Chapter 1. Introduction
1.4 The goal of this thesis
The purpose of this thesis is to build the immunofluorescence analysis and show the mTOR inhibitor Everolimus efficacy for breast cancer cell lines by IF expression.
Chapter 2
Materials and methods
2.1 The experimental structure
The experimental design would be divided into two parts. First part was to do cell viability rate assay and MTT assay for breast cancer cell lines and patient-derived cell culture (PDCC). The second part was to show how the immunofluorescence signals can predict the sensitivity of mTOR inhibitor, Everolimus.
2.2 Cell culture, cell lines, and patient-derived cell culture (PDCC)
Breast cancer cell lines MCF7, MDA-MB-231, BT474, Hs578T and two PDCC ABC-16TX1, ABC-82T were chosen to see whether the IF signals could predict the mTOR inhibitor Everolimus sensitivity or not.
MCF7 cell lines were cultured in 10% FBS DMEM/F12 medium. BT474 cell line were cultured in Hybricare medium, which EGF was added. MDA-MB-231 cell line were cultured in 10% FBS DMEM HG medium. Hs578T cell line were cultured in 10%FBS DMEM HG medium, which insulin was added. Patient-derived cell culture (PDCC) ABC82T and ABC-16TX1 were unique cells, which were derived from breast cancer patient metastatic biopsy samples, and these two PDCC were cultured in IH medium. All cells were incubated in the culture dish
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(704001, NEST) in 37℃, 5% 𝐶𝑂2 incubator. Table 1 and Table 2 list the gene mutation and the characteristics of these cell lines [11] and PDCC.
Table 1 Gene mutation for cell lines and PDCC
Table 2 Characteristics for cell lines and PDCC
2.3 Reagents
In this thesis, some reagents and buffers were used. Cell fixation buffer and Alexa Fluor 488 anti-human CD24 antibody were purchased from BioLegend.
Cell permeabilization kit were purchased from MiltenyiBiotec. Mouse anti-human CD45 conjugated with Alexa Fluor 488 and Hoechst33342 were purchased from Invitrogen. Pho-4EBP1 Thr37/46 antibody, vimentin antibody conjugated with Alexa Fluor 647 and pho-GSK3β Ser9 antibody were purchased from Cell Signaling Technology. Mouse anti-human CD44 antibody were purchased from BD Biosciences. Pho-S6K1 Ser424 antibody, pan cytokeratin C11 antibody, the donkey anti-rabbit and anti-mouse IgG H&L conjugated with Alexa Fluor 488 and 647 secondary antibody were purchased from Abcam. Pan cytokeratin AE1/AE3 antibody were purchased from Novus Biologicals. LKB1 antibody, goat anti-rabbit and anti-mouse IgG(H+L) superclonal secondary antibody, HRP conjugated were purchased from ThermoFisher. Everolimus were purchased from Selleckchem. 10X TGS buffer and 10X TG buffer for western blot were purchased from Omics Bio. MTT powder were purchased from Sigma-Aldrich.
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2.4 Cell viability rate assay
MCF7, MDA-MB-231, BT474 cell lines, and ABC-82T, ABC-16TX1 two PDCCs were used. First, seeded cells in the 3.5 cm culture dish were grown to about 50-60% confluence. Second, 200 nM Everolimus were added into the dish. After about 24 hours, cells were harvested and the number of live cells were counted to determine cell viability rate. The MTT assay was also used to confirm
the cell sensitivity. The cell viability rate is defined as：
𝑐𝑒𝑙𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 𝑎𝑓𝑡𝑒𝑟 24 ℎ𝑜𝑢𝑟𝑠 − 𝑐𝑒𝑙𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑎𝑓𝑡𝑒𝑟 𝑎𝑏𝑜𝑢𝑡 24 ℎ𝑜𝑢𝑟𝑠 𝑐𝑒𝑙𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑎𝑓𝑡𝑒𝑟 𝑎𝑏𝑜𝑢𝑡 24ℎ𝑜𝑢𝑟𝑠 × 100%
2.5 MTT and IC50 assays
About 5000-10000 cells were seeded in 100μl medium into the 96-well and incubated in 37℃, 5% 𝐶𝑂2 incubator overnight before the drug treatment.
Second, the same volume of the culture medium which contained different Everolimus concentration were replaced and were incubated for about 48 hours.
