Research into the cancer biology has taken a step further ahead with the identification of putative cancer stem cells in the tumor masses which are responsible for resistance to conventional therapy. These cells have the self-renewal property of the normal stem cells but loose the strict control on cell division which makes them cancerous. Hence it has been postulated that they are present in various tumor masses and many studies have isolated and characterized such stem cells. The isolation and characterization of stem cells using stem cell markers can be done by various techniques such as flow cytometry and PCR. The project is carried out to characterize the cancer stem cells CT26 from colon cancer which have been subject to various concentrations of doxorubicin.(please put source here or reference)
This will be done by isolation and characterization of cancer stem cells by using chemotherapeutic agents such as Doxorubicin and then culturing Doxorubicin resistant cells in different growth media in order to characterize their growth requirements. Finally, cancer stem cells will be characterized by expression of markers such as CD44, CD133 ,PML,MHC-1,P36 and SOX-2.
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This will be done by using the following techniques:
1-Cell culture technique.
2- Soft agar Assay
3-Proliferation Assay for drug resistance.
4- Characterization of stem cell markers by PCR and RT-PCR
The data obtained will be collected and compiled for further analyses.
A cancer may be defined as a group of diseases characterized by the uncontrolled growth of abnormal cells which have the ability to spread throughout the body or body parts (Bender and Bender, 2005). There are more than 100 types of cancer and can affect any part of the body (WHO, 2006). In 2004, cancer caused 7.4 million deaths all over the world which account for 13% of deaths globally (WHO, 2006). The etiology of cancer is complex and multi factorial. Many factors contribute towards development of cancer such as ultraviolet rays, diet, certain chemicals, drugs, viruses and hormones etc.Despite a number of treatment options devised to manage and treat cancer, researchers have met with only little success with early detection and prevention being the mainstay of dealing with cancer. (source)
2.2 Stem Cells
Stem cells are the progenitor of other cells that produce all other cells of the body. These are themselves undifferentiated and do not perform any other function. They have a capacity to propagate themselves by the process of self-renewal and also produce mature cells of other tissues by carrying out their differentiation to perform various functions (Reya et al, 2001). This property of self-renewal is vital for them as they are required in the production of many cells throughout life. Along with this property of self-renewal and division, the normal stem cells regulate a balance between their self-renewal and differentiation and keep a strict control on their numbers (Sagar et al, 2007). Owing to these properties, stem cells have been widely used in the replacement of immune cells and blood cells which have been damaged by a cancer or some other cause. Moreover, they also find their application in tissue regeneration and as a delivery medium for cancer therapy (Sagar et al, 2007).
2.3 The "Cancer Stem Cell" Concept
The concept of "cancer stem cells" emerged in 1980s which led the scientists to believe that this might be partially responsible for achieving success in cancer treatment (Lin et al, 2008). However as a result of lack of specific markers for their isolation and less advanced technology, not much advancement could be made at that time. Later, Dick and colleagues isolated putative cancer stem cells (CSCs) from acute myelogenous leukemia and led to a series studies for isolation of these cells from various forms of cancer.
Cancer stem cells have been defined as cells within the tumor growth with a tumor which have the potential of initiating a tumor (Sagar, 2007). These cells have the property of self-renewal and form multiple forms of cancer cell lines that form the tumor (Clarke et al, 2006). Hence, other terms such as "tumor initiating cell" and "tumorigenic cells" have also been used to describe these CSCs (Clarke et al, 2006). This small subset of stem cells have the ability to both divide and increase the population of various types of non-tumorigenic differentiated cells in the tumor cells pool which constitute the bulk of tumor. Moreover, these cells have a slow rate of division and proliferation (Pérez-Caro and Sánchez-García, 2006) (Figure 1).
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Figure 1: (Source: Bjerkvig et al, 2005)
A normal stem cell has the properties of self-renewal, strict control on cell division and ability to differentiate. However, the cancer stem cells do not have the ability to have control on cell numbers hence causing unchecked increases in these differentiated non-tumorigenic cell populations (Sagar et al, 2007). Thus a cancer stem cell works in a similar way for the growth and spread of tumor but it is not subject to the intrinsic and extrinsic controls to which normal stem cells are subject to (Pérez-Caro, M. and Sánchez-García, 2006).
2.4 Isolation and Characterization of Cancer Stem Cells
One problem which arose is that was difficult to recognize these cells with tumor initiative potential as they are very few in number with plating efficiency ranging from 1/1,000-5,000 to 1/1,000,000 (Lin et al, 2008). However, identification of stem cell markers led the(you mean stem cell or cancer stem cell ) researchers to isolate these cells from various other tumors such as CD 44, CD 133, Sca1, and Thy1. It must be kept in mind that these markers are not universally present on all cancer stem cells and vary widely from one organ to another (Clarke et al, 2006).
