The document discusses a research project with two main goals: 1) To examine if drug resistant Calu1 lung cancer cells export the chemotherapy drug cisplatin through extracellular vesicles, and 2) To examine if drug resistance can be transferred from resistant to sensitive Calu1 cells through extracellular vesicles. The researchers isolated extracellular vesicles from cisplatin resistant Calu1 cells and found they contained exosomes and microvesicles. XPS analysis did not detect cisplatin in the vesicles, indicating resistance is not from drug export. When vesicles from resistant cells were added to sensitive cells with cisplatin, the cells showed higher viability, demonstrating resistance transfer through the vesicles. Further studies are suggested to identify the molecules transmitting resistance and potential targets to

1. GÖKÇE LARA BODUR
ILGIN KİŞİOĞLU
2017

2. Our project has two main goals.
First goal is to examine whether drug resistant
Calu1 cells excrete cisplatin with extracellular
vesicles.
The other goal is to examine if the drug
resistance is transferred from drug resistant
Calu1 cells to drug-sensitive Calu1 cells
through extracellular vesicles.
AIM OF THE PROJECT

3. Different treatments are used for different types of cancers due to
the differences in their growth, spreading and response to
chemotherapeutics. Nowadays; the most common cancers are
breast, lung, prostate and servical cancer.
In our project, we worked with Calu1, which is
Lung Epidermoid Carcinoma Cell Line.

4. Drug resistance in cancer is a
complication that decreases the effect
of cancer treatment. As a
consequence, it requires increase of
drug dosage. Consequently, this
increases drug’s side effects. In recent
studies, it is shown that extracellular
vesicles (EV) have effective roles in
development and transmission of drug
resistance from cell to cell.
Physical appearances of Extracellular Vesicles

5. Schematic
view of EV
exportation
from drug
resistant cell
to drug-
sensitive cell

7. Start of Calu1 and
Cisplatin Resistant Calu1
Cell Cultures
Transfer of Calu1 cell
suspension to T-25 cell plate.
Isolation of Extracellular
Vesicles from Calu1 and
Cisplatin Resistant Calu1
Cells
Ultracentiruge machine

8. Standart Name C* (μg/ml) PBS (μL) BSA (μL)
A 25 487.5 12.5
B 15 492.5 7.5
C 10 495 5
D 5 497.5 2.5
E 2 499 1
F 1 499.5 0.5
Required solutions to prepare Bradford Protein Assay
*C represents the concentration of protein standarts
Protein Assay of Isolated Extracellular Vesicles from Cisplatin
Resistant Calu1 Cells (Bradford Protein Assay)
We put our colored standarts and unknown sample (Coomasie
compound is used) in ELISA Reader in order to detect their absorbance

9. Absorbance values of standard protein solutions
*Absorbance values are shown by subtraction of baseline value’s
absorbance (which is 0.3467)
Measured
Samples
Concentration
(μg/ml)
Measured Absorbance Value*
Average
1st
Measurement
2nd
Measurement
Standart A 25 0.5249 0.5311 0.5280
Standart B 15 0.4035 0.3973 0.4004
Standart C 10 0.2542 0.2648 0.2595
Standart D 5 0.1340 0.1340 0.1340
Standart E 2 0.0605 0.0598 0.0602
Standart F 1 0.0248 0.0148 0.0198

10. Calibration Curve and Absorbance measurements of Extracellular Vesicle proteins and
calculated protein concentrations
a Absorbance values are shown by subtraction of baseline value’s absorbance
(0.3467).
b Concentration values are calculated considering the dilution factor (muptiplied by
6)

11. Determination of the Extracellular Vesicle’s Sizes
(Dynamic Light Scattering)
Zeta Sizer Nano
ZS Instrument Intensity-size distribution of extracellular vesicles

12. Investigation of Cisplatin Existance in Extracellular Vesicles
(with XPS)
XPS Spectrums of Cisplatin and Extracellular Vesicles

13. Detection of the Cisplatin Toxicity on Calu1 Cells
Cell counting on
Hemocytometer with Trypan
Blue staining under inverted
microscope
Calu1 cells and seeding Calu1
cells in 96 well plate

14. Solution
Number
Dose
(μM)
Sample Solution
(includes
Cisplatin)
(μl)
Medium
(μl)
Total
(μl)
A 1 6 594 600
B 5 30 570 600
C 10 60 540 600
D 50 300 300 600
E 100 600 0 600
Solutions are prepared in
order to apply to Calu1
cells
The Toxicity of various
Cisplatin doses on Calu1
cells

15. Implementation of the Extracellular Vesicles (Isolated from Cisplatin
Resistant Calu1 Cells) to Calu1 Cells with Cisplatin

16. Micrographs of cells implemented with
Cisplatin, Extracellular Vesicles and
Cisplatin+Extracellular Vesicles
The graph demonsrates the vitality percentage of
Cisplatin and Cisplatin+Extracellular Vesicules
implementation on Calu1 cells

