Exosome Purification – Staining of Derived Exosomes – Wound Healing Assay – RNA extraction – Flow Cytometry Analysis of HepG2 Cells – Reverse Transcription and Real-Time Quantitative PCR ( use the same crateria in both of example use your own words to avoid plagiarism.)
8263045
7029BMS Laboratory Book
Overall Aim:
The overall aim of this investigation was to determine whether exosome-mediated
communication between macrophages and HepG2 liver cancer cells can induce changes in
cell phenotype and behaviour. This was done utilising flowcytometry, qPCR and migration
assay techniques.
The hypothesis was that inducing exosome-mediated communication between macrophages
and HepG2 cells would increase cell migration and proliferation. Moreover, sample 2 would
exhibit the largest change in cell phenotype and behaviour as it contained the middle fraction
of exosomes which was the purest, least contaminated and most concentrated fraction.
Session 1:
Exosome Purification
The initial phase of this investigation was the purification of exosomes from THP-1 monocytic,
cell line, throughout which the aim was to isolate a sample of exosomes with maximum yield
and maximum purity (Patel et al., 2019). Furthermore, the aim was to obtain the 3 fractions
containing the highest concentration of exosomes which we estimated to be fractions 9-11.
This was accomplished utilising Sepharose CL-2B column to perform size-exclusion
chromatography (SEC) which involves the use of a porous stationary phase and a mobile
phase (Lobb & Möller, 2017). The nature of the stationary phase allows for the differential
elution of particles from the solution; bigger particles elute first, followed by smaller vesicles
(Sidhom et al., 2020).
Differential ultracentrifugation (dUC) has traditionally been the gold standard for exosome
purification. dUC relies on the sequential separation of particles by density- and sizedependent sedimentation using a series of centrifugation steps (Konoshenko et al., 2018).
Most dUC procedures require up to 3 hours of centrifugation as an increase in centrifugation
time is directly correlated with exosome purity. As such, in recent years other techniques such
as SEC have become preferable as they are less labour intensive and produce purer
samples(Konoshenko et al., 2018).
Although SEC is overall the preferred method of exosome purification, if this investigation
were to be repeated it would be suggested that a Sepharose CL-4B column be used as a 2020
study by Benedikter et al. 2017, found that an increase in the percentage of agarose present
in the 4B column provided better separation of small particles such as exosomes when the
mobile phase was cell culture media (Benedikter et al., 2017).
PKH67 Staining
After the initial purification of exosomes, the three fractions were stained using PKH67, a
lipophilic fluorescent label. The aim of this technique was to successfully label the exosomes
to enable their presence to be detected when reading at the appropriate wavelength.
8263045
PKH67 is a green fluorochrome with excitation at 490nm which functions by intercalating
aliphatic reporter molecules into the lipid bilayer. This mechanism of intercalation is
unspecific therefore, it is imperative that the sample of exosomes be pure to ensure accurate
results (Pužar Dominkuš et al., 2018). Although this technique is currently the most common
method of staining exosomes, there is new evidence that suggests due to its unspecific nature
there are instances where it also stains non-plasma membrane cellular components such as
lipo-proteins (Simonsen, 2019).
If I were to repeat this investigation, I would continue to use the same PKH67 staining
technique. This is because the alternative methods of staining are either not cost effective,
such as transfection of cells to produce exosomes containing a genetically encoded
fluorescent reporter such as CD63-eGFP (Sutaria et al., 2017), or it impairs the functionality
of exosome through the staining of a less dynamic transmembrane-bound exosome proteins
(van der Vlist et al., 2012).
RNA Extraction
The aim of this technique in this investigation was to extract high quality, high yield exosomal
RNA from the isolated fractions in preparation for use in RT-qPCR. In this investigation the
Trisure method of RNA extraction was used, as such thiocynate and phenol compounds were
used to facilitate the disruption of cells during homogenisation. Subsequently, chloroform
was used to separate the homogenate into 3 phases where RNA is found in the upper aqueous
phase. The RNA was then washed in multiple steps to remove impurities.
Although this is a relatively cost-effective method with a high yield, it requires significant
manual processing and as such is time-intensive when compared to simpler techniques such
as the column separation method (Scholes & Lewis, 2020). Subsequently, if I were to repeat
this investigation, I would use the spin column method which is an RNA extraction method
that utilises membranes than contain silica to bind to nucleic acids. Lysates pass through the
silica membrane using centrifugal force with the RNA binding to the silica until it is eluted with
RNase-free water. This method is simple and involves very little manual labour, moreover due
to depleted chances of ethanol contamination samples are of higher quality (Mônica
Ghislaine Oliveira et al., 2016).
Wound Healing Assay
Wound healing assay is an assay performed in 2D cell monolayer where a cell-free region is
created through the deliberate destruction of the confluent cell monolayer, such as by a
pipette tip as in this investigation, which is then available for cells to occupy (Stamm et al.,
2016). The gathering of data across numerous time points is able to demonstrate the
progression of cell proliferation (Cory, 2011). In this investigation the aim of this method was
to determine the effects of exosome-mediated communication on the behaviour of HepG2
liver cancer cells through a change in cell growth patterns.
The wound-healing assay, as performed in this investigation has numerous advantages over
more complicated methods as it is easy to carry-out and all materials are widely available in
any lab. However, utilising a pipette tip to create the cell-free gap causes irregular scratches
and therefore difficulty ensuring reproducibility and comparability of results (Liang et al.,
8263045
2007). As such, if I were to complete this investigation again, I would utilise silicon insert to
ensure uniformity across all cell-free gaps. In addition to this, I would introduce more timepoints at which cell-migration would be measured in order to elucidate greater data trends.
Reflection and Planning for Next Session:
Overall, I feel as if the first session was relatively positive. I was nervous to be back in the lab
after so long, but this led to me being very prepared having read and annotated the protocols
in great detail. Despite working well in a group, with so many people in the lab I felt rushed
to get in and out of the cell culture hood which meant I did not use good aseptic technique
as I would usually. Hopefully this did not impact my results and has not led to any
contamination of my samples. Next session my goal is to stay calm and take my time with the
methods whilst still working efficiently.
