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SOFT AGAR COLONY FORMATION
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Abstract
Soft agar colony formation assay is primarily conducted to test whether cells have undergone
malignant transformation. Recent advances have enabled soft agar colony formation to be carried
out with better quantification accuracy thus enhancing process speed. The use of semisolid agar
media for colony quantification now utilizes fluorometric dyes which have essentially eliminated
traditional manual counting techniques. Detection of colonies through fluorometric techniques
under a microscope has reduced assay times from the previous three to four weeks to a single
week.
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Introduction
Cell transformation refers to the stimulation of specific phenotypic modifications in
cultured cells consistent with tumorigenic cells. Tumorigenic cells have defined characteristics
which include anchorage independent cell development, uninhibited cell development, autocrine
growth production factors, distorted cell morphology, and introduction of tumors in immunity
deprived nude mice (Bose et al. 2013 p 228). Anchorage independent cell development refers to
the situation where no solid substratum such as the glass surface on culture flasks or discs is
necessary for growth of culture cells (Ji et al. 2013 p.6). This implies that cultured cancer cell
development can be propagated in a soft media or suspensions where culture cells can
appropriately form cell colonies. This procedure was first explained half a decade ago by two
medical researchers, namely Manpherson and Montagnier (Steinber, 2013 p. 309).
It is important to note that traditional methods of soft agar assay do not produce
recoverable cancer colony cells that are viable for biological research (Yang et al. 2013 p.2193).
As such, a novel procedure has been formulated and this process is referred to as the advanced
colony formation assay which allows for the efficient and effective of viable altered colony cells
which can allow for more research through progressive culturing and tests (Döppler et al. 2013 p.
7). Results from the experiments conducted by Manpherson and Montagnier will be addressed
throughout this thesis relative to the recent developments that have led to the development of
advanced soft agar colony formation systems.
Anchorage Independent Cell Development
Neoplastic alterations occur as a result of a series of both genetic and epigenetic changes
which allow for the growth of cell populations capable of multiplying independently such that
internal and external indicators of restrained growth are not visible (Fejzo et al. 2013 p. 3100).
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Anchorage independent cell development has been hailed as a significant hallmark with regard to
cell transformation as it has proved to exhibit high degrees of accuracy and stringency in the
detection of cancerous transformations in body organ cells (Suresh et al. 2013 p.5464).
In the contemporary medical environment, soft agar colony formation has been embraced
as a universal means with which to observe anchorage independent development (Steinber, 2013
p. 310). The process serves to enable the measurement of the rate of cancer cell propagation in
semisolid cell culture media in a period of three to four weeks through manual counting of
observable colonies (Yoshida et al. 2013 p. 174). Much literature has been published though the
application of manual counting techniques has been considered as not only being cumbersome
but also complex and time consuming when large sample numbers have to be tested (Steinber,
2013 p. 310). Manual counting is also quite subjective in that the determination of significant
results becomes overly difficult relative to colony size development.
Applications of Soft Agar Colony Formation in Cancer Research
Soft agar colony formation is basically applied in the biological and medical fields in a
number of very significant ways in cancer research (Steinber, 2013 p. 309). Firstly, the procedure
is applied in running chemosensitivity tests of tumor cells with the main aim of establishing
potent antitumor agents (Ho et al. 2013 p. 210). Secondly, the procedure is used in the
development of novel therapeutic approaches aimed at controlling the outgrowth of cancer cells.
This implies that soft agar colony formation has led to limiting the number of animals used in
cancer research studies as this process is most likely to be the most applied procedure in cancer
research (Steinber, 2013 p. 309).
The Role of Hypoxia in Soft Agar Colony Formation Assay
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Most cancerous tumors are intensely hypoxic and these offer a poor degree of diagnosis
compared to non-hypoxic cancer tumors. Hypoxia has been known to promote cell development
in anchorage independent soft agar colony formation. For ESFT cell, cell lines SK-N-MC and
TC252 were seeded in a soft agar experiment and assessed for 14 days noting differences in
normoxic conditions and hypoxic conditions. Results showed little increases in colony size under
hyporexic conditions though under normoxic the colony size was significantly smaller. This is
because hypoxia allows for enhanced glucose uptake, glycolysis, which affects transcriptional
regulation thus stimulating cell motility and subsequent invasions. This is mainly due to the fact
that transcriptional actions consistent with hyporexia inducing factors not only increase but also
stabilize protein levels as the main response consistent with adapting to tumor hypoxia.