The initial evidence for the presence of these cancer stem cells came from haematological tumors but they have been now isolated from a variety of solid tumors such as breast, colon, prostate, lungs, pancreas, head and neck, kidney, liver and brain tumors. (Lin et al, 2008).
The cancer stem cells in all organs share the ability of self-renewal and differentiation however the type of stem cell varies significantly from one organ to the next. For analysis of these cells a serial transplantation in animal models is the gold standard experimental studies. In these studies, the cells are xenografted into a non-obese diabetic mouse with severe combined immunodeficiency syndrome that are then analysed at various time intervals for tumor formation (Clarke et al, 2006).
This analysis for tumor transformation is done by checking for the presence of various cell surface markers. Various studies suggest the presence of cell surface markers on surface of tumor cells. For example CD 133 was initially used with success for isolation of stem cells in(please you mean here stem cell or cancer stem cells) brain tumors but it was revealed that this is present on normal stem cells and other tumors as well such as colon cancer. The definitive characterization of these stem cell markers in various tumors is essential as they will be the mainstay of any future therapies intended to target CSCs (LaBarge and Bissell, 2008). Other markers like CD 44 have also been studied and Liu and colleagues (2007) have suggested that there is a positive expression of CD44 in breast cancer stem cells as compared to the normal breast cells and this showed a significant association between the presence of this gene and the overall survival rate.
Other stemness markers have also been studied such as SCA 1 have been identified in characterization of stem cells in mammary tumors of BLAB-neu T transgenic mice (Grange et al, 2008).
The studies for identification and characterization of these makers commonly employ flow cytometry technique to recognize these cell surface markers either isolated from the mouse models or maintained in a tissue cell line. The methods employ use of cells which have been tagged by antibodies and use of FACS (Grange et al, 2008).
Other technique that can be used for this purpose is PCR and RT-PCR (Haraguchi et al, 2005).
2.4 Resistance to Doxorubicin
If the cancer stem cells theory proves to be true, then it would provide answers to many problems such as lack of response to therapy, resistance to therapy and recurrence (Sagar et la, 2007). Keeping the cancer stem cell hypothesis in view, if such tumor is treated with chemotherapeutic drugs targeting the rapidly dividing cells which will be cancer stem cells in this case and these CSCs are resistant to these drugs then the therapy will prove ineffective and will be followed by relapses (Clarke et al,, 2006) (Figure 2).
Figure 2: (Source: Sagar et al, 2007).
Resistance have been shown against a number of chemoptherapeutic drugs such as Gemcitabine, 5-fluorouracil (5-FU), bevacizumab and doxorubicin.
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A recent study by Zheng et al (2010) showed that thyroid cancer were resistant to doxorubicin and the tumor continued to progress despite therapy with this drug. These thyroid cells were then characterized by FACS for their content of cancer stem cells, their in vitro sphere-forming capacity and their expression of multidrug resistance transporters of the ABC gene family which may confer drug resistance to the cells. The cells were treated with doxorubicin and then analyzed. It was shown that treatment with doxorubicin killed the non-side (stem cell) population of cancer cells derived from anaplastic thyroid carcinoma cells. As a result, there was an advantage to stem cells and they overgrew in the culture. This chemoresistance of these drugs is used to isolate and characterize the stem cells as by their property of showing stem cell markers (Li et al, 2010).
3. Aims and Objectives
The aims of this project are
1. To isolate and characterize cancer stem cells by using chemotherapeutic agents such as Doxorubicin.
2. To culture Doxorubicin resistant cells in different growth media in order to characterize their growth requirements.
3. To observe the expression of markers CD44,CD133 ,PML,MHC-1,P36 and SOX-2 on the stem cells by using PCR and RT-PCR.
4.0 Experimental Approach
Carcinoma cell line CT-26 will be used to obtain multiple clones of cancer stem cells. These stem cells will be differentiated on the basis of surface markers on them such as CD44, CD133 ,PML,MHC-1,P36 and SOX-2. This characterization of cancer stem cells will be done by PCR and real time PCR. (need source)
4.1 Cell Culture and Maintenance
The carcinoma cell lines will be revived from storage vial in liquid media. In order to achieve this revival, DMEM media with 10% FCS and RPMI media with 10% FCS and NEEA will be used. Cells will be incubated for 48-96 hours at 37 Â°Â C in an atmosphere of 5% CO2 and then it will be repeatedly sub-cultured to obtain a confluent growth in culture flasks. This confluent growth will be them washed with PBS and will be then trypsinized and incubated for a few minutes. (need source)
4.2 Treatment of CSCs with chemotherapeutic drugs (Doxorubicin)
Confluent growths obtained in the flasks mentioned above will be used for chemo resistance essay. The trypsinized cells will be counted to obtain about 106 cells. This cell count will be cultured in DMEM and RPMI respectively. Doxorubicin (an anticancer chemotherapeutic drug) will be added in different concentrations to observe the effect of these on growth of cells.