Session 2:
Reverse Transcription
Reverse transcription involves the production of cDNA from an RNA template via the enzyme
reverse transcriptase. Small RNA cDNAs are generated by reverse transcription (RT) utilising
microRNA (miRNA)-specific RT primers which detect only the mature miRNA target and not
precursor molecules (TaqManTM MicroRNA Reverse Transcription Kit, 2018). In this
investigation the aim was to utilise the TaqMan MicroRNA Reverse Transcription Kit to
produce a high yield, high quality cDNA for use in real time quantitative polymerase chain
reaction (RT-qPCR).
The TaqMan MicroRNA Reverse Transcription Kit is highly specific and well suited to miRNAs
due to the inclusion of the RT stem-loop primer and as such is able to accurately convert
miRNA to cDNA while maintaining quality and yield. If I were to repeat this method, I would
use the same TaqMan miRNA kit due to its specialisation for microRNAs (TaqManTM MicroRNA
Reverse Transcription Kit, 2018).
Flowcytometry
The aim of the flowcytometry technique used in this investigation was to detect the
percentage uptake of exosomes and subsequent change in cell phenotype of HepG2 liver
cancer cells. Flowcytometry involves the passage of cells in single file through the path of a
laser. Cell components are fluorescently labelled and excited by the laser to emit light which
can then be detected. The exosomes purified in this investigation were stained with PKH67
fluorescent dye, and as such, cells that displayed exosome uptake exhibited fluorescence at
the FITC-A wavelength (Rim & Kim, 2016).
If I were to repeat this investigation, I would continue to utilise flow cytometry as although
exosomes themselves lie outside of the detection limit for flowcytometry, the purpose of this
study was to observe exosome uptake and change in cell phenotype. I would suggest the
additional use of NanoSight analysis of exosomes as this would elucidate the mean
concentration and mean size of exosomes and thus, further data trends could be interpreted.
8263045
Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)
RT-qPCR was carried out in this investigation with the aim of quantifying the amount of
miRNA, miR-150 within each sample which is commonly found within exosomes derived from
THP-1 cells. Moreover, this technique was used to analyse trends in exosome uptake and
subsequent exosome-mediated communication between macrophages and HepG2 liver
cancer cells and the resultant changes in cell phenotype and behaviour.
Within RT-qPCR, the TaqMan probe method and SYBR Green fluorescent dye method are the
two most common techniques for miRNA detection. SYBR Green is relatively unspecific and
subsequently is unable to recognise non-specific products such as primer dimers which
reduces the accuracy of qPCR quantification. if I were to repeat this investigation I would
continue to use TaqMan probe RT-qPCR (Ye et al., 2019).
Results:
Flow Cytometry
When PKH67 stained exosomes were incubated with HepG2 cells, a change in the FITC-A
fluorescence can be seen. Cells within the M2 gated parameters, as represented in red in
figure 1, align with the phenotype of the negative control (figure 1D) which contained cells
that had both no PKH67 stain and no exosome, as signified by its peak positioned further left
on the X-axis, closer to 103. In contrast, cells within the M3 gated parameters, similarly
represented on in figure 1, align with the phenotype of the positive control (figure 1E) which
contained cells that exhibited FITC-A fluorescence, and thus were positive for both the PKH67
Stain and exosomes. This is signified by a shift in the peak to the right of X-axis closer to 105.
Figure 1B established that sample 2 contained the highest percentage of exosome uptake
with 35.7% falling within the M3 parameters, thus exhibiting FITC-A fluorescence
representative of PKH67 staining. Figure 1A shows that sample 1 had the next highest
percentage with 32.5% falling within the M3 parameters, thus demonstrating FITC-A
fluorescence representative of PKH67 staining and exosomal uptake. Therefore, Sample 3, as
shown in figure 1C displayed the smallest percentage within the M3 parameters with only
25.7% exhibiting exosomal uptake.
8263045
Figure 1: Detection of FITC-A fluorescence in HepG2 cells incubated with PKH67 stained THP1 derived exosomes by flow cytometry. A) example data for a representative flow cytometry
plot of cells treated with fraction 9 exosomes, B) example data for a representative flow
cytometry plot of cells treated with fraction 10 exosomes, C) example data for a
representative flow cytometry plot of cells treated with fraction 11 exosomes, D) Negative
control of unstained cells, E) Positive control of stained exosomes
Wound Healing
The results presented in table 1 demonstrate an exosome-dependent increase in the
percentage reduction in migration area, when compared to the average of T0 values, as seen
by positive percentage difference values in column 4. Sample 2 displayed the greatest
reduction in migration area when compared to the control at T48 with a value of 66.5%.
Sample 1 showed the next greatest percentage reduction in migration area at T48 when
compared to the control at 30.6%, followed by sample 3 with 29.5%.
Sample
1
2
3
Control (%)
26.8
10.6
17
Exosome Treated (%)
57.3
77.1
46.4
Difference between Control and Exosome Treated (%)
30.6
66.5
29.5
Table 1: Percentage reductions (1 d.p) in cell-free area calculated from mean of T0 values for
three samples of example data.
8263045
qPCR
As can be seen from figure 2, the CT value for all three samples differed which suggests a
difference in their miR-150 content. However, due to variances in conditions such as volume
of intact RNA and reaction efficiency, the CT values themselves are unable to accurately
represent the true concentration of miR-150 within each sample. However, as shown by the
data in figure 3, the CT values were processed utilising U6 snRNA CT values and the 2ΔΔCT
equation in order to produce the relative fold changes in each sample. As such, the levels of
miR-150 expression were able to be more accurately analysed and compared.
The ΔΔCT values shown in figure 3 established that sample 2 had a 1.15-fold increase in miR150 expression when compared to sample 1 indicating that sample 2 had the most miR-150
expression. Conversely, sample 3 was determined to have a 2.14-fold decrease in miR-150
expression when compared to sample 1 therefore indicating that sample 3 had the least miR150 expression across all sample.
Figure 2: qPCR Curves for HepG2 cells incubated with 3 separate fractions of purified THP-1
exosomes. All samples were analysed in duplicate for the presence of miR-150 and the
housekeeping gene U6.