Disadvantages of the Traditional Soft Agar Colony Formation Process
There are a number of practical disadvantages in using the traditional soft agar colony
formation assay. One of the most commonly cited disadvantage is the duration required so that
experimental results can start being obtained (Steinber, 2013 p. 310). The period required for the
traditional soft agar colony formation procedure to begin bearing experimental fruits is between
two to four weeks. Another practical disadvantage associated with the procedure is the high rate
of use of laboratory resources. For instance, this procedure requires research technicians to
utilize a relative high amount of plastic disks and flasks as well as laboratory space. The third
practical disadvantage associated with colony formation process is the time consuming manual
counting technique associated with the traditional soft agar colony formation process (Steinber,
2013 p. 310). The fourth practical disadvantage of the traditional process is the need for accurate
temperature control lab apparatus (Xia et al. 2013 p.418). If the soft agar temperature rises above
the recommended degree when adding the semi sold agar into the test cancer cells then the
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likelihood that the cancer colony cells will be destroyed is high (Blaskovich et al. 2013, p.3). If
the soft agar temperature falls below the recommended temperature range then there is a high
probability that the agar will solidify (Taliaferro-Smith et al. 2013 p.23).
Recent Developments in Cancer Research
Research studies carried out over the past few years have sought to eliminate the practical
disadvantages associated with the classic soft agar colony formation procedure (Jaiswal et al.
2013 p.454). Firstly, the classic soft agar colony formation process has been progressively
miniaturized to realize higher throughput. The traditional 6-well design has been decreased
downwards to a 96-well micro-tier plate design (Steinber, 2013 p. 310). This has further been
miniaturized into a 384-well micro-tier format with reference to recent publications. Secondly,
the length of the entire process has been progressively decreased into process with an overall
duration of only seven days. Thirdly, the overly laborious and sometimes inconsistent manual
cell quantification technique has been replaced with innovative, reliable and highly effective
quantification techniques.
The application of automated volume or image analysis techniques has been developed to
quantify colony cells (Luo et al. 2013 p. 15). The use of dyes such as Alamar Blue has also aided
much in reducing the time consumed in the quantification process (Jeong et al. p.1). Plate readers
have also been developed to aid in the quantification of colony cells stained with Alamar Blue
(Steinber, 2013 p. 310). Lastly, the entire soft agar colony formation assay is now automated
enabling high throughput screening further enhancing the ability to observe cell growth in
anchorage independent cell development.
Measuring Cancer Stem Cells Colony Formation Using the 96-Well Design
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Ke, Albers, Claassen, Chatterton, Hu, Meyhack, Wong-Staal and Li in 2004, published a
paper in the Biotechniques journal and it was the first time the 96-well design was applied in the
establishment of soft agar colony formation (Steinber, 2013 p. 310). The 96-well design also
incorporated the fluorometric dye to enhance the quantification of cell propagation (Kanno et al.
2013 p.881). Referred to as the fluorometric readout, the dye enabled the development of a
parameter to better investigate cell colony formation.
HeLa and HeLaHF are sell lines with HeLaHF representing the altered cells and HeLa
representing unaltered cells (Kwun et al. 2013 p. 130). Both were introduced into a liquid
medium and cultured for 24 hours. HeLa and HeLaHF were also introduced into a semi solid
medium and cultured for one week (Steinber, 2013 p. 310). The two cell lines were placed 96well design micro-plates in an effort to investigate anchorage dependent as well as anchorage
independent cell growth (McLaughlin et al. 2013 p .368). The termination of the experimental
duration resazurin also known as Alamar Blue was introduced to stain the cell in both 96-well
design plates so as to conclusively the number of cells in each of the 96 wells for both cell lines
(Steinber, 2013 p. 312). The nonfluorescent nature of resazurin transforms into resorufin with red
fluorescence in living tissue cells. Fluorometric measurements determine the quantity of
resorufin generated in this manner. An assumption was made to the effect that the quantity of
resorufin produced as being directly proportional to living cell numbers in each of the 96 wells.
As such, the fluorescence measured presents an approximation on the extent of cancer stem cell
development in soft agar.