To achieve this, doxorubicin resistance comparative analysis will be done over a range of drug concentrations varying between 100ng/ml to 1000ng/ml. The behaviour and growth patterns of cells will be observed.
Populations of cells resistant to doxorubicin will be re-cultured through another passage. During this passage, various growth nutrients will be added to the different sets of media in which the doxorubicin cells will be cultures. The plates will be incubated again at 37Â°Â C in an atmosphere of 5% CO2 for 48-96 hours.
The steps of can be described as under:
CT-26 cells will be cultured under normal culture conditions for 72 Hrs in culture flask T75 with seeding of 750000 cells.
The culture media for these cells will be DMEM media with 10% FCS containing 1000ng Doxorubicin/ml of the media and incubated for 4 hours.
The medium contents will be then drained off and fresh DMEM will be added. This will again be incubated for 48 hours to allow cells to revive themselves.
The doxorubicin resistant cell population will be divided in two parts after trypsinization.
One population of cells (population I) will be cultured for 96 hours in the normal medium in the flask T75. While population II will be cultured in a corning ultra-low attachment flask T25 for 96 hours in an optimal spheroid generation medium.
For both these populations, growth patterns and behaviour of cells will be analysed.
If the population I is viable, it will be sub-cultured in normal environment and growth medium. The ultra-low attachment flask with population II will be shaken gently and sub-cultured in flask T75 for 72 hours.
Following this, population II will be again divided into population A and population B. Once again, population A will be cultured in flask 75 for 96 hours in normal medium whereas population B will be cultured in a corning ultra low attachment flask T25 for 96 hours in optimal spheroid generation media.
The characterization of SCSs can also be carried out by PCR and RT-PCR effectively (Haraguchi et al, 2005).
PCR is an in vitro procedure or method which helps amplify definite target DNA sequences found within a source of DNA. Generally PCR is developed to allow selective amplification of a particular target DNA sequence/s within a heterogeneous set of DNA sequences. As the newly synthesised DNA strands act as templates for more DNA synthesis in the following cycles PCR acts as a chain reaction (Strachan and Read, 1999).
PCR measures the data at the end-point (plateau) phase. Major advantages of the PCR technique as a cloning method are its rapidity, sensitivity and robustness. However, it does not provide quantification (need reference name)
Real-time PCR has been introduced which provides PCR. This permits the researcher to actually see or view the increase in the quantity of the DNA as it is amplified (Wong and Medrano, 2005). In contrast to the previous traditional PCR, the RT-PCR measures and collects the data when the reacts is actually in progress. It also provides quantification of RNA or DNA to be measured.
Real-time PCR greatly helps simplify the amplicon identification through providing the means to monitor the accumulation of particular products continuously during the cycling process. The Real-time PCR is also beneficial as analysis through this method can be done without opening the tube which can then be discarded without the risk or danger of dissemination of PCR amplicons or other target molecules in the lab circumstances. This is also efficient, cost-effective and economical. Real-time PCR also covers certain drawbacks of standard PCR, such as, standard PCR formats depend on the end-point analysis. These are not quantitative because the final product yield is not mainly reliant on the concentration of the target sequence in the sample. The Real-time PCR triumphs over this demerit of the standard PCR. However, the extreme sensitive nature of RT-PCR contributes to false positive tests which are difficult to avoid due to contamination in the laboratory. . ( need source name or reference)
4.5 Characterization and Isolation of CSCs
The cells grown earlier in the flasks will be analysed for the presence of CD44, CD133, MHC, PML after extraction of RNA by using PCR and real time PCR.
Almost 104 cells will be collected in a microcentrifuge tube with 350 Â Âµl RLT buffer containing 1% 2-merceptoethanol. The total RNA from these cells will be then extracted using the RNA extraction kit and purity and concentration of the RNA will also be determined.
T7 linear amplification will be applied and this amplified RNA will be then transcribed to DNA to be analysed using PCR and real time PCR. ( need source name or reference)
The results and data thus obtained will be analyzed by using statistical tests to obtain quantifiable and dependable results.
tigationMilestones of Inves
The project will commence in April with are ethical approval and literature review. Milestones of this project like basic training in techniques, cell revival, treatment with doxorubicin and characterization of CSC will be done in May, June and July. The data will be analysed and thesis will be compiled in August and September.