8263045
1.6
1.4
ΔΔ CT Value
1.2
1
0.8
0.6
0.4
0.2
0
Sample 1
Sample 2
Sample 3
Sample Number
Figure 3: ΔΔCT values with standard deviation error bars for HepG2 cells incubated with 3
separate fractions of purified THP-1 exosomes.
Discussion and Data Analysis:
Exosomes are small extracellular vesicles composed of lipid bilayer with the primary function
of mediating cell-cell communication. Due to their highly heterogenous nature, they have
recently received significant attention for their role in pathobiological processes and as such,
were the focus of this investigation (Weidle et al., 2017). Throughout this study,
flowcytometry, RT-qPCr and migration assay techniques were used to determine whether
exosome-mediated communication between macrophages and Hep2 liver cancer cells could
induce changes in cell phenotype and behaviour.
Overall, the results of the techniques used in this investigation revealed an exosomedependent change in both cell phenotype and behaviour. The results of the flowcytometry
revealed that all samples exhibited uptake of exosomes which represents a change in cell
phenotype. As shown in figure 1, sample 2 exhibited the most substantial change in
phenotype, followed by samples 1 and 3 respectively.
Although the uptake of exosomes was relatively low, it was significant enough for the results
of the migration assay to reveal a direct correlation between the percentage of HepG2 cells
that exhibited exosomal uptake in each sample in the flowcytometry, and the percentage
reduction in cell-free area in the migration assay, with sample 2 having the largest reduction
followed by samples 1 and 3 respectively. Therefore, it can be concluded that exosomalcommunication between macrophage-derived exosomes and HepG2 liver cancer cells causes
an increase in cell migration and proliferation and thus a change in cell behaviour. This is
supported by a 2021 study by Wu et al., which established that macrophage derived
exosomes facilitated tumorigenesis and metastasis by transferring αMβ2 integrin to tumor
cells (Wu et al, 2021).
The results of the RT-qPCR carried out in this investigation similarly supported the correlation
between the percentage of HepG2 cells that exhibited exosomal uptake in each sample in the
flowcytometry and the fold changes seen in miR-150 expression. miRNAs are small non-
8263045
coding RNAs which function as regulators of gene expressed that are usually encapsulated in
exosomes to ensure the molecules stability (MacFarlane & R. Murphy, 2010). miR-150
normally functions in haematopoiesis where it regulates genes that have key roles in in
differentiating stem cells towards becoming megakaryocytes. However, in malignancies, this
control over cell differentiation implicates miR-150 as a key promotor in tumour development
and cancer progression. Therefore, the increase in migration and cell proliferation seen in the
wound-healing assay in addition to the changes in cell phenotype can be attributed to the
transport of miR-150 into cells via exosomes. (Zhang et al., 2010)
Taken as a whole, the results of this investigation emphasise the range of biological effects
associated with exosomes which is a current focus within scientific literature. However, there
is significant research required to elucidate both the specific pathways, and the roles that
these molecules play in cancer cells. The outcomes of this research have the potential to aid
in the development new treatments for previously fatal malignancies (Pritchard et al., 2020).
Reflection:
I am satisfied with my performance within the lab as I was able to gain experience in both
Flowcytometry and RT-qPCR which are both techniques that I haven’t previously carried out.
In addition to this, I feel as if I worked more calmly and efficiently in the second lab which was
aided by my understanding of the protocol.
I was disappointed that I was unable to obtain any viable wound-healing assay results as the
cells in my exosome-treated well had become contaminated due to my poor aseptic
technique and rushing from the previous lab. In the future, when completing labs such as
these again, I will endeavour to stay calm and not rush to maintain proper aseptic technique
and avoid sample contamination.
Overall Discussion:
Overall, the results of this investigation support the proposed hypotheses of an exosomedependent change in cell phenotype/behaviour and that the sample treated with the second
fraction of exosomes would result in the greatest change in cell phenotype/behaviour.
Moreover, the study fulfilled its aims of demonstrating that exosome-mediated
communication between macrophages and HepG2 liver cancer cells would initiate changes in
cell phenotype such as; uptake of exosomes and an increase miR-150 expression and changes
in cell behaviour such as; increase in proliferation and migration.
The conclusion that exosome-mediated communication influences liver cancer progression
as shown in studies such as Wu et al., who demonstrated that cell growth and proliferation
and subsequently tumour metastasis increased in liver cancer when exposed to macrophagederived exosomes both in vivo and in vitro. However, due to the heterozygous nature of
exosomes, the exact pathways through which exosomes cause these outcomes is still
unknown (Wu et al., 2021). Exosomes also have the potential to occupy key roles in new
treatments as an in vitro study by Liang et al. 2018, found that engineered exosome delivery
of active miR-26a to HepG2 liver cancer cells resulted in decreased rates of cell migration and
proliferation (Liang et al., 2018).
Word Count: 2193
8263045
References:
Benedikter, B. J., Bouwman, F. G., Vajen, T., Heinzmann, A. C. A., Grauls, G., Mariman, E. C.,
Wouters, E. F. M., Savelkoul, P. H., Lopez-Iglesias, C., Koenen, R. R., Rohde, G. G. U.,
& Stassen, F. R. M. (2017). Ultrafiltration combined with size exclusion
chromatography efficiently isolates extracellular vesicles from cell culture media for
compositional and functional studies. Scientific Reports, 7(1).
https://doi.org/10.1038/s41598-017-15717-7
Chen, Y., Song, Y., Du, W., Gong, L., Chang, H., & Zou, Z. (2019). Tumor-associated
macrophages: an accomplice in solid tumor progression. Journal of Biomedical
Science, 26(1). https://doi.org/10.1186/s12929-019-0568-z
Cissell, K. A., & Deo, S. K. (2009). Trends in microRNA detection. Analytical and Bioanalytical
Chemistry, 394(4), 1109–1116. https://doi.org/10.1007/s00216-009-2744-6
Cory, G. (2011). Scratch-wound assay. Methods in Molecular Biology (Clifton, N.J.), 769, 25–
30. https://doi.org/10.1007/978-1-61779-207-6_2
Konoshenko, M. Yu., Lekchnov, E. A., Vlassov, A. V., & Laktionov, P. P. (2018). Isolation of
Extracellular Vesicles: General Methodologies and Latest Trends. BioMed Research
International, 2018, 1–27. https://doi.org/10.1155/2018/8545347
Liang, C.-C., Park, A. Y., & Guan, J.-L. (2007). In vitro scratch assay: a convenient and
inexpensive method for analysis of cell migration in vitro. Nature Protocols, 2(2),
329–333. https://doi.org/10.1038/nprot.2007.30
Lobb, R., & Möller, A. (2017). Size Exclusion Chromatography: A Simple and Reliable Method
for Exosome Purification. Methods in Molecular Biology, 1660, 105–110.