17. To sum up with, we isolated Extracellular Vesicles from Cisplatin Resistant Calu1
and detected their protein quantities in order to use in following cell culture works.
We analysed the particule sizes of Extracellular Vesicles to determine the type of vesicles.
According to our analyses, we found out that we have exosomes and microvesicles in our
isolate.
In our XPS results, we didn’t come across with signals (that are specific to Cisplatin) in
Extracellular Vesicles isolated from Cisplatin Resistant Calu1. This showed us that the drug
resistance doesn’t depend on the exportation of Cisplatin with Extracellular Vesicles.
In our experiments to show whether drug resistance is transferred from Cisplatin Resistant Calu1
cells to Calu1 cells with Extracellular Vesicles; we observed that Cisplatin+Extracellular
Vesicules implemented cells show more viability than only Cisplatin implemented cells. The
viability of only Extracellular Vesicules treated cells was not decreased.
As a result, it demonstrated that «Extracellular Vesicles (which are isolated from Cisplatin
Resistant Calu1 Cells) implemented Calu1» cells became more resistant to Cisplatin.

18. In order to specify the
molecules (DNA, mRNA,
miRNA, etc.) that transfer drug
resistance with Extracellular
Vesicles (which were exported
from Cisplatin Resistant Calu1
cells) may be investigated in
further studies.
Mechanisms which prevent the
Cisplatin resistance
transportasion with
Extracellular Vesicles may be
investigated.
New drugs that target
resistance transfer
mechanisms should be
investigated.
Drug resistance transfer with
Extracellular Vesicles can be
investigated with other cancer
drugs. Also this can be
investigated in both Calu1 and
other cancer cell lines.
FUTURE
PROSPECTS

19. SPECIAL THANKS TO…
We thank everyone supported us from the very beginning of our
project. Special thanks to Dr. Mustafa Kotmakci from Ege University
Faculty of Pharmacy Department of Pharmaceutical Biotechnology,
to our school’s Scientific Committee Co-Head Dr. Onur Akpinar, to
our Biology teacher Ilkem Sonmez, and last but not least to our
school principals for supporting us in scientific research projects.

20. REFERENCES
• 1. Cesi, G., Walbrecq, G., Margue, C., & Kreis, S. (2016). Transferring intercellular signals and traits between cancer cells:
extracellular vesicles as “homing pigeons.” Cell Communication and Signaling, 14(1), 13. doi:10.1186/s12964-016-0136-z
• 2. Chen, W. X., Liu, X. M., Lv, M. M., Chen, L., Zhao, J. H., Zhong, S. L., Tang, J. H. (2014). Exosomes from drug-resistant breast
cancer cells transmit chemoresistance by a horizontal transfer of MicroRNAs. PLoS ONE, 9(4).
doi:10.1371/journal.pone.0095240
• 3. Sousa, D., Lima, R. T., & Vasconcelos, M. H. (2015). Intercellular Transfer of Cancer Drug Resistance Traits by Extracellular
Vesicles. Trends in Molecular Medicine, 21(10), 595–608. doi:10.1016/j.molmed.2015.08.002
• 4. Osteikoetxea, X., Balogh, A., Szabó-Taylor, K., Németh, A., Szabó, T. G., Pálóczi, K., Buzás, E. I. (2015). Improved
Characterization of EV Preparations Based on Protein to Lipid Ratio and Lipid Properties. PloS one, 10(3), e0121184.
doi:10.1371/journal.pone.0121184
• 5. Kotmakçı, M., & Çetintaş, V. B. (2015). Extracellular Vesicles as Natural Nanosized Delivery Systems for Small-Molecule
Drugs and Genetic Material : Steps towards the Future Nanomedicines. Journal of Pharmacy and Pharmaceutical Sciences,
18(3), 396–413.
• 6. Altuğ T.(2009), Hayvan deneyleri etiği. Sağlık Bilimlerinde Süreli Yayıncılık, sayfa 53 – 68.
• 7. Alper Akçalı.(2010), Araştırmalarda tanımlanmış hücre hatlarının kullanılmasının önemi. Türk Onkoloji Dergisi. 25(3). sayfa
119-123.
• 8. Vildan Bozok Çetintaş, Zuhal Eroğlu.(2013),Cisplatin direncinde etkili moleküler mekanizmalar. Süleyman Demirel Üniversitesi
Tıp Fakültesi Derisi. 20(2)/ sayfa 72-79.
• 9. Xiao, X., Yu, S., Li, S., Wu, J., Ma, R., Cao, H., Feng, J. (2014). Exosomes: decreased sensitivity of lung cancer A549 cells to
cisplatin. PloS one, 9(2), e89534. doi:10.1371/journal.pone.0089534
• 10. Cetintas, V. B., Kucukaslan, A. S., Kosova, B., Tetik, A., Selvi, N., Cok, G., Eroglu, Z. (2012). Cisplatin resistance induced by
decreased apoptotic activity in non-small-cell lung cancer cell lines. Cell Biology International, 36(3), 261–265.
doi:10.1042/CBI20110329