Download A Validation of the Clonogenic Assay: A Study of Glioblastoma in Vitro

A Validation of the Clonogenic Assay: A Study of Glioblastoma in Vitro
REU student: Jacqueline Sugar
Graduate Student Mentor: Nathan Withers
Faculty Mentor: Dr. Marek Osinski
Activities:
A. Introduction
B. Background
C. Research Objective
a. Materials
D. Methodology
E. Descriptions of Experiments
Findings:
F. Findings
a. Results
b. Conclusions
c. Future work
Activities:
A. Introduction
Glioblastoma is currently the deadliest of brain cancers; there is no effective treatment. Most
patients live 3 months after diagnosis. The removal of brain tumors may extend life expectancy
to one year. My project is the validation of the clonogenic assay, which is used to study the
effects of irradiation in glioblastoma [1].
The cells are from a line of U-87, which are cancerous human glial cells in vitro. Glial cells are
the glue-like material surrounding neurons in the brain. The brain has a dynamic and fluctuating
ratio of glial cells and neurons, which adds to the uncertainty of the behavior of cancer. The glial
cells are also akin to an immune system in the brain, which may contribute to why glioblastoma
is so invasive. Glial cells are also linked to the blood brain barrier [2], and tumor vasculature is
part of the pericyte landscape that must be navigated by drug delivery treatments [3].
Originally, two assays were to be compared. One was the clonogenic assay, which results in
genetic clones, and the other would have been the MTT assay, which uses 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole[4]. Unfortunately the
summer has ended before completing the MTT assay, and so in lieu of a comparison of assays,
there is a validation of one of the assays.
A pivotal moment in my REU experience was my attendance at a lecture at the UNM Cancer
Center, given on July 23, 2012 by Dr. Andre Nel. I am infinitely grateful for the exposure to the
following ideas. These concepts of the applications of nanomedicine have been developed since
my attendance at the lecture, and have truly inspired me to continue to research the roles of
nanoparticles in cancer treatments. I had been a skeptic until this lecture, and it has galvanized
my confidence in the future of nanotechnology. There is most definitely a secure future for this
branch of science, and nearly unbounded possibilities for potential applications.
Nonetheless, there has been a tremendous amount of hype surrounding nanoscience [3]. The
phase of hype must be transcended by experimental evidence. Nanomaterials have proven
highly effective in biological processes. Engineered nanostructures can readily interact with biomolecules, such as protein corona, receptors, etc. Nanocrystals can be used not only with
regenerative medicine and tissue engineering, but also with delivery and interaction with
biological systems. The difficulty lies in the prevention of agglomeration of nanocrystals, and
also in the encapsulation, circulation half-life and retention of particles at the tumor site, but as
stated earlier, nanoparticles can reduce drug and radiation toxicity, even hydrophobic drugs [3].
Nanocrystals can also act as multi-functional delivery systems with tumor targeting ability.
Often there is poor lymphatic drainage at the tumor site, as well as hypoxia and other somatic
disorders that prevent active drug delivery. Much of the cancer drug will be processed by the
body via the kidneys, liver, lungs and spleen. These renal and hepatic macrophage processes
filter out the drug before it can target the tumor, without effective delivery. Nanocrystals
provide the capability to probe the tumor microenvironment, and to understand and capitalize
upon biochemical signatures. This can allow passive uptake of drugs, linked to specific cell
mechanisms. An example of this is systemic delivery of small interfering RNA (siRNA), also
known as silencing RNA. This has proven to be an effective therapeutic measure, by interfering
with gene expression for cells that have blocked apoptosis, oncogenes, and other undesirables. It
uses a complementary nucleotide sequence, 20-25 nucleotides in length. The siRNA tricks the
cell into translating its information to the proteins, in lieu of the undesirable nucleotides of the
cells’ own mRNA [3].
Mesopourous silica is an example of a nanomaterial that has worked well in experimental drug
delivery for mice with human tumor xenografts. This library of silica functions as a platform for
controlled delivery through turbid suspension. Nanovalves can also be placed within the
material, which open and close on demand, usually when encountering the signaling antibody.
Some are magnetically activated, opening within an oscillating field [3].
The figure below shows the multifunctional platform of a nanoparticle with specified delivery
systems.
Figure 1. A schematic of nanovalves [5].