https://doi.org/10.1007/978-1-4939-7253-1_9
MacFarlane, L.-A., & R. Murphy, P. (2010). MicroRNA: Biogenesis, Function and Role in
Cancer. Current Genomics, 11(7), 537–561.
https://doi.org/10.2174/138920210793175895
Mônica Ghislaine Oliveira, A., Pérez-Sayáns, M., Padín-Iruegas, M.-E., Reboiras-López, M. D.,
Suarez-Peñaranda, J. M., López-López, R., Faig Lima Carta, C., Scholz Issa, J., GarcíaGarcía, A., & Dias Almeida, J. (2016). Comparison of RNA Extraction Methods for
Molecular Analysis of Oral Cytology. Acta Stomatologica Croatica, 50(2), 108–115.
https://doi.org/10.15644/asc50/2/2
Patel, G. K., Khan, M. A., Zubair, H., Srivastava, S. K., Khushman, M., Singh, S., & Singh, A. P.
(2019). Comparative analysis of exosome isolation methods using culture
supernatant for optimum yield, purity and downstream applications. Scientific
Reports, 9(1), 1–10. https://doi.org/10.1038/s41598-019-41800-2
Pritchard, A., Tousif, S., Wang, Y., Hough, K., Khan, S., Strenkowski, J., Chacko, B. K., DarleyUsmar, V. M., & Deshane, J. S. (2020). Lung Tumor Cell-Derived Exosomes Promote
8263045
M2 Macrophage Polarization. Cells, 9(5), 1303.
https://doi.org/10.3390/cells9051303
Pužar Dominkuš, P., Stenovec, M., Sitar, S., Lasič, E., Zorec, R., Plemenitaš, A., Žagar, E.,
Kreft, M., & Lenassi, M. (2018). PKH26 labeling of extracellular vesicles:
Characterization and cellular internalization of contaminating PKH26 nanoparticles.
Biochimica et Biophysica Acta (BBA) – Biomembranes, 1860(6), 1350–1361.
https://doi.org/10.1016/j.bbamem.2018.03.013
Rim, K.-T., & Kim, S.-J. (2016). Quantitative Analysis of Exosomes From Murine Lung Cancer
Cells by Flow Cytometry. Journal of Cancer Prevention, 21(3), 194–200.
https://doi.org/10.15430/jcp.2016.21.3.19
Scholes, A. N., & Lewis, J. A. (2020). Comparison of RNA isolation methods on RNA-Seq:
implications for differential expression and meta-analyses. BMC Genomics, 21(1).
https://doi.org/10.1186/s12864-020-6673-2
Sidhom, K., Obi, P. O., & Saleem, A. (2020). A Review of Exosomal Isolation Methods: Is Size
Exclusion Chromatography the Best Option? International Journal of Molecular
Sciences, 21(18), 6466. https://doi.org/10.3390/ijms21186466
Simonsen, J. B. (2019). Pitfalls associated with lipophilic fluorophore staining of extracellular
vesicles for uptake studies. Journal of Extracellular Vesicles, 8(1), 1582237.
https://doi.org/10.1080/20013078.2019.1582237
Stamm, A., Reimers, K., Straub, S., Vogt, P., Scheper, T., & Pepelanova, I. (2016). In vitro
wound healing assays – state of the art. BioNanoMaterials, 17(1-2).
https://doi.org/10.1515/bnm-2016-0002
Sutaria, D. S., Badawi, M., Phelps, M. A., & Schmittgen, T. D. (2017). Achieving the Promise
of Therapeutic Extracellular Vesicles: The Devil is in Details of Therapeutic Loading.
Pharmaceutical Research, 34(5), 1053–1066. https://doi.org/10.1007/s11095-0172123-5
TaqManTM MicroRNA Reverse Transcription Kit. (2018, August). Www.thermofisher.com.
https://www.thermofisher.com/order/catalog/product/4366596#/4366596
van der Vlist, E. J., Noltet Hoen, E. N. M., Stoorvogel, W., Arkesteijn, G. J. A., & Wauben, M.
H. M. (2012). Fluorescent labeling of nano-sized vesicles released by cells and
subsequent quantitative and qualitative analysis by high-resolution flow cytometry.
Nature Protocols, 7(7), 1311–1326. https://doi.org/10.1038/nprot.2012.065
Weidle, U., Birzele, F., Kollmorgan, G., & Ruger, R. (2017). The Multiple Roles of Exosomes in
Metastasis. Cancer Genomics & Proteomics, 14(1), 1–16.
https://doi.org/10.21873/cgp.20015
8263045
Ye, J., Xu, M., Tian, X., Cai, S., & Zeng, S. (2019). Research advances in the detection of
miRNA. Journal of Pharmaceutical Analysis, 9(4), 217–226.
https://doi.org/10.1016/j.jpha.2019.05.004
Wu, J., Gao, W., Tang, Q., Yu, Y., You, W., Wu, Z., Fan, Y., Zhang, L., Wu, C., Han, G., Zuo, X.,
Zhang, Y., Chen, Z., Ding, W., Li, X., Lin, F., Shen, H., Tang, J., Zhang, Y., & Wang, X.
(2021). M2 Macrophage–Derived Exosomes Facilitate HCC Metastasis by
Transferring α M β 2 Integrin to Tumor Cells. Hepatology, 73(4), 1365–1380.
https://doi.org/10.1002/hep.31432
Zhang, Y., Liu, D., Chen, X., Li, J., Li, L., Bian, Z., Sun, F., Lu, J., Yin, Y., Cai, X., Sun, Q., Wang,
K., Ba, Y., Wang, Q., Wang, D., Yang, J., Liu, P., Xu, T., Yan, Q., & Zhang, J. (2010).