After Everolimus treatment for about 48 hours, the culture medium were replaced by 12mM MTT about 100μl and were placed for 3 hours in 37℃,
5% 𝐶𝑂2 incubator. Finally, the same amount DMSO were replaced for 20 minutes at 37℃ and the absorbance of DMSO at 570 nm were determined, which stood for the number of live cells by the plate reader (MQX 200, BioTek). the IC50
values were also determined for every cell lines. The accuracy of the plate reader would be done before the MTT assay.
Figure 3 The calibration Curve of plate reader from MTT assay.
2.6 Immunofluorescence staining
For immunofluorescence staining, cells were harvested first, the fixation buffer 200μl were added,incubated for 20 minutes at 4℃and washed with running buffer. Second, cells were permeabilized by adding the inside perm buffer, incubated for 20 minutes at 4℃ and washed with running buffer (some antibody did not use this process).
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After cells were fixed and/or permeabilized, the primary antibodies were added, incubated for 25 minutes at 4℃ and washed with running buffer. When primary antibodies were bonded at cells, the secondary antibodies conjugated to Alexa Fluor 488 or 647 channels were added, incubated for 25 minutes at 4℃ and washed with running buffer. Immunofluorescence results were taken by the microscope.
2.7 Western blots
First, 105 cells were boiled with sample buffer for about 15 minutes and the cell samples which contained proteins were loaded into the wells of the Mini-PROTEAN Precast Gels which were purchased from Bio-Rad. Second, the gels were run with 200 volts for about 30 minutes. When the gels were run completely, proteins were transferred to PDVF membranes which were purchased from Merck Millpore with 100 volts for 30 minutes.
After running gels and blotting, the PDVF membranes were stained with Ponceau S solution for about 5 minutes to show the total proteins expression to check the equal amount protein and were washed with PBS-T three times. After this, the PDVF membranes were blocked with milk at room temperature for about 2 hours. After blocking, the primary antibody (1:1000) were added into the milk,
incubated at 4 ℃ overnight and were washed with PBS-T three times. The secondary antibody which HRP conjugated (1:5000) were added into the milk, incubated at room temperature for about 1 hours and washed with PBS-T six times.
Proteins were detected using UVP BioSpectrum 500 Imaging System.
2.8 Statistical analysis
The immunofluorescence pictures of the thesis were taken from Leica DMI 6000 B microscope and analyzed by MetaMorph software. In MetaMorph software, there were the counts nuclei function, which can count the cells IF intensity automatically. After taking the photos and counting the IF intensity, it was compiled statistics the by SigmaPlot 12.5 software and expressed as mean ± s. e. m.
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Chapter 3
Results and discussion
3.1 Immunofluorescence marker for Everolimus resistance in cell lines and PDCC
3.1.1 Gene mutation and drug sensitivity
The goal of this work is to determine Everolimus sensitivity for these cell lines and PDCC by immunofluorescence (IF) markers. It is important to understand the relation between IF signals and the Everolimus sensitivity.
Doctors also can use them for patients to make the judgment on usage of Everolimus.
Although the expression of mTOR pathway is affected by regulation of p53, KRAS, PI3CA and PTEN gene mutation, these mutations do not provide reliable marker characteristics to predict Everolimus sensitivity. Hence, this work aims to use immunofluorescence method towards this end. And from Table 2 and figure 4, ABC-16TX1 cells show the stem cell expression, which CD44 expression is high and CD24 expression is low by immunofluorescence, stands for the high ability of drug resistance.
Figure 4 ABC-16TX1 PDCC exhibits stem cell characteristics (CD44 is high and CD24 is low), which will likely lead to the ability of drug resistance.
3.1.2 Cell viability and IC50 value of cell lines and PDCC
For cell viability asssay, the number of cells will be counted before and after adding Everolimus 200nM for 24 hours. Figure 5 represents cell viability rate for BT474, MCF7, MDA-MB-231 cell lines, and ABC-82T, ABC-16TX1 PDCC. The cell viability rate of BT474 and MCF7 are negative on sensitive to Everolimus, while the cell viability rate of MDA-MB-231, 82T and ABC-16TX1 are positive on resistant to Everolimus.