Yet, even if we can package a drug, can it be delivered? What
is the therapeutic efficacy? In vitro we may study the drug
uptake, localization, release, hazard screening, apoptosis, and
intercellular payload. Does this compare to in vivo? Using
nanoparticles to enhancepermeability improves biodistribution
and enhances electron paramagnetic resonance (EPR) effects.
50-200 nanometers is the best size, with 100nm optimal. [3]
Most cancer drugs are highly toxic. The animal’s liver enzymes increase, cardiac enzymes, etc.
This has not been as severe with nanoparticle encapsulation. For example, SiO2 breaks down
after 5 days in live mice, expelled by the body’s filtration system [3]. The graph and photograph
on the following page shows how effective nanoparticles can be in shrinking a xenograft tumor
in mice by delivering doxorubicin.
Doxorubicin delivery in mice:
Figure 2: Tumor growth inhibition of doxorubicin-loaded NP3 in tumor-bearing nude mice. (A)
Comparison of the tumor inhibition effect of doxorubicin-loaded NP3 (Dox-NP3) versus free drug (free
Dox), empty particles, and saline in the KB-31 xenograft model. The tumor-bearing mice were
intravenously injected with 120 mg/kg doxorubicin-loaded NP3 weekly for 3 weeks. This particle dose is
equivalent to 4 mg/kg doxorubicin being delivered to each animal. The animals receiving the free drug
were injected with the same amount of doxorubicin weekly for 3 weeks. To compare the effect of NP3
alone, empty particles were intravenously injected at 120 mg/kg, weekly for 3 weeks. The saline group
received intravenous saline administration at the same time points. Tumor size was accurately measured
twice a week by the same observer. Tumor weight was calculated according to the formula Tumor weight
(mg) = (length in mm) × (width in mm)2/2. *p< 0.05, compared to saline; $p< 0.05, compared to free
doxorubicin. (B) Atthe end of this experiment, tumor tissue was collected from each sacrificed animal, and
a photograph of the tumor tissue was obtained [6].
As discussed, targeted nanoparticles have the potential to overcome the toxicity and efficacy
limitations associated with traditional cytotoxic agents and newer-generation molecularly
targeted drugs by trafficking a greater fraction of the administered drug directly to cancer cells in
a controllable and tunable manner [7].
The potential for the application of nanotechnology is intoxicating. There are far too many
examples to cite in this paper. I have only begun to understand the very periphery of this
expansion of human knowledge. I am fortunate in that my project for this summer has been not
only to validate the clonogenic assay as a tool for analyzing the effects of radiation therapy of
glioblastoma. I have also been attempting to understand the role of nanoparticles in alternate
modalities for the treatment of cancers, as well as the reduction of collateral damage during
radiation treatment to healthy surrounding tissues.
B. Background
In assays, cell survival curves are analyzed. When mammalian cells are irradiated, the cells are
affected differently, depending on dose, dose rate, and the stage of the cell cycle [8]. The assays
help to determine if the treated cells are able to reproduce.
Treated cells are incubated and then examined for metabolic activity. In the clonogenic assays,
the cell colonies typically form within 10-14 days [9]. The MTT assays would show metabolic
activity within 2-4 days. The reason for comparing the two is to determine whether the MTT
assays are a reliable and universal test. Clonogenic assays are a widely accepted practice in
cancer research, but MTT assays would prove to be a quicker and very useful practice, if
reliable.
Cells that retain all reproductive as well as functional activities after irradiation are classified as
having
survived.
Cells
characterizeed as nonfunctional
when
Figure 3. Cell survival curve
differentiated, or losing reproductive capacity when undifferentiated, are indicative of cell death
[8].
On the previous page, Figure 3 is a classic example of in vivo cell survival illustrating the linear
tail (on a log-linear plot) of the cell survival curve at high dose where cell killing becomes purely
exponential [10].
The goal of my mentor is to use a linear-quadratic model or α/β model, instead of a log-linear
graph of surviving colonies in vitro.
The linear component of the curve is parameterized by α, which has units of inverse-dose.
Single-hit survival is given by: SF1 = exp(-αD). The quadratic component of the curve is
parametrized by β, which has units of inverse-dose-squared. The quadratic component of the
curve is used to account for two-hit cell killing. Two-hit survival is given by SF2 = exp(-βD2).
Assuming that the single-hit and two-hit mechanisms are independent, the overall expression for
the curve is SF = (SF1)(SF2) = exp-(αD + βD2)… If α D = β D2, then D = α/β. Thus we can
use this information to solve for D, or the optimal dose of radiation [11].