In the liquid culture medium, the intensity generated as a result of resazurin staining is
considered to be directly proportional to HeLaHF as well as HeLa cell numbers in each of the 96
wells (Lennartsson et al. 2013). Subsequent results show that HeLa cells proliferate at a rate that
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is relatively faster compared to HeLaHF cells. In the soft agar, a linear correlation between the
cell numbers in each well and the Alamar Blue staining extent was evident for HeLa cells
(Steinber, 2013 p. 310, 311). For the HeLaHF cells, the intensity of staining in each well was
observed to remain in background levels such that this was independent of the cell numbers
contained in each of the wells.
The soft agar test design developed by the team of Ke et al. enabled the determination as
to whether or not cancer cell line development is anchorage independent in a seven day period
with regard to the 96-well micro-plate design (Sumida et al. 2013 p.141). This seven day
incubation period has proved to be effectively efficient in the investigation of A549, DU145,
MCF7, HeLa, DLD1, HCT116 and U87 cells, the procedure has also provided satisfactory
results for cancerous colon cell lines DLD-2, MIP-101 and HT-29 (Mumby, 2013). To further
investigate as to whether this can apply to other cancerous cell lines, individualized tests need to
be carried out for every specific cell line.
KE et al. test system can also be applied in the analysis of gene transfection which causes
cancer cell behavioral modifications. The cell line referred to as DLD-1 for colon carcinoma is
known to contain an active gene known as K-RAS. In the instance where a lentiviral vector
inclusive of siRNA against a mutated K-RAS is introduced into DLD-1 line cells and the
resultant transduced cells expressed as mutated K-RAS siRNA, K-RAS mRNA levels were found
to significantly diminish (Steinber, 2013 p. 312). These diminished levels result in an associated
reduction of the cells ability to realize continued development in soft agar. The experiment was
also known to be appropriate for the study on the outcomes of transitory expressed siRNA aimed
against K-RAS or alternatively PLK a threonine type protein kinase exhibited in the HeLa cells
compared to the unaltered HeLaHF. In the two cases, a reduction in the prevalence of specific
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genes resulted in a significant decrease in HeLa cell development in soft agar (Rohle et al. 2013
p.630).
Measuring Cancer Stem Cells Colony Formation Using the 384-Well Design
Anderson, Towne, Burns and Warrior were the first team of researchers to offer a
description of the vigorous 384-well throughput soft agar colony formation assay (Steinberg,
2013 p. 311). The study investigated compounds with the ability to inhibit the development of
lung carcinoma in human beings more so with regard to the cancerous HCC827 cell line. The
first step in this procedure was to introduce 10µL of a 0.6% agar solution into cell culture
medium and introduced to each of the 384 wells (Steinberg, 2013 p. 311). Alamar Blue was used
to stain the wells after one week incubation duration for which the temperature was maintained
at 37ºC (Singh et al. 2013 p.8962).
Preliminary results provided a basis for the conclusion that for this type of assay, each of
the 384 wells needed to be introduced with 5000 cells. After the seven day incubation duration
Alamar Blue staining was performed in a six to 24 hour long period (Lin et al. 2013 p). The
results further indicated that values for IC150 for compounds such as gefinitib, staurosporine,
vandetanib and erlotinib present a development inhibiting effect in HCC827 cell line ranging
between o.4 and 5nM. Lapatinib on the other hand appeared not to inhibit cell development for
lack of potency (Pires et al. 2013 p.1). The same was observed for imitanib (Steinber, 2013 p.
312). The assay procedure developed by Anderson et al. showed results consistent with results
which have been published in earlier scientific literature this served to approve the 384-well
high-throughput soft agar colony formation as a working model in studies relevant to cancer
research.
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Conclusion
Soft agar colony formation has been critical to the development of cancer related research
studies. Since the inception of the procedure over fifty years ago, research scientists have
proactively sought to develop more conclusive soft agar colony formation procedures that will
shed more light into the behaviors of altered cell colonies. Protein and DNA analysis is one
application where procedures have made it possible to envisage the future development of cancer
vaccines. The developments of the 96-well and 384-well soft agar colony formation protocols
have played a significant role in the development of treatments and therapies for cancer patients
in a duration that is shorter and with more conclusive results.
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