Figure 6 represents results for the MTT assay and IC50 values for breast cancer cell lines and PDCC. Figure 6(a) shows cell viability for Hs578T, BT474, MCF7 cell lines under different Everolimus concentration. Results show that the inhibition rate are over 50% when these cells are less than 100 nM, which can be regarded as Everolimus-sensitive cells. Figure 6(b) shows cell viability for ABC-16TX1 cells under different Everolimus concentration. Results show that
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cell viability is still about 80% under high Everolimus concentration. It cannot find IC50 values which means high resistance of Everolimus. Figure 6(c) shows IC50 values for breast cancer cell lines and PDCC, which means the resistant characteristics for these cells. Every experiment is tested in triplicate.
Figure 5 Cell proliferation under the 200 nM Everolimus after about 24hours. Some
cells are Everolimus-sensitive cells, the others are Everolimus-resistant cells.
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Figure 6 Cell viability for cell lines and PDCC by MTT assay (a) Hs578T, BT474 and MCF7 (b) ABC-16TX1 (c) IC50 values of Everolimus.
3.1.3 Immunofluorescence expression of mTOR markers and Everolimus sensitivity
Figure 7 shows the baseline IF intensity for the cell lines and PDCC prior to adding of Everolimus. Figure 7(a) and 7(b) means the images of pho-S6K1 Ser424 and 4EBP1 Thr37/46 respectively. For ABC-16TX1 cells, both pho-4EBP1 and pho-S6K1 have high IF intensity.
Figure 7(c) and 7(d) show the normalized IF intensity (by the intensity of BT474) for pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 respectively. Data
suggest variation in IF intensity among these cells for both protein markers. In light of the results in figure 5 and 6, where sensitivity and resistance of each cell type is known, IF intensities for each of the protein marker do not correlate with known response to Everolimus. Results also display functional heterogeneity as mentioned in the reference [17] for some cells, like ABC-82T and MDA-MB-231, i.e., when IF signals of pho-4EBP1 are high, IF signals of pho-S6K1 are low, or when IF signals of pho-4EBP1 are low, IF signals of pho-S6K1 are high.
Results from figure 7(c) and 7(d) can be divided into three group of cells.
The first group contains Everolimus sensitive cells Hs578T, BT474 and MCF7, which both pho-S6K1 and pho-4EBP1 have low IF intensities. The second group is middle or resistant Everolimus sensitivity cells MDA-MB-231 and ABC-82T, which pho-S6K1 have low intensities and pho-4EBP1 have high intensities or pho-S6K1 have high intensities, and pho-4EBP1 have low intensities. The third group is ABC-16TX1 cells, in which both the pho-S6K1 and pho-4EBP1 are high, thus demonstrate high Everolimus resistance. Three these groups are listed in Table 3.
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Figure 7 IF expression for BT474, MCF7, ABC-82T, and ABC-16TX1 cells without Everolimus and the normalized IF intensity. (a) pho-S6K1 Ser424 (b) pho-4EBP1
Thr37/46 (c) Pho-S6K1 Ser424 (d) Pho-4EBP1 Thr37/46.
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Table 3 The group of cells by pho-S6K1 and pho-4EBP1
Cell lines and PDCC Respone to Everolimus
Pho-S6K1 low, pho-4EBP1 low Hs578T, BT474,
MCF7
Pho-S6K1 high, pho-4EBP1 high ABC-16TX1 Strongly resistant
Figures 8(a) and figure 8(b) display the combined IF intensities of pho-S6K1 and pho-4EBP1 by adding results of figure 7(c) and 7(d). Unlike figure 7(c) and 7(d) for individual marker, combined intensities suggest a clear trend.
Hs578T has the lowest relative intensity of all, followed by BT474 and MCF7 with higher intensity. These three cell lines are sensitive to Everolimus therapy (Figure 5 and 6). MDA-MB-231, ABC-82T and ABC-16TX1 all have high relative intensity and they are resistant to Everolimus. Figure 8(c) represents the relationship between combined IF intensity and IC50 value (Figure 6). Results indicate that combined IF intensity is proportional to IC50 value in term of response to Everolimus. The high expression of pho-4EBP1 and pho-S6K1 in some cells may reflect mutation of mTOR. Two references [25,26] also provided
evidence that the expression of mTOR downstream marker will increase when the mTOR genes are mutated. Further studies suggested that high expression of 4EBP1 Thr37/46 due to resistant to rapamycin [32,33]. Moreover, pho-S6K1 Ser424 is also related to the MAPK/ERK pathway, which is another pathway in cell proliferation. In a gastric cell line study, expressions of pho-S6K1 Ser424 and pho-AKT Ser473 in rapamycin-resistant cells were higher than that in rapamycin-sensitive cells [31].