Interphase death refers to cell death in any phase of life cycle. We are interested in mitotic
death, which is at a specific time of a cells reproductive phase. Immortality is a feature of cancer
cells, due to the absence of apoptosis-inducing genes such as P53, also known as the kill switch.
Apoptosis is the programmed cell death [12], which is a natural phase in a cell’s life cycle.
Mitotic death is signified by the slow death of cell colonies because they have no future, or way
to reproduce. Mitosis is shown in the figure below.
Figure 4. A cell cycle [13]
The schematics below show the interactions of chromosomes as they line up during mitosis, as
well as the location of DNA within each chromosome.
Figure 5. Mitosis [14]
Figure 6. DNA [15]
Genetic instability can be a part of this process. Exposure to free radicals and radiation can
cause breakages of double strand DNA. This, as well as an incalculable number of carcinogenic
factors, contributes to the beginnings of genetic mutations.
Figure 7.
DNA
damage
[16].
On the previous page, figure 10 shows damage to DNA from free radicals and other sources of
radiation exposure [15].
As shown, chromosomes align during mitosis and attach at the centromere before dividing.
DNA is contained within these chromosomes. Fragmentation and translocation of chromosomes
results in deletion and inversion of genetic coding material. If P53, or another gene linked to
programmed cell death is part of this
fragment, it remains unexpressed.
P53 is a gene linked to tumor
suppression, and without genes such
as P53, there is dynamic, unmediated
cell reproduction, without apoptosis.
Thus, there is rapid and chaotic
evolution of immortal cells. This
depletes precious oxygen and forms
necrotic tumors, as cells in the interior
die from lack of nourishment. [17]
Above, figure 8. Acentric fragments of chromosome [18].
Left, figure 9.
Dicentric chromosomal
fragmentation, resulting in
deletion and inversion of
genetic material [19].
One way to arrest the unmediated growth is with doses of radiation. Figure 10 shows applied
radiation damage to DNA.
Figure 10. Radiation is used to break a strand of rapidly evolving DNA. [20]
Post-treatment biomarkers then present themselves such as H2A.X – a repair gene which
becomes ΓH2A.X once it phosphorylates to serine 139. Phosphorylation is the addition of a
phosphate group to a protein, which occurs within DNA at the strand breaks [21]. ΓH2A.X is
especially useful because it appears in the body within one hour of treatment, which is much
faster than the usual 24 hours. Biomarkers are essential in metabolic control, signal transduction,
gene regulation, etc. Antibodies acting as biomarkers are important due to their high affinity and
high specificity, such as the epidermal growth factor receptor. There are 20-25,000 genes in
human genome, but over 1,000,000 proteins in human proteome. Below, figure 8 shows the
appearance of biomarkers at
the site of DNA damage.
Figure 11. The appearance
of biomarkers [22].
In the following diagram, a tumor is exposed to multiple beams of radiation. Below this,
brachytherapy is imaged using a radioactive source inside the lesion. But, tumors are not
perfectly isolated and spherical. This diagram demonstrates damaged healthy tissue that
surrounds the tumor, as well as undertreated lesions surrounding the target. Within a
nanocontext, the crystals can be used to selectively target irregular tumors, thus reducing
collateral damage.
Radiation Therapy in Cancer Treatment
External beam radiation therapy enters patient at several different angles.
Tumors are not
Spherical chickens!
Damaged
healthy tissue
Under-treated
Lesion
Brachytherapy:
Radioactive sources
Placed inside lesion for
Set time period.
14
Image courtesy of Nathan Withers
Figure 12. A schematic of multiple beams of radiation (top) and brachytheraphy (bottom).
The work of my graduate mentor has involved lanthanum fluoride nanocrystals in targeting
tumors with irradiation. Due to the high atomic number of certain types of lanthanum, radiation
energy is more efficiently absorbed by this. Gamma rays are photons, and usually shoot outside
of the tumor volume when used in radiation therapy, occasionally even overshooting the patient
body. When these photons are changed into electrons, there is a short and effective path length
of energy [11]. This utilizes the photoelectric effect in lieu of Compton scattering or pair
production. Thanks to the higher atomic number of the nanocrystals deposited into the tumor,
more energy is deposited directly into the tumor. Nanoparticles act as a radio-attractor, utilizing
the photoelectric effect. If radiation is absorbed, ionization and excitations occur that are not
distributed at random, but localized along tracks of charged particles. This deposits more energy
into target and preserves healthy tissue.
