It has been found that the development of diseases can be
linked to a change in the mechanical properties of a living cell 1,2.
Diseases have many different properties, some show signs such as muscular
dystrophies 3, whereas other require experimental methods to be observed such
as cancer 2. Although some cases show clearly observable signs of cancer,
there are many cases where further testing has been required. This paper looks
into a new promising technique for cancer diagnosis that can be applied
alongside common methods such as mammography 4 at the present time.
Atomic Force Microscopy (AFM) has been used over recent
years for imaging and testing the properties of biological samples 5.
Recently it has been found that cancerous cells appear to be softer than that
of normal cells 6,2. This paper looks into applying these recent finding
with AFM to create a new diagnostic method. The paper reveals that cancerous
cells show a unique fingerprint at each stage of cancer progression which will
help define the stage of cancer development patients are at.
With the aid of ultrasound imaging, the Swiss team managed
to take five biopsies from each patient, in which one of the biopsies was used
for the IT-AFM procedure whereas the others underwent the standard procedure.
Each of the biopsies was placed into an ice-cold isotonic Ringer solution to
minimize tissue degradation before the procedures took place. The biopsies were
then immobilized onto a plastic dish and were the procedure took place under
Indentation-AFM is a certain method of AFM used to determine
the elasticity of the sample. AFM system made around the cantilever, which has
a very thin probe or tip, which is used to determine the samples properties. A
laser is set up to reflect off the top of the cantilever onto a quadrant
photodiode, which records the light hitting the surface. This will record the
deflections of the cantilever down to the smallest increment. Now by
quantifying this deflection by Hooke’s law 7 we can calculate the Force
applied to the sample. The system will be calibrated before the experiment so
an accurate value of Hooke’s constant, k, will of been calculated.
The principle of AFM Indentation is illustrated in Figure 1.
The AFM cantilever approaches the tissue from a few micrometers above; makes
contact with the cell; indents the cell so that cantilever deflection reaches a
preselected set point, and pulls away from the cell. As discussed before, the cantilever
deflection is recorded via the reflection of a laser as a function of its
Before making contact with the tissue, the cantilever moves
through a medium that will not cause any deflection, which will show better
readings when the tissue is indented. Normally this procedure is done in the
air however they wanted to mimic physiological conditions. While indenting the
tissue, the cantilever bends, and the deflection signal increases. This is due
to the probe applying an impinging force on the tissue causing it to deform and
a deflection to be measured. The cantilevers
are modeled as elastic beams so their deflection is proportional to the force
applied (Hooke’s law) to the tissue. The maximum deflection is set so that this
limits the force applied, hence avoiding too much damage to the tissue.
From this, the cantilever deflection, the load force applied
to the tissues can be calculated and a graph of load force vs indentation. While the probe is retracted a negative
deflection greater than the expected trend is experienced, which is shown at
part d in figure 1. This is down to the
probe-tissue adhesion, which means that the probe pulls the tissue upwards with
it until the force of retraction is greater than the friction between them,
causing them to separate. The maximum
load force applied to the tissue was 1.8nN, which gave an indentation of depths
around 150 to 3000nm. The force curves are then analyzed using the Oliver and
Pharr technique 9, which is a method for calculating Young’s modulus of the
The Young’s modulus is a measure of the stiffness of a
material, hence showing us if the cells become softer as cancer develops 10. The
report shows that there was significant softening of the cancer tissue compared
with its counterpart.
They found that there were significantly different stages of
cancer development as shown in Figure 2. It can be seen that for normal tissue,
the dominant peak was found at 1.1 to 1.8kPa. For benign tissues, it was found
at 1.9-3.7kPa which is significantly higher. For cancer cells, the peak was
found between 0.3 and 0.8kPa. This hence shows there are three clear stages of
cancer development. This reveals that
cancer progression is not limited to the matrix stiffening 12 that was
previously thought to be.
The results shown were found in agreement with the
measurements done on single cells 13 and were also backed up by the results
gathered on transgenic mice. It can be seen that there are also second and
third peaks shown in the results, which is common among stiffer materials.
These results gathered are for breast tissue, however, there
is no doubt that there will be similar results produced with other cancer
tissues, giving identifiable fingerprints. Despite the results, it still
doesn’t tell us the cause of the observed increase in stiffness and how this
result might contribute to cancers’ progression. Furthermore due to complexity of solid tumors
which can exhibit molecular, cellular and architectural alterations 14,15,
at present this method should be performed alongside mammography, providing an
additional step in the classification of cancerous tissue.