Introduction SciX Atomic Force Microscopy Assessment Answer

Abstract

This report aims at examining the effect of duration of the DOX treatment on the cell stiffness. Various cell lines were used in this study, but the work was proceeded with MCF 7 cell line. Doxorubicin hydrochloride (DOX) is an anti-cancerous agent which is used in this study for providing treatment to the cells. Cell stiffness is measured by the Young’s modulus to measure the cell’s elasticity. The major theory for measuring the cell stiffness in response to anti cancerous treatment is that the cells change in elasticity and mechanical structure while undergoing apoptosis. Therefore, the effectiveness of the DOX treatment can be measured by measuring the stiffness of cells. Atomic Force microscope is one of the most advanced technologies that can detect the changes in the mechanical behaviour of the cells. In this study, AFM has been used to detect the changes in the cell elasticity and hence, evaluating the effectiveness of DOX treatment. Different concentrations of DOX (0.5 and 1.0 µM) have been used for different duration (4 and 24 hours). This report can be helpful in determining the optimal concentration and the duration for treating the cancerous cells with DOX and thus, helping in treatment of cancer. 

Literature review

Cancer has been known to be a leading cause of deaths in the world accounting for almost 10 million deaths in 2020. Most of the new cases were due to breast cancer and lung cancer while lung cancer and colon and rectum cancer accounted for maximum deaths in 2020 (WHO, 2022). Doxorubicin, also known as DOX or Adriamycin is an anticancerous drug of anthracycline family. It is a DNA topoisomerase II inhibitor and is used in treatment of various cancer types such as breast cancer, lung cancer etc (Chen et al., 2018). DOX works by intercalating into the DNA and inhibiting the topoisomerase I and II which causes damage to the DNA and forms reactive oxygen species. This results in activation of caspases which leads to programmed cell death or apoptosis (Franco et al., 2018). The biomechanical properties of cells can help in assessing their viability. The stiffness of metastatic cells can act as a potential biomarker of their viability. Integrins, extracellular matrix and the mechanical processes of cell can tell about their survivability (Bergman et al., 2021). Changes in cell elasticity have been implicated in progression of many diseases such as kidney disease, Alzheimer’s, malaria, cataracts, etc. The cells stiffness is generally measured by apparent Elastic modulus. Atomic Force microscope is one of the best methods that can be used for evaluating the cell stiffness by using quantitative parameter of Young’s modulus. Atomic Force Microscopy is a high- resolution imaging technique of first demonstrated in 1985. It is a powerful tool for doing the surface analysis. AFM uses non- disruptive measures for accurate magnetic, electrical, optical, mechanical, topographical and chemical analysis of the properties of the sample surface with very high resolution in vacuum, liquid or air. AFM has been one of the most advanced technologies to be used in the field of science especially in medical Sciences (Figure 1). AFM studies have shown that cancerous cells are generally softer and have less cell stiffness then the non- malignant cell types (Wullkopf et al., 2018). However, the study by Nguyen et al. (2016) contradicts this paradigm suggesting that stiffer pancreatic ductal adenocarcinoma (PDAC) cells are more invasive than the compliant cells and are more efficient in deforming through the narrow gaps and invading surrounding tissues thus, transiting to distance sites. 

Figure 1. The figure shows general outline of working of atomic force microscopy. (a) The sample is probed with very sharp tip that is attached to a flexible cantilever and laser is focused on the cantilever and reflected through a mirror on the photodiode. The force exerted by the sample on the tip which causes the deflection of the cantilever. The changes in path is detected by the photodiode and is recorded on the computer. (b) graph showing the force generated versus time. (c) the force distance curve can be used for analysing the elasticity and the addition force of the cell (Viljoen et al., 2021).

Scientific research question.

Does the duration of DOX Treatment Have an effect on cell stiffness in cancer cells? 

Scientific hypothesis

DOX is an anti- cancerous drug and prolonged treatment on the cells can cause apoptosis of the cancerous cells. The death of the cells can be evaluated by checking the cell stiffness using AFM.

Methodology

Cell lines 

Four cell lines MCF- 7, MRC-5, HT- 1080 and MIA PaCa were used in this study for checking the effect of DOX treatment on the cell stiffness. The details of the cell lines used in this study are given in Table 1. MCF- 7 stands for Michigan Cancer Foundation and is a breast cancer cell line. MCF-5 stands for Medical Research Council cell strain 5. 

 Cell culturing and preparation of sample. 