Furthermore, the law of Bergonie and Tribondeau states that ionizing radiation is more effective
against cells that are:
1. Undergoing a high division rate
2. Undifferentiated
3. Have a long dividing future [23]
As cancer cells continue mutating without regulation or apoptosis, they lose their differentiation,
enabling somatic mobility and spreading though a patient’s body. This lack of differentiation
aids the cancer in becoming more radiosensitive, and radiation therapy can be enhanced through
the use of nanoparticles.
C. Research Objective
a. List of Materials:
∙Biological Safety Cabinet (BSC), incubator,
microscope, micropipetters, centrifuge;
∙Tyrpan blue for staining cells in order to count
them with the hemocytometer;
∙6-well plates, glutaraldehyde and crystal violet
for clonogenic assay;
∙MTT kit, including 96-well plates, 96-well
plate reader;
∙Cell growth media, phosphate-buffered saline
(PBS);
Figure 13. Clonogenic Assay Plate [9]
The assay comparison is to determine a level
of consistency in these tests for radiation
damage. In clonogenic assays, the question
is whether the cells are able to reproduce.
We looked for colonies within 10-14 days.
In the MTT assays, the treated cells are
incubated with a chemical and we would, in
theory, have lookd for an absorption line using a spectrophotometer. These assays may be
testing the same information, but how universal are the results?
Right, Figure 14. MTT Assay Plate [4].
D. Methodology
The first order of business for the summer was to get
acquainted with a Biological Safety Cabinet (BSC). In order
to properly feed and care for the cells, a tremendous amount
of subtleties must be considered. After warming the growth
media in the incubator and sterilizing all surfaces with a
70% ethanol spray, the spent media is carefully removed
from the flasks and plates using a vacuum that only touches
the side of the well. The glioblastoma cells adhere to the
bottom of the vessels, and it is vital to not disturb them when
changing media. The fresh media is then added in a similar
fashion, without touching the bottom of the flasks and wells,
in addition to a proportionate amount of antibiotic and
antimycotic.
Figure 15. A scientist using the BSC.
The density of cells was determined by diluting cells to known concentrations. Using the
cells/mL calculated via the hemocytometer, we calculated bow many mL of cells was to be
mixed with cell growth media to arrive at a concentration of 0.25x105 cells/mL per 12mL of
fluid. The following formula is used to make a 10mL stock solution:
[
24]
Our formula was as follows:
0.25x10
Cells mL12mL
3x105 Cells 

 _________ mL cell solution to add
_____________ Cells mL _____________ Cells mL
5
Then, using a 75 cm2 culture flask, we added 78.72 mL of cell growth media and 480 μL of the
0.25x105 cells/mL stock solution to make a solution of 150 cells/mL. Then, using the 100-1000
μL pipette with a new tip, we added 800 μL of antibiotic solution to all of the wells of the 4 sixwell plates.
E. Description of Experiment
Once the cells were growing
and stable at appropriate
concentration, we drove
them to the gamma irradiator
and exposed the flasks to
varying levels of radiation.
The clonogenic assay is
performed by removing the
cell growth media after
irradiation, and then washing
the plate wells with
phosphate buffered saline
(PBS).
Above, Figure 16. Cells being washed by PBS.
The colonies were
then stained with a dye
and allowed to absorb
it for 30 minutes.
Figure 17. Colonies being stained with crystal violet.
Figure 18. The plates are
dipped into a water bath to
rinse the crystal violet,
after 30 minutes of sitting
and absorbing the dye.
Figure 19. The stained 6well plates after being
washed by PBS, dyed
with crystal violet, rinsed
and air-dried. They are
now ready for colony
counting!
Findings:
F. Findings
a. Results
The majority of the ten weeks have been spent learning how to care for glioblastoma.
There have been many details to attend to concerning using the cryogen for storing frozen
cells, the process of thawing the cells to use in the lab, and plating them into wells or
flasks.
Above, Figure 20. The cryogen.
We made our own growth media out of Eagle’s Essential Minimum and bovine fetal serum. My
mentor and I learned that the best way to thaw the U-87 growth media is by taking it from the
freezer and allowing it to sit at room temperature for ten minutes before placing it into the
incubator . As it warms, it must be agitated every ten minutes in order to further prevent
formation of precipitates [25]. When we added an intermediary step of thawing in the
refrigerator, the salts in the growth media would crystallize and the pH would become unreliable.