Secreted Monocytic miR-150 Enhances Targeted Endothelial Cell Migration.
Molecular Cell, 39(1), 133–144. https://doi.org/10.1016/j.molcel.2010.06.010
Table of Contents
21.Overall Aim
22.Session1
22.1Objectives
22.2Procedures
22.2.1Exosome Purification
32.2.2Staining of Derived Exosomes
42.2.3Wound Healing Assay
42.2.4RNA extraction
52.3Reflection and Planning for the Next Session
53.Session2
53.1Objectives
53.2Procedures
53.2.1Flow Cytometry Analysis of HepG2 Cells
63.2.2Reverse Transcription and Real-Time Quantitative PCR
64.Results
64.1Flow Cytometry
74.2Wound healing assay
84.3qPCR Results
95.Data Analysis and Discussion
106.Reflection on Session 2
107.Overall Discussion
12References
1|P ag e
1.Overall Aim
Exosomes are extracellular microvesicles (30±200 nm diameter) secreted by
most types of cells and employed for intercellular communication (Wortzel et
al., 2019). Exosomes are surrounded by a lipid bilayer and contain various
biomolecules, such as proteins, metabolites, lipids, glycans, and small DNAs
and RNAs (Mathieu et al., 2019), that carry critical information and play an
essential role in intercellular communication. However, exosomes, particularly
macrophage-derived exosomes, can also promote tumour generation and
development and thus play a significant role in tumour progression, including
metastasis, invasion, and tumour immunity (Han et al., 2021). Thus, the
following experiments investigate the effect of exosomes derived from THP-1
cells(phagocytic cells from a patient with acute monocytic leukaemia) on the
human hepatocellular carcinoma(HCC) cell line HepG2 to assess whether the
exosomes induce behavioural or phenotypic changes liver cancer cell
proliferation.
2.Session1
2.1Objectives
The first session sought to achieve five objectives: exosome purification, which
made to discharge the organic waste and unwanted element to obtain optimal
results, exosomes staining to be labelled within the tubes, incubation of HepG2
cells with THP-1 exosomes using two treatment processes to obtain the optimal
conditions for flow cytometry(FC) analysis and scratch wound healing assay
and incubated to assess cell migration, and RNA extraction to purify the RNA
sample and prepared for reverse transcription quantitative PCR(RT-qPCR).
2.2Procedures
2.2.1Exosome Purification
Phorbol myristate acetate was used to trigger THP-1 cells to secrete exosomes
(Bai et al., 2018). Among the available purification methods based on density,
2|P ag e
shape, or surface proteins, the size-exclusion chromatography(SEC) protocol
developed by the Böing group was followed (Böing et al., 2014). It is a singlestep isolation procedure and depends on separation based on molecular size
variations(diameter larger than 75 nm) rather than chemical characteristics;
moreover, it is inexpensive, simple, and quick(Barth et al., 2012; Böing et al.,
2014). Thus, it is considered an ideal candidate method to isolate exosomes.
However, it does not differentiate between the starting fractions 9±12 and other
material; moreover, it does not isolate vesicles less than 70 nm in diameter or
separate vesicles between 70 and 500 nm (Böing et al., 2014). In addition, the
efficiency of exosome isolation by SEC is still debated (Baranyai et al., 2015).
Furthermore, human error can occur and increase the risk of contamination,
which can be avoided by developing an automated process.
Other possible isolation methods include ultracentrifugation, which is most
commonly utilised and considered the Stander method(Witwer et al., 2013).
However, its protocols vary among users, leading to inconsistencies in the
recovery of microvesicles; moreover, several articles report that this technique
is time consuming and that the efficiency of exosome isolation is poor (MomenHeravi et al., 2012; Rekker et al., 2014). Another alternative technique is
exosome precipitation, which is rapid and efficient and does not require
expensive specialised equipment (Murakami et al., 2012). Nevertheless,
exosomes obtained using this technique contain many contaminating proteins
that substantially affect subsequent proteomic analysis (Cheng et al., 2019).
2.2.2Staining of Derived Exosomes
Staining promotes the visibility of cells during FC analysis. In this experiment,
exosomes were stained with the green fluorescent dye PKH67, the most widely
used labelling method for exosomes, which readily and non-specifically
incorporates into any lipid structure (Takov et al., 2017). Although it is a simple
and easy method to label exosomes, it carries the risk of exosome aggregation;
moreover, it labels both lipoproteins and lipid micelles (Yi et al., 2020).
HepG2 cells will be incubated overnight with stained THP-1±derived exosomes
for FC analysis next session.
3|P ag e
2.2.3Wound Healing Assay
The wound healing assay is easy, cheap, and considered a standard method
for probing collective cell migration in vitro (Jonkman et al., 2014). Automated
microscopy is utilised in wound healing assays for cancer cell analysis, drug
testing, and other similar tests due to its reproducibility (Kauanova et al., 2021).
For this experiment, the fraction was added to a well of a 6-well plate of HepG2
cells. A straight line was then scratched down the middle of each
well(test+control) with a sterile 100 µl pipette tip.
A disadvantage of this method is that it is challenging to create reproducible
wounds because the scratch is performed manually. Applying intense pressure
or introducing the pipette at the wrong angle can harm the extracellular matrix,
affecting migration rates (Jonkman et al., 2014). However, this method can be
improved by instead inserting a silicone tool designed for this purpose into the
wells to stop cell growth. By removing this tool, a cell-free area is created (Planz
et al., 2018).
2.2.4RNA extraction
Complete RNA isolation from DNA was carried out using TRIzol, which is an
effective reagent for isolating microRNA due to its effectiveness in protein
denaturing(Rio et al., 2010). Although RNA pellets can be difficult to resuspend
when using TRIzol, the quality is still excellent for primer extension analysis. In
addition, according to Adilakshmi et al. (2009), the TRIzol method of RNA
extraction is less costly and more reliable than other methods.
Alternatively, commercially available extraction kits that are faster and easier to
use are commonly employed in RNA isolation. In a study published last year,
the miRNeasy kit(Qiagen) was found to be the best option for RNA isolation
compared to other six kinds of extraction kits(Wright et al., 2020). This
technique depends on combining the selective binding properties of a silicabased membrane with microspin technology(Laurent & Alexander, 2015).
