Effects of Doxorubicin on In Vitro Breast Cancer Model with Lymphocyte and Monocyte Cells

Güneş ÖZEN-EROĞLU1, Ayşe EROL2,, İlhan YAYLIM1, D. Serap KURUCA3

1Istanbul University, Aziz Sancar Institute of Experimental Medicine, Department of Molecular Medicine, İstanbul-TURKEY

2Istanbul University, Faculty of Medicine, Department of Medical Biology, Istanbul-TURKEY

3Istanbul University, Faculty of Medicine, Department of Physiology, Istanbul-TURKEY

*Corresponding Author: gozen.88@hotmail.com

Introduction and Aim:• Breast cancer is most common malignancy in females worldwide. With the prominence of

immunotherapy as an alternative to chemotherapy in cancer treatment, examining theinteraction of cancer cells and immune cells has gained great importance. (1, 2).

• Doxorubicin (Dox), an effective chemotherapeutic agent, is widely used in various types of canceras well as in the treatment of breast cancer.

• In this study, it was aimed to determine the effect of lymphocytes and monocytes on thechemotherapy efficacy and drug resistance of Dox in breast cancer cells.

Material and Methods:• In our study, immune cells were cultured in Iscove′s Modified Dulbecco′s Medium (IMDM)

supplemented with 10% Fetal Bovine Serum (FBS), 1% Penicillin/ streptomycin (P /S). Breastcancer cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with10% FBS, 1% P/S. Cells incubated in a humidified atmosphere at 37 °C with 5% CO2.

• To test the effects of immune cells on breast cancer cells, two different co-culture models weregenerated: lymphocyte cells: breast cancer cells and monocyte cells: breast cancer cells. Thecombination of immune cells and breast cancer cells was tested in different cell ratios, and a cellratio of 5:1 was selected according to the MTT test and microscope images. In the model createdwith lymphocyte cells, two groups that were stimulated and not stimulated withPhytohemagglutinin M (PHA-M) (10 µg/mL for 72 hours) were examined. Cells used in the modelswere Jurkat as lymphocte cell, THP-1 as monocyte cell, and MDA-MB-231 cells as breast cancercell.

• Dox-induced cytotoxicity was determined by MTT assay in both all of cells and co-culture models.

• Apoptosis analysis was performed by flow cytometry according to the determined IC50

concentration using Annexin V/PI staining.

• In addition, MDR-1 protein expressions, which is multi-drug resistance protein, were evaluatedflow cytometrically.

• Statistical analysis in the study was carried out using Graphpad Prism 5.03 Software.

Results:

• When the MTT results of the Dox were evaluated, higher IC50 values were determined in the co-

culture model.

• However, no significant difference in IC50 values was observed between co-culture models of PHA-

M-stimulated Jurkat cells and unstimulated Jurkat cells.

• Cell death rates were assessed by Annexin V-propidium iodide (PI) staining assay with Dox 0,2 µm

for 72h using flow cytometry.

• Additionally, a significant increase in MDR-1 protein expressions was detected with Dox treatment

at the same concentration.

Figure 2 : Representative microscopic images (10X) of all type cells were treated with Doxtreatment 0,2 µM for 72 hours.

Discussion and Conclusion: • In many studies, it has been reported that the communication between cancer cells and

immune cells and the response to drugs vary. With the emergence of immunotherapy as analternative to chemotherapy in cancer treatment, it is of great importance that futurestudies focus not only on cancer cells but also on immune cells (1-3).

• In our study, relatively high IC50 values in the 5:1 (immune cells: breast cancer cells) ratio invitro co-culture model were associated with drug resistance. Especially, a significantincrease in MDR-1 protein expressions was detected with Dox treatment in the Jurkat-MDA-MB-231 co-culture model.

• Consequently, we think that the number and functionality of immune cells and drugresistance may be related in cancer. As the importance of the association of cancer cells andimmune cells is understood, we hope that it will be easier to adapt the results of the studiesto cancer patients. This perspective will shed light on many cancer studies.

• As the next step, it is planned to perform the PD-1 and PD-L1 related drug resistance studieswith the generated in vitro model.

Cells IC50 (µM) Co-culture models IC50(µM)

MDA-MB-231 0,22 MDA-MB -231:stimulated Jurkat 0,2

Jurkat 0,09 MDA-MB-231: Jurkat 0,3

THP-1 0,11 MDA-MB-231: THP-1 0,3

Table of IC50 values in Dox treated cells and co-cultures.

AcknowledgementThis study is supported by Istanbul University-Support Programme for Scientific Research Projects-numbered TDK-2020-35486.I would like to thank you for being supported by YOK 100/2000 Doctoral Scholarship Program and TUBITAK 2211-C Domestic Priority Fields Doctoral Scholarship Program within the scope of this thesis project.

MDA-MB-231 control MDA-MB-231 Dox 0,2 µM

Jurkat control Jurkat Dox 0,2 µM

THP-1 control THP-1 Dox 0,2 µM

Stimulated Jurkat-MDA-MB-231 Dox 0,2 µM

Stimulated Jurkat-MDA-MB-231 control

THP-1-MDA-MB-231 control THP-1-MDA-MB-231 Dox 0,2 µM

MDA-MB-231

2 4 6

-50

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Figure 3: Dox-induced total apoptotic cellratios in MDA-MB-231, Jurkat, THP-1 cellsand co-culture models was examined byAnnexin V-propidium iodide (PI) stainingassay with Dox 0,2 µm for 72h using flowcytometry.

Figure 1: Cell viability in MDA-MB-231, Jurkat, THP-1 cells and co-culture models by treatment ofDox (0-10 µm) for 72 h by MTT assay.

References:1. Galli, F., Aguilera, J. V., Palermo, B., Markovic, S. N., Nisticò, P., & Signore, A. (2020). Relevance of immune cell and tumor microenvironment imaging in the new era of immunotherapy. Journal of Experimental & Clinical Cancer Research, 39, 1-21.2. Black, M., Barsoum, I. B., Truesdell, P., Cotechini, T., Macdonald-Goodfellow, S. K., Petroff, M., ... & Graham, C. H. (2016). Activation of the PD-1/PD-L1 immune checkpoint confers tumor cell chemoresistance associated with increased metastasis. Oncotarget, 7(9), 10557.3. Saraiva, D. P., Matias, A. T., Braga, S., Jacinto, A., & Cabral, M. G. (2020). Establishment of a 3D co-culture with MDA-MB-231 breast cancer cell line and patient-derived immune cells for application in the development of immunotherapies. Frontiers in oncology, 10, 1543.

Figure 4: Dox-induced drug resistancein MDA-MB-231 and co-culturemodels was investigated by MDR-1protein expressions with Dox 0.2 µMfor 72 h using flow cytometry.

Cell Viability

Cell Images

Assessment of Apoptosis

Assessment of Drug Resistance