Soft Agar Colony Formation

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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.

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). 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

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 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

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 96-well 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 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 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.

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.

Bibliography

Blaskovich, M. A., Yendluri, V., Lawrence, H. R., Lawrence, N. J., Sebti, S. M., & Springett, G. M. 2013. Lysophosphatidic Acid Acyltransferase Beta Regulates mTOR Signaling,PloS one,8(10).

Bose, R., Kavuri, S. M., Searleman, A. C., Shen, W., Shen, D., Koboldt, D. C., ... & Ellis, M. J. 2013. Activating HER2 mutations in HER2 gene amplification negative breast cancer,Cancer discovery,3(2), 224-237.

Döppler, H., Liou, G. Y., & Storz, P. 2013. Downregulation of TRAF2 mediates NIK-induced pancreatic cancer cell proliferation and tumorigenicity,PloS one, 8(1), e53676.

Fejzo, M. S., Anderson, L., von Euw, E. M., Kalous, O., Avliyakulov, N. K., Haykinson, M. J., ... & Slamon, D. J. 2013. Amplification Target ADRM1: Role as an Oncogene and Therapeutic Target for Ovarian Cancer,International journal of molecular sciences,14(2), 3094-3109.

Ho, V., Yeo, S. Y., Kunasegaran, K., De Silva, D., Tarulli, G. A., Voorhoeve, P. M., & Pietersen, A. M. 2013. Method summary,BioTechniques,54(4), 208-212.

Jaiswal, A. S., Panda, H., Pampo, C. A., Siemann, D. W., Gairola, C. G., Hromas, R., & Narayan, S. 2013. Adenomatous Polyposis Coli-Mediated Accumulation of Abasic DNA Lesions Lead to Cigarette Smoke Condensate-Induced Neoplastic Transformation of Normal Breast Epithelial Cells,Neoplasia (New York, NY),15(4), 454.

Jeong, Y. T., Cermak, L., Guijarro, M. V., Hernando, E. & Pagano, M. 2013, FBH1 protects melanocytes from transformation and is deregulated in melanomas,Cell Cycle,12(7): 0-1.

Ji, T., Lin, C., Krill, L. S., Eskander, R., Guo, Y., Zi, X., & Hoang, B. H. 2013. Flavokawain B, a kava chalcone, inhibits growth of human osteosarcoma cells through G2/M cell cycle arrest and apoptosis,Molecular cancer,12(1), 1-11.

Kanno, H., Sato, H., Yokoyama, T. A., Yoshizumi, T., & Yamada, S. 2013. The VHL tumor suppressor protein regulates tumorigenicity of U87-derived glioma stem-like cells by inhibiting the JAK/STAT signaling pathway, International journal of oncology,42(3), 881.

Kwun, H. J., Shuda, M., Feng, H., Camacho, C. J., Moore, P. S., & Chang, Y. 2013. Merkel Cell Polyomavirus Small T Antigen Controls Viral Replication and Oncoprotein Expression by Targeting the Cellular Ubiquitin Ligase SCF< sup> Fbw7</sup>,Cell host & microbe,14(2), 125-135.

Lennartsson, J., Ma, H., Wardega, P., Pelka, K., Engstrom, U., Hellberg, C. & Heldin, C. H. 2013, The Fer tyrosine kinase is important for platelet-derived growth factor-BB-induced Stat3 phosphorylation, colony formation in soft agar and tumor growth in vivo,Journal of Biological Chemistry.

Lin, C. H., Guo, Y., Ghaffar, S., McQueen, P., Pourmorady, J., Christ, A. & Hoang, B. H. 2013. Dkk-3, a Secreted Wnt Antagonist, Suppresses Tumorigenic Potential and Pulmonary Metastasis in Osteosarcoma,Sarcoma, 2013.

Luo, Y., Kaz, A. M., Kanngurn, S., Welsch, P., Morris, S. M., Wang, J. & Grady, W. M. 2013. NTRK3 is a potential tumor suppressor gene commonly inactivated by epigenetic mechanisms in colorectal cancer,PLoS genetics, 9(7), e1003552.

McLaughlin, S. K., Olsen, S. N., Dake, B., De Raedt, T., Lim, E., Bronson, R. T., ... & Cichowski, K. 2013. The RasGAP Gene,< i> RASAL2</i>, Is a Tumor and Metastasis Suppressor,Cancer cell,24(3), 365-378.

Mumby, M. 2013. Characterization of the Role of the PP2A-AB Gene, a Putative Tumor Suppressor, in Cell Growth and Tumorigenesis.

Pires, M. M., Hopkins, B. D., Saal, L. H., & Parsons, R. E. 2013. Alterations of EGFR, p53 and PTEN that mimic changes found in basal-like breast cancer promote transformation of human mammary epithelial cells,Cancer biology & therapy,14(3), 0-1.

Rohle, D., Popovici-Muller, J., Palaskas, N., Turcan, S., Grommes, C., Campos, C., ... & Mellinghoff, I. K. 2013. An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells,Science,340(6132), 626-630.

Singh, B., Bogatcheva, G., Washington, M. K., & Coffey, R. J. 2013. Transformation of polarized epithelial cells by apical mistrafficking of epiregulin, Proceedings of the National Academy of Sciences,110(22), 8960-8965.

Steinberg, P. 2013, Automated Soft Agar Colony Formation Assay for the High‐Throughput Screening of Malignant Cell Transformation,High-Throughput Screening Methods in Toxicity Testing, 309-316.

Sumida, T., Murase, R., Onishi-Ishikawa, A., McAllister, S. D., Hamakawa, H., & Desprez, P. Y. 2013. Targeting Id1 reduces proliferation and invasion in aggressive human salivary gland cancer cells,BMC cancer,13(1), 141.