4|P ag e
2.3Reflection and Planning for the Next Session
The protocols were very clear to follow. Although some essential elements for
the experiments were missing and the pipette left on my bench was not working
properly, the staff team were friendly and helpful in assisting me with the
experiments. The scratch technique for the wound healing assay was
challenging, and I did not do it properly. I would need to repeat it several times
to improve my skills and obtain a well scratch. The queue for the NanoDrop
was long; thus, waiting without placing the samples in ice could have caused
degradation of RNA.
For the next session, I plan to read articles related to exosome purification and
quantification to increase my knowledge and better understand the principles
of techniques in order to help my progress and allow me to predict and interpret
results. I will also observe the wound healing wells and prepare to perform FC
and qPCR.
3.Session2
3.1Objectives
The main objectives of this session were to analyse the diffusion rate of
exosome-treated HepG2 cells and compare them with non-exosome-treated
cells via FC, evaluate the levels of miR-150 in THP-1±derived exosomes using
RT-qPCR via TaqMan Small RNA Assays to identify the RNA gene expression
levels, and determine the results of the wound healing assay by imaging the
gaps.
3.2Procedures
3.2.1Flow Cytometry Analysis of HepG2 Cells
FC is utilised in exosome surface protein characterisation to measure sizes and
structures, and it was used here to investigate the diffusion rate of exosometreated HepG2 cells. FC is the most popular method utilised in extracellular
vesicle(EV) examination due to its ability to identify single EVs(Gurunathan et
al., 2019). In addition, FC is the most suitable and reproducible method for
5|P ag e
examining different cell properties in clinical samples, which differentiates FC
from all other techniques(De Rond et al., 2019). Nevertheless, FC can be
improved using Advanced Imaging FC, which is a powerful image-based
technique that enables multiparametric, high-throughput, and vesicle-byvesicle characterisation(Mastoridis et al., 2018).
3.2.2Reverse Transcription and Real-Time Quantitative PCR
Using the RNA isolated in session 1, qPCR was performed utilising TaqMan
Small RNA Assays using miR-150 RT and U6 snRNA primers to assess the
miR-150 levels in the exosomes. Sequence similarity, stability issues, and the
short length of miRNA molecules present challenges in quantification by RTqPCR. Nevertheless, sensitivity and specificity can be improved to obtain
accurate results when combined with stem-loop RT-based TaqMan Small RNA
Assays; additionally, this method provides other benefits such as easy workflow
and extensive dynamic range(Chen et al., 2011).
4.Results
4.1Flow Cytometry
HepG2 cells were treated with THP-1±derived exosomes and incubated
overnight. Both FITC(identifying cell populations by determining the intensity)
and FSC were measured, and 10,000 events were assessed in each run. The
FC results are presented in Figure 1 as fluorescein isothiocyanate(FITC)
histograms after applying gating parameters and forward scatter(FSC) without
gating.
6|P ag e
Figure 1
Flow cytometry results
Panels A±E: fluorescein isothiocyanate(FITC) histogram data from HepG2 cells
with or without treatment with THP-1±derived exosomes: (A)positive control,
(B)negative control, (C) sample1, (D) sample2, and (E) sample3. Panels F±J:
forward scatter(FSC) data: (F)positive control, (G)negative control, (H)
sample1, (I) sample2, and (J) sample3.
4.2Wound healing assay
Figure 2 shows the impact of THP-1±derived exosomes on HepG2 cell
migration.
Figure 2
Wound healing assay
(A)Microscopic image of scratch at 0 hrs. (B,C) Microscopic images of (B) nontreated HepG2 cells(control) and(C) THP-1±derived exosome-treated HepG2
cells 48 hrs after scratch generation.
7|P ag e
ImageJ was employed to measure the gaps in the images in Figure 2. After 48
hrs, cell migration by THP-1±derived exosome-treated HepG2 cells had
occurred, reducing the wound gap. In contrast, the wound area remained in the
control plate(Table 1).
Table 1
Gap measurements
Condition
Area
Min
Max
Mean
Control
322508
133.898
109
191
Sample
168119
133.737
109
184
Table1 presents measurements of the area of gaps, mean, min, and max for
both exosome-treated and non-treated(control) HepG2 cells.
4.3qPCR Results
No qPCR results were obtained for any of the samples. Nevertheless, expected
results are presented in Figures 3 and 4.
Figure 3
PCR amplification
The three panels (A) sample1, (B) sample2, and (C) sample3 each contain
duplicates of miRNA-150 and U6. The green horizontal line is the cycle
threshold(ct).
8|P ag e
Figure 4
Bar chart of the predicted fold changes of samples 1,2, and 3
Figure4 presents the fold change for each sample.
5.Data Analysis and Discussion
The uptake of stained exosomes by HepG2 cells was assessed via FC after
overnight incubation, and the expected results were obtained. A clear
significant shift between the positive and negative controls was observed. In all
three samples, approximately 2/3 of the population was shifted to the left
(dead/alive), with a high number of events in the first sample(Figure-1C)
compared to the other samples.
In the wound healing assay, the gap in the control (non-treated) sample after
48 hrs was approximately similar to the gap at 0 hrs(Figure-2). In contrast, the
exosome-treated cells exhibited substantial cell migration, which reduced the
wound area(Figure-2). This result was expected, as THP-1±derived exosomes
have been shown to induce the migration of HepG2 cells-(Chiba et al., 2016;
Wang et al., 2018), and in this experiment, the treated cells reduced the wound
area approximately 2x faster than the non-treated cells.
All of the qPCR samples failed to produce results. There are several possible
reasons for the qPCR failure, including calibration and temperature set-up and
instrument faults. Figure 3 depicts the graphs of accumulated PCR products
over time in the examples provided. Sample 2(Figure-3B) had the highest cycle
threshold, increasing approximately 1.2-fold, which indicates more miRNA-150.
9|P ag e
In contrast, sample 3 exhibited a 2.1-fold decrease in miR-150. High levels of
miRNA-150 inhibit liver cancer cell migration and invasiveness; notably, the
survival rate in liver cancer patients with low miR-150 levels in plasma
exosomes was significantly lower than that of patients with high miR-150
levels(Zhang et al., 2012; Yugawa et al., 2021).
