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Cancer can be defined as a medical disorder characterised by uncontrolled growth, invasion and metastasis. The slow progression and conversion of benign tumours in to active malignant tumours relates to the degree of this disease. Cancer stem cells are present in these cancerous tissues and possess an ability to differentiate to generate matured cells. One of the therapeutic procedures employed is conventional drug therapy which inhibits the division by killing differentiating and differentiated cells, however during this time, the presence of cancer cells cause relapse of the disease as the drugs do not act upon the cancer stem cells as it does on the tumours. Research investigations gained importance in treating the cancer along with the cancer stem cells to treat cancers effectively and as a part of this research isolation and characterization of cancer stems have gained immense importance which help in studying the behaviour of the cancer stem cells.
In this research project, isolation and characterization of the cancer stem cells from murine cell lines is performed by the following procedures:
Maintenance of cell lines and culture medium.
Chemo resistance assay by using doxorubicin.
Immunochemistry by using biological markers which help in characterization of stem cells
Flow cytometry technique used in isolation of desired cancer stem cells.
Later analysis is done using raw data and results from FACS analysis which aid further to decipher many therapeutic interventions using set of standard statistical procedures prodded with extensive research work.
BACKGROUND- LITERATURE REVIEW
2.1. Cancer: Cancer is a medical term used to describe group of cells displaying uncontrolled growth by regular and repetitive division beyond normal limit. Cancer cells are characterized by invasion, a process of destroying adjacent tissues while division and metastasis, a process of spreading infection to other locations in the body. The process thus leads to conversion of benign tumours to malignant tumours. Increased number of cancer patients in the USA and UK is forcing new medication and treatment strategies to improve health condition. Use of stem cells in cancer treatment is one of the latest developments in treating cancer. Many research works are being conducted in order to treat cancer using cancer stem cells. Stem cells act by replacing the immune system of unhealthy patient with that of a healthy donor (Steinberg, 2000).
2.2. Stem cells: Stem cells are defined as those which have ability to divide indefinitely by self renewal to generate mature cells of a tissue through differentiation (Spangrude et al.,1998). Stem cells do not occur in all the tissues and these must be identified and purified to study their properties. One of the examples of stem cells which are isolated from humans is haematopoietic stem cells and is responsible for generation of haematolymphoid systems (Morrison and Weissman, 1994). Stem cells possess potential merits which enable it to be used in various therapies. The two types of mammalian stem cells identified are embryonic stem cells and progenitor cells. The embryonic stem cells arise from adult tissues whereas the progenitor cells repairs the system and is responsible for maintaining turn over of regenerative organs like blood, skin and intestine (Tuch, 2008). Properties of the stem cells include self renewal, a tendency to replicate several times by numerous cycles and potency, a capacity to differentiate into totipotent or pluripotent cell types (Tuch, 2008). Stem cell research is used to treat many disorders like cancer, Parkinsonâ€™s disease, Spinal cord injuries, multiple sclerosis and many more (Stem cell therapy, 2005).
2.3. Cancer stem cells: Cancer stem cells (CSCs) are those which are found in tumours or cancerous cells and possess the characteristics similar to that of stem cells. These cells are tumorigenic and exhibit characteristics of self renewal and differentiation. These cells give rise to new tumours by relapse and metastasis (Spangrude et al., 1998). Cancer stem cells were initially studied on animal models which could not provide therapeutic use. Although conventional cancer therapies kill differentiated and differentiating cells which halt the regeneration of new cells, the CSCs remain in their usual state of division and cause relapse of disease (Gupta et al., 2009). Investigations are still in research to establish effective treatment strategy for cancer using cancer stem cells.
The presence of cancer stem cells poses a challenge in treating cancer which include identification of disease and stage of disease progression, drugs used against the target, strategies to prevent metastasis etc (Clarke et al., 2006). The cancer stem cells originate by mutation of normal stem cells which make them highly resistant towards chemotherapeutic agents. Cancer treatment could be advanced by targeting the cancer stem cells and preventing them from metastasis (Clarke et al., 2006). The diagrammatic illustration represented below shows difference between conventional therapy and therapy against targeted CSCs.
Source: (Clarke et al., 2006)
2.4. Isolation and characterization of cancer stem cell: Isolation and characterization techniques are used to separate cancer stem cells from the bulk of tumours in the human body. Stem cell markers, which are protein products are employed to aid isolation and identification of stem cells (Petrenko et al., 1999). Functional assays mark the beginning of characterization and markers promote characterization by attaching to the surface proteins of the cells.
CD44 cells are used extensively as cell surface markers to isolate many breast and prostrate cancer stem cells (Li et al., 2007). It has also served as indicator to increase time of survival in ovarian cancer patients (Silanpaa et al., 2003).