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Figure 8 Combined IF expression of pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 for cell lines. (a) Everolimus-resistant cells will have high IF intensity. (b) the average expression of combined IF intensity of pho-4EBP1 Thr37/46 and pho-S6K1 Ser424. (c)
Comparison between IC50 values, and combined signals.
Figure 9 shows ROC curves by for individual and combined protein markers of IF intensity. The area of ROC for pho-4EBP1 only is 0.69 and the area of ROC for pho-S6K1 only is 0.64.The area of ROC curve for combined protein markers is 0.85. This method has a better efficiency than pho-4EBP1 and pho-S6K1 individually to predict the Everolimus sensitivity. From results, we could make the conclusion that we can choose the better one to use. Every experiment is tested in triplicate.
Figure 9 The ROC curve for pho-S6K1, pho-4EBP1 and the two combined markers.
The combined IF intensity has a better efficiency than pho-4EBP1 and pho-S6K1
individually.
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3.2 Pho-4EBP1 and pho-S6K1 do not have high specificity for AKT/mTOR
Although the IF approach can distinguish Everolimus sensitivity by
combining pho-4EBP1 Thr37/46 and pho-S6K1 Ser424, some studies suggest that if the cells were resistant for Everolimus, the expression of pho-4EBP1 would low and the expression of PI3K would be high [16]. This result, particularly the former part, is opposite from our experimental results (Figure 7(d)).
Figure 10 indicates other neighbor pathways and targets which might affect pho-4EBP1 and pho-S6K1 [27,28]. We suggest that mTOR pathway downstream targets pho-4EBP1 and pho-S6K1 might not have high specificity for AKT/mTOR. In other words, pho-4EBP1 and pho-S6K1 might be controlled not only by AKT/mTOR.
To test our hypothesis, another AKT downstream marker, pho-GSK3β Ser9 antibody, which stands for the activation of pho-AKT, and LKB1 antibody were chosen to observe whether pho-4EBP1 would be impacted only by AKT/mTOR or not.
Figure 11(a) represents the IF relative intensity (FITC) for pho-GSK3β.
Figure 11(b) and figure 11(c) show western blot for pho-GSK3β. ABC-16TX1,
ABC-82T, and BT474 cells have strong band corresponding to pho-GSK3β at 46 kDa. In contrast, MDA-MB-231 and MCF7 cells have weak band for pho-GSK3β. From results, the expression of pho-AKT cannot represent the resistance
of Everolimus. One reference [27] also indicated that the higher expression of pho-GSK3 in lung cancer cell lines, the higher the resistance of Everolimus. The results of this work confirm this phenomenon and show that the mTOR resistance might be impacted not only by the AKT/mTOR but also by LKB1 or another pathway like for instance, the MAPK/ERK pathway.
Figure 12(a) also shows the LKB1 IF signals (APC) for MCF7 and ABC-16TX1 cells, where figure 12(b) quantifies intensities for all cell types studied.
Figure 12(c) represents the western blot for LKB1. Everolimus sensitive cells MCF7 is higher than Everolimus resistant cells ABC-16TX1 [29,30], means that LKB1 can affect Everolimus efficacy.
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.
Figure 10 Pho-4EBP1 and pho-S6K1 might be impacted not only by
AKT/mTOR but also by another target or pathway.
Figure 11 The expression of pho-GSK3β (a) IF relative intensity (b) western blot with ponceau s solution, which shows the total protein (c) western blot with beta-actin. It
indicates that the expression of pho-AKT cannot represent the resistance of
Everolimus.
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Figure 12 LKB1 expression for cell lines and PDCC (a) The IF image for MCF7 and ABC-16TX1 cells (b) quantifies intensities for IF signals. (c) The western bolt expression of LKB1 for cell lines. It shows that Everolimus-sensitive cells has the
higher LKB1 IF intensity than Everolimus-resistant cells.
3.3 Combined IF expression of pho-4EBP1 and pho-S6K1 before and after adding Everolimus
When staining some cell lines, for instance, MCF7, it will have the fundamental IF signals of pho-4EBP1 Thr37/46 and pho-S6K1 Ser424. It is important to check these signals are the right signals we want. To prove this result, we add Everolimus 500 nM after about 24hours and stain with pho-4EBP1 and pho-S6K1 antibody.