6.Reflection on Session 2
The results varied among samples 1-3 and controls in the FC assay in my
group. This was also noted by most other groups despite the same specimen,
materials, and methods being used for each group. This could be due to
differences in individualV¶ skills and ability to follow instructions and protocols
properly. For the wound-healing assay, it was easy to compare and measure
the gap between treated and control samples. The fact that qPCR failed for all
students LQGLFDWHGWKDWLWZDVQRWGXHWRLQGLYLGXDOV¶ errors; all steps and devices
need to be reviewed to determine what caused the failure.
7.Overall Discussion
These experiments aimed to determine the phenotypic impacts of THP-1±
derived exosomes on HepG2 cells besides investigating the hypothesis that
exosomes work to prevent cancer metastasis and proliferation. Overall, the
results were expected based on previous studies. HCC is a common type of
primary liver cancer, and exosomes derived from a tumour were found to
contribute to successive tumour progression and development-(Wang et al.,
2021). In addition, Wu et al. (2021) established that HCC development and
progression relies on the local microenvironment, with TAMs as the primary
factor contributing to tumour aggressiveness. Moreover, macrophage-derived
exosomes are described in HCC regulation-(Bai et al., 2020).
Several studies have shown that THP-1±derived exosomes increased cell
migration in wound healing assays, in line with the results of the experiment
described here(Figure-2; Wang et al., 2018; Qu et al., 2019). A significant shift
in dead and live cell populations was observed in the FC assay(Figure-1).
miRNA-150 plays an important role in cell migration and is observed in
10 | P a g e
exosomes; thus, exosomes can enhance cell migration-(Rahbarghazi et al.,
2019; Yugawa et al., 2021).
Further investigation on THP-1±derived exosomes could be employed to
understand their secretion and function. For example, their protein profiles
could be analysed using label-free liquid chromatography-tandem mass
spectrometry (Yao et al., 2018). In addition, signalling pathways activated could
be
identified
using
a
phospho-kinase
antibody
array
on
HepG2
exosomes(Wang et al., 2018). Furthermore, morphological characterisations of
exosomes could be performed using nanoparticle tracking analysis and
scanning electron microscopy.
Finally, exosomes are considered a safe means to delivery therapy, including
for HCC (Han et al., 2021). Therefore, further investigation and determination
of the functional roles of exosomes could be utilised to prevent cancer from
spreading, enabling its control and treatment.
11 | P a g e
References
References
Adilakshmi, T., Soper, S. F., & Woodson, S. A. (2009). Structural analysis of RNA in
living cells by in vivo synchrotron X-ray footprinting. Methods in Enzymology, 468,
239-258.
Bai, G., Motoda, H., Ozuru, R., Chagan-Yasutan, H., Hattori, T., & Matsuba, T. (2018).
Synthesis of a cleaved form of osteopontin by THP-1 cells and its alteration by
phorbol 12-myristate 13-acetate and BCG infection. International Journal of
Molecular Sciences, 19(2), 418.
Bai, Z., Li, H., Li, C., Sheng, C., & Zhao, X. (2020). M1 macrophage-derived exosomal
MicroRNA-326 suppresses hepatocellular carcinoma cell progression via mediating
NF-ț%VLJQDOLQJSDWKZD\ Nanoscale Research Letters, 15(1), 1-14.
Baranyai, T., Herczeg, K., Onódi, Z., Voszka, I., Módos, K., Marton, N., Nagy, G., Mäger,
I., Wood, M. J., & El Andaloussi, S. (2015). Isolation of exosomes from blood
plasma: Qualitative and quantitative comparison of ultracentrifugation and size
exclusion chromatography methods. PloS One, 10(12), e0145686.
Barth, H. G., Saunders, G. D., & Majors, R. E. (2012). The state of the art and future
trends of size-exclusion chromatography packings and columns. LC GC North
America, 30(7)
Chen, C., Tan, R., Wong, L., Fekete, R., & Halsey, J. (2011). Quantitation of microRNAs
by real-time RT-qPCR. PCR protocols (pp. 113-134). Springer.
Cheng, H., Fang, H., Xu, R. D., Fu, M. Q., Chen, L., Song, X. Y., Qian, J. Y., Zou, Y. Z.,
Ma, J. Y., & Ge, J. B. (2019). Development of a rinsing separation method for
exosome isolation and comparison to conventional methods. Eur Rev Med
Pharmacol Sci, 23(12), 5074-5083.
12 | P a g e
Chiba, M., Watanabe, N., Watanabe, M., Sakamoto, M., Sato, A., Fujisaki, M., Kubota,
S., Monzen, S., Maruyama, A., & Nanashima, N. (2016). Exosomes derived from
SW480 colorectal cancer cells promote cell migration in HepG2 hepatocellular
cancer cells via the mitogen-activated protein kinase pathway. International Journal
of Oncology, 48(1), 305-312.
De Rond, L., Libregts, S., Rikkert, L. G., Hau, C. M., Van Der Pol, E., Nieuwland, R., van
Leeuwen, T. G., & Coumans, F. (2019). Refractive index to evaluate staining
specificity of extracellular vesicles by flow cytometry. Journal of Extracellular
Vesicles, 8(1), 1643671.
Gurunathan, S., Kang, M., Jeyaraj, M., Qasim, M., & Kim, J. (2019). Review of the
isolation, characterization, biological function, and multifarious therapeutic
approaches of exosomes. Cells, 8(4), 307.
Han, C., Zhang, C., Wang, H., & Zhao, L. (2021). Exosome-mediated communication
between tumor cells and tumor-associated macrophages: Implications for tumor
microenvironment. Oncoimmunology, 10(1), 1887552.
Harding, C., Heuser, J., & Stahl, P. (1983). Receptor-mediated endocytosis of transferrin
and recycling of the transferrin receptor in rat reticulocytes. Journal of Cell
Biology, 97(2), 329-339.
Jonkman, J. E., Cathcart, J. A., Xu, F., Bartolini, M. E., Amon, J. E., Stevens, K. M., &
Colarusso, P. (2014). An introduction to the wound healing assay using live-cell
microscopy. Cell Adhesion & Migration, 8(5), 440-451.
