37
D10
D0
D0 usually lies between 1 to 3 Gy.
D50 the dose to reduce survival to 50%.
Number of DNA Damage after D 0
1.Base Damage MAXIMUM
2. Single strand breaks
3.Double strand breaks MINIMUM

19. Mammalian Cell
Survival Curve
Tested on
1.Hela Cell of Human cervical
cancer.
2.Hamster cells from lung of a
newborn chinese hamster.
Initial portion of mammalian cell survival curve is non exponential,rest portion is exponential

20. Mammalian Cell Survival Curve
LOW DOSE OF RADIATION:Initial part
of the curve is constantly bending
untill it reaches Point B.
HIGH DOSE OF RADIATION: Curve
becomes flat again.

21. Mammalian Cell Survival Curve compared with Bacterial
Cell Survival Curve
BACTERIAL MAMMALIAN
linear throughout. Initially Curved then
straight.
Equal increment of
dose gives equal
proportion decrease in
SF.
Initial part
Equal increment of
dose WONT GIVE
equal proportion
decrease in SF.
Exponential. Initial non exponential
then exponential.
As it can be seen untill point B, even with giving
same dose,the survival fraction will not reduce to
SAME PROPORTION. ,so initially cell kill is not
possible wit D 10.
B

22. Inference
• LOW DOSE OF RADIATION:
• Non Exponential relationship of survival fraction and dose of radiation
• Gives rise to shoulder effect.
• HIGH DOSE OF RADIATION
• Exponential realtionship maintained.
• Same dose causes equal reduction of survival fraction.
If there is no shoulder effect, efficiency of radiation to kill cells increase. ie, Alpha Particles.

23. Models Explaining Cell Survival Curve
•Multitarget Model
(NOT USED CLINICALY)
•Linear Quadratic Model
(USED IN CLINICAL PRACTICE)

24. Multi Target Model
• Older model.
• A cell is supposed to have multiple target.
• Radiation should eliminate all the targets to bring cell death.
• if any of the target is left behind then the cell survives.
• ie. If a cell consists of 100 targets and 99 is killed by radiation,then the
cell survives.

25. Cell Kill
SHE MHE
Single Hit Event Multiple Hit Event
Only 1 hit is enough to
cause cell death.
Mulltiple hits are
required.
Lethal Damage Sub lethal Damage
Initial Part of the
mammalian cell survival
curve is solely
contributed by SHE
As the dose increases
along with single hit
event,multiple hit events
also come into action.
• LOW DOSE REGION: SHE>>>>
MHE
• MIDDLE REGION: SHE= MHE
• HIGH DOSE REGION:
MHE>>>> SHE

26. Quasithreshold Dose (Dq)
• A dose below which radiation does not show any effect.
• But in reality any ammount of radiation has an effect.
• so the dose is seemingly resembles thresold value,but it is not in
actuality.
so term QUASI THRESHOLD.
HOW TO GET Dq IN CELL SURVIVAL CURVE?
The quasi-threshold dose must be extrapolated from the curve:
Begin by drawing a horizontal line (i.e. parallel to x-axis) from S=1.
Now extend the straight portion of the survival curve back to the
horizontal line from unity surviving fraction.
The point of intersection is Dq.
Dq

27. Extrapolation Number
• In the intercept of the extrapolated
multitarget survival curve with the
vertical logarithmic axis specifying
the survival fraction.
• The extrapolation number gives the
number of targets that each must be
hit at least once to have a lethal
effect on the biological system under
study.
Extrapolation number

28. Summary of Multi Target Model
• Theoretical model.
• This model can not be used in clinical practice.
• Main draw back of the model is the concept of Threshold,as
radiation has no threshold.Some ammount of damage is
always present in cell.
• Another important draw back is ,this model can not identify
the pattern the early and late reacting tissue.

29. Linear Quadratic Model
• Postulated by LEA & READ
• Clinically being used.
• Resolves the drawbacks of the MULTI TARGET MODEL.
• Cell is considered one Target instead of Multiple.
• Target is DNA which has double strand.