Figure 13 shows the combined expression of pho-4EBP1 and pho-S6K1 for Everolimus-sensitive cells (MCF7, BT474) and Everolimus-resistant cells (MDA-MB-231, ABC-16TX1) for both control group and with Everolimus 500nM after 24 hours. Figure 13(a) represents the signals of MCF7 are decreased after adding Everolimus. Figure 13(b) represents the signals of BT474 cell line in Everolimus 500nM group are higher than control group. Figure 13(c) shows that the signals of Everolimus-resistant cells are the same and it is obvious that
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MDA-MB-231 cell line and ABC-16TX1 cells are Everolimus-resistant cells.
BT474 cell line also has the higher resistance than MCF7 cell line. These results also maybe be related to the expression of pho-GSK3β and negative feedback of pho-AKT. If cells have some characteristics of resistance in Everolimus-sensitive cells, the dynamitic change for pho-4EBP1 and pho-S6K1 will happen.
For Everolimus-resistant cells, the expression of pho-4EBP1 and pho-S6K1 will almost unchanged. These results also show that signals are right in our research.
Figure 13 Combined expression of pho-4EBP1 and pho-S6K1 for both control group and Everolimus 500nM group for 24 hours (a) MCF7 cell line (b) BT474 cell line (c)
MDA-MB-231 cell line and ABC-16TX1 PDCC.
3.4 Epithelial-mesenchymal transition state and Everolimus sensitivity
The efficacy of Everolimus is believed to be affected by the cellular program EMT [12]. We used an EMT marker (vimentin) to interrogate this understanding.
Figure 14 represents results for four cell types. The expression of vimentin is negative for BT474 and MCF7, which are sensitive to Everolimus. The expression is positive for the other cells such as MDA-MB-231 and ABC-16TX1 which are resistant to Everolimus. This results explain the positive relation between EMT state and the efficacy of Everolimus. However, this vimentin results still do not list the sensitivity of Everolimus in order.
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Figure 14 The IF expression of vimentin for cell lines. Red ones represent Vimentin signals and blue ones represent the nucleus signals. The positive relation between
EMT state and the efficacy of Everolimus.
3.5 The arrangement of IF marker in rare cancer cells detection
In order to use the pho-4EBP1 and pho-S6K1 IF marker into the application of rare cancer cells detection, we arrange the channels for these markers. In this section, MCF7 cell line will be the model of rare cancer cells.
The CD45 antibody will be placed in the location of the FITC channel, which distinguishes the white blood cells and rare cancer cells, meaning that the signals of white blood cells will be positive. The pan cytokeratin C11+AE1/AE3 antibody will be placed in the location of the PE channel, where it detects rare cancer cells. Pho-4EBP1-Thr37/46 add pho-S6K1 Ser424 antibody will be placed in the location of the APC channel. The stained Hoechst will also be used
to stand for the signal of the cell nucleus. Figure 15 shows the situation for MCF7 cell line in the blood and compares that for the white blood cells in healthy people. For white blood cells in healthy people, CD45 expression is positive, pan cytokeratin C11+AE1/AE3 expression is negative and the expression of pho-4EBP1 and pho-S6K1 combined markers is positive. For MCF7 cell line, CD45 expression is negative, pan cytokeratin C11+AE1/AE3 expression is positive and the expression of pho-4EBP1 and pho-S6K1 combined markers is positive.
Figure 15 The arrangement of IF expression. (a) White blood cells in healthy people (b) MCF7 cell line. The expression of CD45 will be arranged in FITC channel, the expression of pan cytokeratin C11+AE1/AE3 will be arranged in PE channel and the
expression of pho-4EBP1+pho-S6K1 will be arranged in APC channel.
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Chapter 4 Conclusions
This study shows that immunofluorescence analysis can be used to indicate the efficacy of mTOR inhibitor Everolimus on breast cancer cells (Hs578T, MCF7, BT474, MDA-MB-231) and patient-derived cell culture (ABC-82T, and ABC-16TX1). Immunofluorescence intensities of pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 indicated that unlike each individual marker which does not correlate with efficacy in Everolimus, the combined IF intensity of these two proteins is representative of Everolimus efficacy. It also indicates that mTOR resistance might be affected not only by AKT/mTOR but also by LKB1 or MAPK/ERK pathway. In addition, the expression of LKB1 and pho-GSK3β might also be potential markers for efficacy of the therapeutic agent. In
many treatment cases, mTOR therapy is combined with hormonal drug e.g.
Tamoxifen. Hence it is important to understand the effect of Tamoxifen in the present IF analysis.
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