Kauanova, S., Urazbayev, A., & Vorobjev, I. (2021). The frequent sampling of wound
VFUDWFKDVVD\UHYHDOVWKH³2SSRUWXQLW\´ZLQGRZIRUTXDQWLWDWLYHHYDOXDWLRQRIFHOO
motility-impeding drugs. Frontiers in Cell and Developmental Biology, 9, 391.
Laurent, L. C., & Alexander, R. P. (2015). RNA isolation using the miRNeasy micro kit.
13 | P a g e
Mastoridis, S., Bertolino, G. M., Whitehouse, G., Dazzi, F., Sanchez-Fueyo, A., &
Martinez-Llordella, M. (2018). Multiparametric analysis of circulating exosomes and
other small extracellular vesicles by advanced imaging flow cytometry. Frontiers in
Immunology, 9, 1583.
Mathieu, M., Martin-Jaular, L., Lavieu, G., & Théry, C. (2019). Specificities of secretion
and uptake of exosomes and other extracellular vesicles for cell-to-cell
communication. Nature Cell Biology, 21(1), 9-17.
Momen-Heravi, F., Balaj, L., Alian, S., Trachtenberg, A. J., Hochberg, F. H., Skog, J., &
Kuo, W. P. (2012). Impact of biofluid viscosity on size and sedimentation efficiency
of the isolated microvesicles. Frontiers in Physiology, 3, 162.
Murakami, Y., Toyoda, H., Tanahashi, T., Tanaka, J., Kumada, T., Yoshioka, Y., Kosaka,
N., Ochiya, T., & Taguchi, Y. H. (2012). Comprehensive miRNA expression analysis
in peripheral blood can diagnose liver disease. PloS One, 7(10), e48366.
Peterson, M. F., Otoc, N., Sethi, J. K., Gupta, A., & Antes, T. J. (2015). Integrated
systems for exosome investigation. Methods, 87, 31-45.
Planz, V., Wang, J., & Windbergs, M. (2018). Establishment of a cell-based wound
healing assay for bio-relevant testing of wound therapeutics. Journal of
Pharmacological and Toxicological Methods, 89, 19-25.
Qu, Z., Feng, J., Pan, H., Jiang, Y., Duan, Y., & Fa, Z. (2019). Exosomes derived from
HCC cells with different invasion characteristics mediated EMT through TGF-ȕVPDG
signaling pathway. OncoTargets and Therapy, 12, 6897.
Rekker, K., Saare, M., Roost, A. M., Kubo, A., Zarovni, N., Chiesi, A., Salumets, A., &
Peters, M. (2014). Comparison of serum exosome isolation methods for microRNA
profiling. Clinical Biochemistry, 47(1-2), 135-138.
Rio, D. C., Ares, M., Hannon, G. J., & Nilsen, T. W. (2010). Purification of RNA using
TRIzol (TRI reagent). Cold Spring Harbor Protocols, 2010(6), pdb. prot5439.
14 | P a g e
Ruocco, N., Costantini, S., Zupo, V., Romano, G., Ianora, A., Fontana, A., & Costantini,
M. (2017). High-quality RNA extraction from the sea urchin paracentrotus lividus
embryos. PLoS One, 12(2), e0172171.
Sidhom, K., Obi, P. O., & Saleem, A. (2020). A review of exosomal isolation methods: Is
size exclusion chromatography the best option? International Journal of Molecular
Sciences, 21(18), 6466.
Takov, K., Yellon, D. M., & Davidson, S. M. (2017). Confounding factors in vesicle uptake
studies using fluorescent lipophilic membrane dyes. Journal of Extracellular
Vesicles, 6(1), 1388731.
Vaswani, K., Mitchell, M. D., Holland, O. J., Qin Koh, Y., Hill, R. J., Harb, T., Davies, P.
S., & Peiris, H. (2019). A method for the isolation of exosomes from human and
bovine milk. Journal of Nutrition and Metabolism, 2019
Wang, S., Xu, M., Li, X., Su, X., Xiao, X., Keating, A., & Zhao, R. C. (2018). Exosomes
released by hepatocarcinoma cells endow adipocytes with tumor-promoting
properties. Journal of Hematology & Oncology, 11(1), 1-14.
Witwer, K. W., Buzás, E. I., Bemis, L. T., Bora, A., Lässer, C., Lötvall, J., Nolte-µW+RHQ
E. N., Piper, M. G., Sivaraman, S., & Skog, J. (2013). Standardization of sample
collection, isolation and analysis methods in extracellular vesicle research. Journal
of Extracellular Vesicles, 2(1), 20360.
Wortzel, I., Dror, S., Kenific, C. M., & Lyden, D. (2019). Exosome-mediated metastasis:
Communication from a distance. Developmental Cell, 49(3), 347-360.
Wright, K., de Silva, K., Purdie, A. C., & Plain, K. M. (2020). Comparison of methods for
miRNA isolation and quantification from ovine plasma. Scientific Reports, 10(1), 111.
15 | P a g e
Wu, J., Gao, W., Tang, Q., Yu, Y., You, W., Wu, Z., Fan, Y., Zhang, L., Wu, C., & Han, G.
(2021). M2 Macrophage±Derived exosomes facilitate HCC metastasis by
WUDQVIHUULQJĮ0ȕLQWHJULQ to tumor cells. Hepatology, 73(4), 1365-1380.
Yao, Z., Jia, X., Megger, D. A., Chen, J., Liu, Y., Li, J., Sitek, B., & Yuan, Z. (2018).
Label-free proteomic analysis of exosomes secreted from THP-1-derived
macrophages treated with IFN-ĮLGHQWLILHVDQWLYLUal proteins enriched in
exosomes. Journal of Proteome Research, 18(3), 855-864.
Yi, Y. W., Lee, J. H., Kim, S., Pack, C., Ha, D. H., Park, S. R., Youn, J., & Cho, B. S.
(2020). Advances in analysis of biodistribution of exosomes by molecular
imaging. International Journal of Molecular Sciences, 21(2), 665.
16 | P a g e
Order an Essay Now & Get These Features For Free:
Turnitin Report
Formatting
Title Page
Citation
Outline