30. Linear vs Quadratic Kill
linear Quadratic
One quanta of radiation
damages two strands with one
hit.
Two quanta of radiation each
breaking a single strand,
produce a one strand is
damaged with each quanta.
One radiation event can cause
LETHAL damage.
Each one causes Sublethal
Damage.
Proportional to Dose (αD)
eg.double-strand break
Proportional top Square of
Dose
(βD2)
More dose needed as sublethal
injury.
eg.2 strand breaks

31. LQ Model
The probability of survival is equal to the exponential of this linear and
quadratic component.
S = e−(αD+βD2)
Alpha
Alpha is the intrinsic radiosensitivity
of the cells, defined as how many logs
are killed per gray.
Beta
Repair capacity of tisssues,different
tissues have different RC.
Combination of both shows the final killing effect.

32. Alpha and Beta Ratio
• the point where linear and
quadratic kill becomes equal.
• αD =βD2
• so D = α/β
Measures the repairing capacity of
particular tissue type.
And also defines the curviness of the
survival curve.

33. Clinical application of α/β
High alpha/beta ratios
(around 10)
Low alpha/beta ratios (1-
3)
Straighter the
curve,narrower the
shoulder.
More Curved,Wide
shoulder.
It exhibits irreparable
damage.
high capability of repair.
It refers to high
radiosensitivity of cells
It refers to
radioresistance of cells
Acute responding tissues Late responding tissues

34. REPRESENTATIVE α/β RATIO FOR HUMAN NORMAL TISSUES AND TUMOURS
Tissue Type Alpha/Beta
Tumor and Early Effect 10
Late Complications 3
Late CNS Effects 2
Prostate 1.5

35. Comparison of the L-Q and MT models
• High doses
the LQ model predicts a survival curve that bends
continuously, whereas the M-T model become linear.
• Low doses
LQ model describes a curve that bends more than a
M-T curve, whereas the M-T model is linear.

36. Limitation of LQ model
• An overly simplistic assumption that an effect in a tissue directly corresponds to an
survival of particular cell type. many parameters play an important role in survival.
• No provision for the influence of cell cycle,Tumor micro environemt,Concept of
Oxygenation Injury.
• No way to account for differences in repair rates b/w different tissues
• Uncertainity surrounding the models applicability for extremes of fractionation.
• Limited understanding of how to apply the model in patients receiving multimodality
therapy.

37. Factors affecting Cell Survival Curve
1.LET(Linear Energy Transfer):
describes the energy an ionizing
radiation transfers to the material
traversed per unit distance.
High LET Low LET
Example Alpha Particles XRay
Shape Of Curve More steep.
Linear
No Shoulder.
More curved.
Less steep
Shoulder present.

38. Factors affecting Cell Survival Curve
• 2.Proliferating Capacity Of Cell:
-higher proliferating cells are more
radiosensetive ,straighter curve like high
alpha beta ratio.
-well differentiated cells are more
radioresistant, More curved CSC like low
alpha beta ratio.
• 3.Intrinsic radiosensitivity:
-Mammalian cells are significantly more
radio-sensitive than microorganisms.
-More DNA means more amount of target
for radiation.

39. Factors affecting Cell Survival Curve
4.Fractionation and CSC (Elkind and Sutton
Theory)
• If the total dose is delivered in fractions with sufficient time in
between fractions, repair of sub-lethal damage occurs.
• Cell act as fresh target.
• The shoulder reappeared.
• Both ‘n’ and ‘Dq’ increases.
• Increased Fractionations:
• The separation of the survival curves between tumor and normal
late responding cells increases.
• Tumors are then preferentially killed.
• Increased Dose /Fraction:results in more injury to late responding
normal tissues and less repair.

40. Factors affecting Cell Survival Curve
• 5. Dose Rate Effect
• 6.Oxygen Effect
Hypoxic cells are more resistant to radiation than aerobic cells.
Dose rate : Amount of radiation absorbed per unit time.
At low dose rates, DNA repair processes are able to repair sub lethal
damage during the radiation treatment itself.
Reduction in dose rate reduces survival curve slope (D0 increases)