CD144 is a glycoprotein cell surface marker present in humans as PROM1 (Corbeil et al., 2001). This glycoprotein attaches and localises to the protrusions of cell membrane. They are most commonly expressed in cancer stem cells to isolate them (Corbeil t al., 2000).
The figure below depicts the surface Stromal cells of the stem cells which help in characterization of stem cells (Petrenko et al., 1999).
Characterization is followed by isolation of these stem cells from the matrix. Flow cytometry is used in isolation and counting of stem cells by suspending them in fluid stream and passing them through electronic apparatus for detection (Loken, 1990). In this process, the tumour cells derived from murine cell lines are labelled with specific antibiotics with use of cell surface markers like CD44. FACS is used to separate labelled and unlabeled populations of cells. The cell populations are then assayed for their potential to initiate tumor growth (Hope et al., 2004).
2.5. Chemo resistance by Doxorubicin:
Treatment of cancer by conventional drugs induces resistance along with elimination of differentiated and differentiating cells. Although the cancerous cells are killed, the cancer stem cells remain unaffected due to mutation, leading to relapse of the disease (Gupta et al., 2009). Cancer stem cells originate from mutation of normal cancer cells and this forms the reason behind resistance to chemotherapy (Clarke et al., 2006). Antineoplastic drugs often portray drug resistance in murine cell lines and the factor attributed to this mechanism is associated with over expression of P-glycoprotein. Doxorubicin chemo resistance is studied by analysing P-glycoprotein expression, topoisomerase II activity and related enzymes in murine cell lines (Mestdagh et al., 1994). Research revealed that uptake of drug, glutathione mechanism and activity of topoisomerase II accounted for drug resistance (Mestdagh et al., 1994).
Chemo resistance assay is performed as a part of this experiment to study and analyse isolation and characterization of cancer stem cells from murine cell lines. Doxorubicin rug is used as a drug of choice to induce chemo resistance.
2.6. Future prospects for treatment of cancer stem cells
Cancer stem cells play a major role in relapse after use of conventional drug therapy. New treatment strategies focus upon eradicating tumor cells with destruction of cancer stem cells without affecting normal stem cells. Chemo resistance further enhance the growth and survival of the stem cells. Some of the studies reveal a hypothesis that treatment against cancer would also fight against the cancer stem cells as they possess similar pathology (Max et al., 2006). Work is still being investigated in this regard. Another hypothesis is that normal stem cells different apparently from cancer stem cells which could form one of the treatment strategy, however studies are yet to confirm the hypothesis (Max et al., 2006).
Future prospects for treating cancer stem cells are based on some of the hypotheses mentioned earlier. In addition to hypotheses, research is focussing greatly on different types of assay procedures like functional, marker, genetic and epigenetic assays (Clark et al., 2006). The biological implications of the cancer stem cells elicits important biological implications to develop animal models in understanding key processes like epithelium-stoma interactions, metastasis and biological markers (Max et al., 2006). The biological implications aim to study metastasis and tumor dormancy in murine cell lines (Clark et al., 2006).
Quantity of the cancer stem cells present in the murine cell lines is direct determinant of cancer risk. The quantity could therefore be used as a tool for cancer assessment and prevention of spreading could be started as soon as it is detected. Cancer stem cells also have implications on the markers. The markers are formed by the tumours as response to the infection and these molecular expressions aid in clinical and prognostic treatment of the patient (Max et al., 2006). The cancer stem cells also aid in therapeutic efficacy and development of new therapeutic strategies in treating cancer. An intermediate end point is one such example which helps in estimating patient survival. This is used in many clinical trials as a progression tool of tumours after a therapeutic agent is delivered to target cancer stem cells (Max et al., 2006). Therapeutic efficacy is one of the other implication which helps in estimating the best therapeutic treatment in relation to the stage of cancer.
New therapeutic opportunities arise from cancer stem cell models by initial elimination and eradication of cancer stem cells. Certain signalling pathways like Notch require Î³ secretase and recent studies showed that inhibitors of this enzyme act against breast cancer which have tendency of over expressing Notch 1 (Clark et al., 2006). Another example of this kind includes hedgehog inhibitor which is used to treat many xenografts associated with the tumours (Max et al., 2006). These examples serve as basis of treating many of the advanced cancers.
To isolate and characterise of cancer stem cells using stem cells drug resistance properties
Selection of stem cells by treatment with doxorubicin
To isolate cancer stem cell and identify the specific cell surface markers like CD44, CD133 from the rest differential population by using flow cytometery analysis.
Mural carcinoma cell lines (CT-26 and Renca cells) will be used as sources to obtain clones of CSCs used in further research. Techniques of isolation and characterization are performed by the use of markers like CD44 and CD133. Flow cytometry is used to differentiate cells base on surface properties. All the experiments are performed according to protocols to ensure that the highly tumorigenic and clonogenic CSCs are separated from the bulk of tumor cells.