For routine culturing, the cells were grown in high glucose Dubelcco’s Modified Eagle’s Medium – High glucose (DMEM) ThermoFisher (#D6429-500ML) in flask with L- glutamine and phenol red (Thermo Fischer), supplemented with 1% Penicillin/Streptomycin and 10% of Foetal Bovine Serum (FBS); ThermoFisher (#10099133). The cells were maintained at 37 º C with 95% relative humidity and 5% CO2 conditions till they reach 70% confluence. For the atomic force microscopy imaging, the cells were harvested from the culture of 70% confluence and doxorubicin treatment was given at a concentration of 0.5 µM and 1 µM. This condition was maintained in the incubator for 4 h and 24 h before the indention and imaging. The cells were washed in phosphate buffer saline (PBS ThermoFisher (#10010049). Cells are fixed to preserve the tissues of the sample permanently in the life like stage so that the expected changes can be prevented by preserving the physical and chemical characteristics of the cell for further studying. It forms covalent cross links between the molecules and effectively adheres them together into insoluble framework. In this study, 2% and 4% formaldehyde was used as fixative agents and the cells were treated for 10 minutes at room temperature. Before the AMF procedure, the cells were washed twice in phosphate buffer saline (PBS ThermoFisher (#10010049). Cells without DOX treatment were used as controls. 

Atomic force microscopy (AFM) 

The experiment of atomic force microscopy was carried out using a JPK Nanowizard 4 XP (JPK instruments, Germany) along with a CellHesion module mounted on an inverted microscope (Axio Observer Z1, Zeiss, Jena, Germany). For the measurement of cell, the probe speed of 5 μm s−1 and 1 nN of force setpoint was used for recording the motion for same length. When reached at the setpoint, the tip was kept for 10 seconds at constant height before retracting in similar speed and recording the motion for similar length.

Statical Analysis

All the preliminary analysis was done using the excel software. The AFM analysis software was used to collect the data of atomic force microscopy. 

Results

The elasticity or the young modulus was noted for all the cell lines in the cells fixed at PFA 2% and PFA 4% (Table 2). As the results of MCF-7 looked promising therefore, the statistical analysis of Young modulus for this cell line was also noted for the control, 0.5 µM (Table 3) and 1 µM DOX (Table 4) treated cells at 4 hours and 24 hours. Since, the experiment was conducted by taking MCF 7 cell line, therefore we are presenting the result of MCF-7 cell line only. The young modulus of MCF-7 cells were noted on elasticity value of hundreds of Pa. Few other studies have also reported the measurements in this range (Hao et al., 2020; Gavara, 2017). The mean Young modulus in the control cells after 4 hours was 388.24 Pa ± 28.93 while the value of the cells treated with 0.5 µM show 559.40 ±27.94 Pa of elasticity. The 1.0 µM DOX treated cells after 4 h of treatment showed 638.83 ±31.54 Pa which showed that at 4 hours treatment increase in concentration has increased the cell stiffness. The impact of DOX treatments at different concentration (0, 0.5 and 1.0 µM) for 4 h on the cell stiffness is shown in Figure 2. The graph clearly indicates that the cell stiffness increased with the increase in the concentration of DOX when treated for 4 hours. On the other hand, a reverse trend has been seen in the cells treated for the 24 hours time duration. The mean value of the control cells after 24 h treatment was 9743.87 ± 828.16 Pa, while the value decreased (5080.174 ± 373.62 Pa) in the cells treated with 0.5 µM after 24 h and further decline was noticed in the 1.0 µM DOX treated cells showing 4639.71 ± 623.71 Pa. Figure 3 shows the impact of DOX treatments for 24 hours in the control 0.5 and 1.0 µM treated cells. The DOX treatment after 24 hours drastically reduced the stiffness of the cells when compared to the control in both 0.5 and 1.0 µM. The F test two-samples for variance of the MCF- 7 cell line 4 the control, and the treated cells at 4 h and 24 hours are given in Table 5. No significant change in the height and width of the cells were noticed after the DOX treatment at different concentrations for different durations (4 and 24 hours) on the cells (figure 4). The Atomic force microscopy images of the height of the cell measured at various concentrations have been illustrated (Figure 5).

Table 2 describes the statistical analysis of Young modulus in the MCF-7 cell line in control, PFA 2% in PFA 4% cells.

Table 3 describes the statistical analysis of Young modulus in the MCF-7 cell line in control, DOX treated cells 0.5 µM and 1.0 µM at 4 h. 

Figure 5 shows the AFM image of the height measured of the control (a), DOX 0.5 µ at 4 h (b), DOX 0.5 µM at 24 h (c) and Dox treated 1.0 µM at 24 h. The left images are the edge enhanced images, while the right ones are height measured images. 

Table 5 summarises F test two samples for variance of the MCF- 7 cell line for the control, and the treated cells at 4 h and 24 hours

Figure 2. The graph shows impact of DOX treatment on control (0 µM), 0.5 µM and 1.0 µM on cell stiffness after 4 h duration. As depicted from the graph, the stiffness of the cell increased with increase in concentration of DOX.

Figure 3. The graph depicts the impact of DOX treatment on the control (0 µM), 0.5 µM and 1.0 µM on cell stiffness after 24 hours treatment. As clearly seen in the graph, the cell stiffness decrease drastically after 24 hour treatment in both the 0.5 µM and 1.0 µM concentration. 

Figure 4 shows the effect of DOX treatment at various concentrations and different durations. No significant change in the height and width of the cells were noticed after the treatment. 

