cytokinetics and tumour growth

Question 1

What is cytokinetics?

Answer 1

This involves the consideration of the rate of entry into S phase, mitosis/cell division and apoptosis

Question 2

Why is it important to measure tumour growth?

Answer 2

To asses patient response to therapy

This can be done through Response evaluation criteria in solid tumours (RECIST)

Question 3

How is tumour growth measured?

Answer 3

If the tumour is large and visible then calipers or rulers may be able to be used
Xrays
Computed tomography
Magnetic resonance imaging
More invasive methods may also be used such as endoscopy, colonoscopy
Tumour markers can be used, these may also allow personalisation of treatment though the markers used may also be present in non-cancer conditions
Cytology and histology to differentiate between residual benign versus and malignant lesion

Question 4

How can mammography be performed?

Answer 4

Through use of a low intensity X-ray which is used to examine the human breast
This detects the presence of characteristic masses and microcalcification
Ultrasound, ductography and MRI may potentially also be used to as adjuncts to mammography

Question 5

What is computed tomography?

Answer 5

This is when computer processed Xrays are used to produce 3D images this is a more sophisticated method of measuring tumour volume

Question 6

What is the definition of tomography?

Answer 6

Imaging by sections or sectioning through the use of some kind of penetrative wave it provides high contrast resolution between different tissues that differ in physical density

Question 7

How can magnetic resonance imaging be used to detect tumours?

Answer 7

Uses strong magnetic fields and radiowaves to from images of the body
It does not use any ionizing radiation and hence is safer than X-ray based methods
It can be used in the staging of rectal and prostate cancer as well as playing a role in the diagnosis, staging and follow up of other tumour types

Question 8

What is the tumour volume doubling time?

Answer 8

This is the rate at which the tumour doubles and takes into account tumour volume and the increase between measurements
It has large patient inter-individuality with times ranging from 88 days to 523 days with an average of 280 days
This suggests an average of 18 years is required from the first tumour cell to produce a tumour with a diameter of 2mm`

Question 9

What is the average volume doubling time of individual human tumours?

Answer 9

These vary widely but the average is about 9 months

Question 10

What is the mitotic index?

Answer 10

The proportion of mitotic cells can be measured and cell cycle time can be calculated assuming that mitosis takes 30 minutes

Question 11

How can the S phase index be measured?

Answer 11

3H thymidine used to be injected into patients then a tumour sample was taken and a cell suspension made and subjected to autoradiography
Cell cycle time may also be able to be calculated from this if it is assumed that cell cycle lasts about 8 hours

Question 12

What is the issue with using the mitotic index, S-phase index or histological methods like Ki-67 labelling and gene expression signatures?

Answer 12

These measure proportions rather than kinetic parameters where as to measure kinetic it is usually necessary to do some type of time course
Early work used multiple tumour biopsies in the same patient together with injection of radioactive thymidine but these experiments had major ethical issues and can now be replaced with flow cytometry

Question 13

What do the different light scattering properties of cells tell us about them?

Answer 13

Diffraction is proportional to cell surface area and is detected by forward scatter
Refraction is proportional to granularity and complexity and is detected as side scatter at right angles to the incidence light axis

Question 14

What does propidium iodide do/

Answer 14

It intercalates DNA and fluoresces red

Question 15

How can antibodies be used to measure S phase cells?

Answer 15

If cells are exposed to bromodeoxyuridine or iododeoxyuridine the analogue is incorporated instead of thymidine
If a single stranded DNA is prepared from these cells is used to immunise a rabbit then antibodies to the substituted DNA will be produced
The antibody can then be used to identify cells that contain the analogues
These cells will be in their S-phase cells
This method allows us to distinguish S-phase cells from cells at other phases in cell cycle including
Conjugation to biotin for later recognition by avidin linked to a marker or direct conjugation of the antibody to fluorescin
Tumour cells can then be permeabilised allowing entry of the antibody and S-phase cells can be identified

Question 16

What is the potential cell doubling time?

Answer 16

This measures the cell population doubling time assuming no cell death

Question 17

What is the in vivo method of measuring the potential cell doubling time?

Answer 17

Admister a single dose of bromodeoxyuridine to the cancer patient, this specifically labales the DNA of S-phase cells
Wait a discrete timeand take a tumour biopsy the delay allows labelled S-phase cells to move through the cell cycle
Expose the cells in vitro to anti-BUdr antibody with green fluorescence which can identify the original pre-labelles S-phase cells
Label the total DNA with propidium
Analyse by two colour flow cytometry

Question 18

How can two colour flow cytometry allow determination of the potential cell doubling time?

Answer 18

The percentage of green cells still left in S phase after 6 hours gives Sphase duration while the percentage of red cells with intermediate DNA dives S phase fraction
the combination of these two values give the potential cell doubling time

Question 19

How does the potential cell doubling time compare to volume doubling times?

Answer 19

They are much shorter as there is a considerable amount of cell turnover in tumour tissue

Question 20

What are the implications of the current model of tumour growth?

Answer 20

Human tumours are characterised by a high degree of turnover, the concept that individual tumour cells grow with a cell cycle time of 4 months is incorrect
Apoptosis can be detected in tumour sections, allowing measurement of an apoptotic index
Tpot values give an indication of the potential of a tumour to repopulate
There is also a relationship between Tpot and clinical outcome with tumours with the shortest Tpot having the worst prognosis

Question 21

How does tumour growth in vivo compare to that in vitro?

Answer 21

The most common method of measurement tumour growth involves counting cells in a culture flask after different times in culture however this does not take into account cell death leading to an under estimation of the cell cycle time making this method useless for cultured clinical samples
Instead a simplified method known as the stathmokinetic method can be used
For established cell lines cell cycle times are sus ally the range of 1-3 days, shorter than the Tpot values that have been determined in from surgical samples
However cell cycle times for freshly cultures tumour material cover a similar range to that of Tpot values with it varying widely

Question 22

What is the principle of the stathmokinetic method?

Answer 22

A drug like paclitaxel is added to arrest the cell cycle at a particular stage
The proportions of cells in one or more phases can be observed at different times and can be compared to those seen in untreated cells
Analysis pf the data provides an estimate of cell cycle parameters that is largely independent of cell death

Question 23

What can be learned from analysis of G1-phase content of tumour samples/

Answer 23

This is of particular interest as it is not linear with time
The initial slope gives the rate of entry into S phase and an estimate of the average G1-phase transit time
The shape also points to a fundamental property of cycling tumour cells which is an intrinsic variability in individual G1- phase transit times which implies that entry into S phase is determined by a probability function