4.1. Maintenance and cell culture: Mural cell lines (CT-26 and Renca) are stored in DMEM media after extracting from vials stored in liquid nitrogen. DMEM media consists 10% FCS and RPMI for CT-26 cell lines and10%FCS and 10%NEEA for Renca cell lines. These cells will be incubated at 370C for 48-96 hrs in 5%CO2. This activity will be continued following the growth of these cell lines in the culture flasks. During sub culturing of cell lines, the flasks would be washed thoroughly with PBS for two times and then exposed to trypsinization and incubation of cell lines for 3-4 minutes. The cells obtained will be quantified and used for further research.
4.2. Treatment of the cell culture by therapeutic drug (Doxorubicin): After quantification, the cell culture will be used for the chemo-resistance assay. Doxorubicin is used for this purpose. Doxorubicin is an anthracycline antibiotic drug used in chemotherapy of cancer. Doxorubicin elicits its activity by intercalating DNA.
In the chemo-resistance assay, confluent T-75 flasks of Parental CT26 and Renca which are already cultured, trypsinized and counted will be taken. 106 cells of the T75 culture flask are cultured in DMEM and RPMI media. To these cells, doxorubicin is added in the concentrations of 1000ng/ml and 300ng/ml of culture media for CT26 and Renca growth medium respectively. Later on, effective concentrations of Doxorubicin (ranging from 100ng/ml to 1000ng/ml) to be administered will be calculated using the dose response curve for both CT26 and Renca. The Dose-response curves will enable us to study the cell growth pattern to be followed until the next passage. FACS analysis will be done at this stage of experiment which helps in identification and characterization of the sub-population. Chemo resistance assay will be done in three stages as described below:
Stage 1: Confluent flask containing passage 6 of CT26 cells, which are already exposed to normal culture media for 72 hours in T75 flask, will be trypsinized and the cells will be cultured in DMEM media along with 10% FCS which will contain 1000ng Doxorubicin/ml.
Stage 2: During this stage, incubation medium will be drained off and replaced with fresh DMEM media containing 10% FCS.
Stage 3: In this stage, the cells will be trypsinized after 48 hours of incubation and split in two fractions which will be named as population 1 and population 2.
Stage 4: The population 1 will be then cultured in adherent flask (T75) in normal media for 96 hours. Simultaneously, population 2 will be cultured in optimal spheroid generation media for duration of 96 hours in a corning Ultra-low attachment flask (T75). Growth pattern of the cells and their behaviour is analysed for both the populations. Following the observations, if the population 1 is still viable, this will be sub-cultured in normal media and growth condition. Culture flask containing population 2 would be shaken gently and cultured in adherent flask (T75) for 72 hours.
Stage 5: The cells obtained from population 2 are split again to population A and population B. The procedure will be repeated again and population A will be cultured in T25 adherent flask whereas population B in a corning ultra low attachment flask (T25) in optimal spheroid generation media for 96 hours. Cell growth of both the populations will be observed again. This assay results in yielding spheroids and raw data which will aid in analysis.
4.3. Use of labelled antibiotics against CD44: The cells obtained by subsequent assays are used against the murane CD44 cells. After the chemotherapeutic assay, the cells from different populations will be washed with phosphate buffered saline (PBS). These cells will be then harvested with 0.05% trypsin/0.025% EDTA. The detached cells after this will be washed again with 1%FCS and wash buffer containing 1% penicillin/streptomycin. These cells will be re-suspended in the concentrations of 105 cells/100 Î¼l. Flurochrome conjugated monoclonal antibodies will be used against the human CD44 cells or their isotopes in the cell suspension. The resulting cell culture will be incubated for 30- 40 minutes at 4Â°C in dark room. The labeled cells will be washed in the wash buffer and then fixed in PBS which contains 1% Para formaldehyde. The cells surfaces are isolated as a result of this marker CD44. Later analysis is done using flow cytometry.
4.4. Characterization and Isolation of cancer stem cell by the use of Flow cytometry: The cancer stem cells are isolated and characterised later by using flow cytometry. In these process two types of FACS tubes are taken for analysis.
In one of the FACS tubes, single cells obtained after spheroid yield is subjected to trypsinization and quantification will be taken.
In another FACS tubes, cells with labelled antibody and FACS buffer are taken together.
The isotopes corresponding to the cells will be employed along with the suspended cells which will be subjected to FACS analysis. Flow cytometry is the best analytical method since it allows rapid, quantitative and detailed analysis of all the subpopulations within the suspension. The major disadvantage of flow cytometry is the rate of throughput and losses of the cell yield.
The raw data and results obtained from the FACS analysis are further studied using statistical procedures to obtain more accurate results. These results are expected to be used to study isolation and characterization of cancer stem cells in human.
MILESTONES OF INVESTIGATION:
Milestones of the research work are analysed to be seeking ethical approval, various experimental steps, analysis and thesis submission. These are recorded in the GNATT chart as follows.