Discussion

In the present study the ability of DOX treatment in decreasing the cell stiffness has been depicted. A clear trend of increase in cell stiffness at 4 hours of treatment was seen in the cells. However, the cell is stiffness decreased after 24 hours of treatment. Ren et al. (2021) studied the membrane tension in the cancer cells with help of AFM and found that the decrease in the membrane tension increases the softening of cancer cells. This approach can be used for studying the behaviour of cancerous cells and can help in regulation. Levi et al. (2021) also studied the effect of DOX treatment at 12, 24 and 48 hours on two mammary tumor cell lines. Both the cell lines were able to take up DOX treatment. Proliferation of cells decreased at 48 hours in both the cell lines. In the above study also, the MCF-7 cell line showed decreased cell stiffness which is correlated with decreased cell activity after 24 hours treatment. Hence it can be said that MCF cells underwent process of apoptosis after 24 hours of treatment. The findings are supported by the work of Pilco-Ferreto and Calaf (2016) who studied the effect of DOX on the breast cancer cell lines MCF-10F, MCF-7 and MDA-MB-231. Expression of anti- apoptotic Bcl- 2 was decreased and oxidative stress was increased due to increase in production of hydrogen peroxide gas. This decreased the NF-κB gene and protein expression in MCF 7 cell line. Caspase- 8, caspase- 3, Bax were upregulated and down regulation of expression of Bcl-2 protein was noted which points towards the induction of apoptosis. Zhang et al. (2014) studied the effect of PDOX and DOX on the MCF- 7 cancer cell lines in vitro . Dose and time dependent cytotoxicity were found in both the PDOX and DOX treated cells which resulted in reduction of the cell viability. Research has also been going on to study the effect of co-treatment of DOX with other known anti- cancerous drugs. Chueahongthong et al. (2021) reported enhanced cytotoxicity and inhibited proliferation in the leukemia cells by Flt 3 protein expression. The authors suggested that DOX treatment can be used for treating leukemia patients. 

Recent study by Chen et al. (2018) have reported that cancer cells are emerging as DOX resistant and creating a major hurdle in effective treatment of the cancer. Similar study in 2010 was conducted by Lupertz et al. by using human colon cancer cells and studying the dose and time dependent effects of doxorubicin on the cell viability and cytotoxicity. The treatment was done for 3 hours with doxorubicin which was followed by incubation in drug free medium for 24 hours. On other hand, a comparative treatment of 24 hours continuous treatment of doxorubicin was done on cell lines. The 3 hours treatment led to the apoptosis of the cells will increase in p53 phosphorylation at the serine residue 392 along with inducing p21, arresting G2 phase and increasing the proapoptotic protein Bax. The continuous treatment for 24 hours showed arresting of cell cycle in G0/G1 phase with no activation of p53. Authors concluded that the doxorubicin can induce apoptosis at a particular treatment conditions and optimal dosage. The effect of concentration of DOX has also been studied for treatment of hepatocellular carcinoma. The authors used HepG2, Huh7, and SNU449 liver cancer cell lines and studied the responses to chemotherapy. While the HepG2 cell line showed hypoxia, the other two cell lines Huh7, and SNU449 were found to be resistant to the to the treatment. The authors suggested that different tumor cell lines can respond differently to the doses of the treatment and suggested that combination of chemotherapy and embolization can be used for future studies (Dubbelboer et al., 2019). The investigators of this study can hence investigate the effect of doxorubicin while applying approach used by Lupertz et al. (2010) and optimise the duration and concentration for assessing the cell viability in this study. Also, as suggested by Dubbelboer et al. (2019), the investigators of the study can take a co-treatment approach to check the cell stiffness and viability and check the effect of cotreatment after 4 h. The above study could be used as an approach for treatment of cancer cells as the cell stiffness decreased after 24 hours. However, the increase in the cell stiffness at 4 hours of treatment can be bothersome for the cancer patients as it can result in further deterioration of the condition and further investigation is needed in this direction. 

Conclusion

In conclusion, it can be said that the research question has been answered by doing the relevant experiments in this report. The effect of duration of DOX treatment on the cells stiffness was needed to be investigated in this report. It was observed that the longer duration of the treatment (24 hours) was more effective in decreasing the cell stiffness of the cancerous cells and hence, indicating the apoptosis in them. The results can be concluded that prolonged treatment with the anti-cancerous drug DOX will be helpful in preventing the cancer but long duration of treatment can cause side effects on the patients as well as will not be cost effective. Therefore, it is important to optimise the dosage and duration of treatment of the DOX. Higher concentration of DOX reduced the width of the cells, but not significantly. Optimization of concentration and the duration of DOX can also be used to analyse this aspect of the effect of treatment on the cell lines. This parameter can also be optimised by. Increasing the. By optimising the. Concentration of the treatment. This investigation can be further done by taking evaluating the cell stiffness using the images of atomic force microscopy. The atomic force microscope can easily detect the changes in the mechanical structure including cell stiffness and hence, can be a useful tool in this study. The investigators of this study can use the co-treatment technique along with other anti-cancer drugs such as curcumin or artemisinin to investigate the effect on the cancer cell lines.

References

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