Learning how to handle the media was an important part of the project.
We also noticed a significant leak in the incubator, which was using a full 25 kilogram tank of
CO2. Since no other groups were sharing the incubator, and there were only 2-5 flasks or plates
in the incubator at a time, this was an alarming amount of carbon dioxide. The incubator was
able to keep the environment at 5% CO2, but was leaking gas out of the back of the machine.
We also needed to update the way that the tank was connected to the hoses by using a silicon
washer. Many details of the setup needed to be addressed before beginning to irradiate the cells.
We also needed to order gluteraldehyde and crystal violet for the clonogenc assays, as well as a
96-well plate reader, and MTT assay kit for the MTT assays.
From the protocols, I also wrote the procedures for the clonogenic and MTT assays, and ordered
the required chemicals for the experiments. I learned how to subculture the flasks of cells, and to
use the hemocytometer, in order to count the number of cells in a volume of growth media, and
thus seed an appropriate concentration. It is important to have 80-90% confluence before
irradiating the cells and performing a post-treatment assay.
Our 6-well plates for the clonogenic assay were plated at 300 cells per well.
b. Conclusions
The plating efficiency (PE) is the ratio of colonies to cells.
PE =
x 100%
The plating efficiency of our control group was 20%. PE =
x 100% = 20%.
The number of colonies that arise after treatment of cells is called surviving fraction (SF).
SF =
The surviving fraction after 3.33 Gray was
= .5667 = 56.67%
The surviving fraction after 6.66 Gray was
= .1625 = 16.25%
The surviving fraction after 9.99 Gray was
= .0472 = 4.72%
Below, figure 21 is the survival curve of irradiated glioblastoma cells.
2
SF= exp-(D+D )
Surviving Fraction
1
 model fit:
 = 0.12
 = 0.02
2
0.1
R fit = 0.992
0.01
0
2
4
6
8
10
Dose [Sv]
Thus, the clonogenic assay works! The plating efficiency is useful information for future
experiments in which the cell density will be manipulated. Also the
87 will be useful for future passages of the cells.
c. Future work
and β parameters for U-
The direction that this project could continue in would be to confirm the reliability of the MTT
assay as compared to the already widely used clonogenic assay. These tests can then be used to
validate the use of nanoparticles in radiation enhancement and targeted drug delivery.
The reproductive capacity of cells, both in vivo and in vitro, is known as the clonogenic
potential.[10] The loss of clonogenic potential may be a very limited definition of cell death, but
it is what defines cell survival in this case. Even if the cell can carry out protein synthesis and
other functions after exposure to radiation therapy, sustained replication is the crucial aspect that
signifies mitotic death.
An improvement of the clonogenic assay would be through the use of agar when growing the
colonies, in order to prevent diffusion. The counting of colonies can be a subjective endeavor,
and this would potentially clarify any misgivings about what constitutes a colony under a
microscope.
The future applications of the clonogenic assay could also be used in conjunction with testing for
cytotoxicity. Radiation is known to damage cells, thats why it is used to treat cancer. But how
toxic are the nanoparticles used to attract the doses? Even if it allows for a lower, more effective
dose rate, what is the effect of lanthanum fluoride on healthy glial cells? And for targeted drug
delivery systems, how does the body process the nanoparticles?
The lack of observable toxicity of the doxorubicin-laden particles in the liver and spleen is an
interesting finding that has not been resolved as yet. One possibility is that the traditional
biomarkers used for following liver injury are ineffective in reflecting RES [Reticuloendothelial
system] damage, but another explanation is that the RES and organs like the liver are quite
resilient in dealing with doxorubicin toxicity when the drug is encapsulated. This constitutes
another important safety feature of a nanocarrier that either could be degraded in situ into
cellular subcomponents or could be excreted from the body once the carrier has served its
therapeutic purpose [6].
Even if a mesoporous silica nanoparticle can slip past the malformed blood vessel fenestrations
of a xenograft tumor [6], studying cancer in vivo is a highly complex system in comparison to in
vitro. Validation of the clonogenic assay is a part of a vast approach to the methodology of
cancer research, and hopefully it will continue to prove an effective tool for analysis of
treatments.
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