AIP Conference Proceedings 2344, 040010 (2021); https://doi.org/10.1063/5.0049155 2344, 040010
© 2021 Author(s).
Investigating different type of ovary cancer cell line for NK cell in vitro co-culture
cytotoxic assay
Cite as: AIP Conference Proceedings 2344, 040010 (2021); https://doi.org/10.1063/5.0049155 Published Online: 23 March 2021
Lady Feren Pangjaya, Sanya Khaerunnisa, Nuzli Fahdia Mazfufah, Retno Lestari Budiman, and Radiana Dhewayani Antarianto
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Investigating Different Type of Ovary Cancer Cell Line for NK Cell in vitro Co-culture Cytotoxic Assay
Lady Feren Pangjaya
1,2, Sanya Khaerunnisa
1,2, Nuzli Fahdia Mazfufah
3, Retno Lestari Budiman
3, Radiana Dhewayani Antarianto
2,4,a)1Biology Undergraduate Program, Faculty of Mathematics and Natural Science, Universitas Indonesia, Kampus UI Depok, West Java, 16424, Indonesia
2Stem Cell and Tissue Engineering Research Cluster, Indonesian Medical Education and Research Institute (IMERI), Universitas Indonesia, Salemba Raya, Jakarta, West Java, 10430, Indonesia
3Department of Biology, Faculty of Mathematics and Natural Science, Universitas Indonesia, Kampus UI Depok, West Java, 16424, Indonesia
4Department of Histology, Faculty of Medicine, Universitas Indonesia, Salemba Raya, Jakarta, West Java, 10430, Indonesia
a)Corresponding author: [email protected]
Abstract. Ovarian cancer is one of the gynecologic cancers with a high mortality rate and increased incidence. Surgery and therapies are used to treat ovarian cancer patients. Therapies such as chemotherapy and radiotherapy can cause resistance therefore immunotherapy using natural killer (NK) cells can be an alternative therapy. NK cells have shown an important role in anticancer activity because it is able to kill target cells without prior sensitization and can acquire immunology memory. However, NK cells as immunotherapy have low capacity and efficacy on infiltrating ovarian cancer’s tumor microenvironment. To overcome these NK cells need to be induced with certain antigen resulting in enhanced NK cells. Enhanced NK cells display a higher cytotoxic activity that can be seen by co-culturing enhanced NK cells with ovarian cancer cell lines. In this mini-review, the selection of ovarian cancer cell lines was displayed to offer reliable consideration to obtain the expected research endpoint.
Keywords: ovarian cancer; NK cells; co-culture; ovarian cancer cell lines; cytotoxic assay
INTRODUCTION
Ovarian cancer which occurs in women has a high mortality rate and increased incidence [1]. Based on the study conducted by Global Cancer Observatory in 2018, there were 295.414 new cases with 184.799 death in the world [2].
The high mortality rate from ovarian cancer is caused by late-stage diagnosis in almost 70% of ovarian cancer patients [3]. Ovarian cancer treatment differs in every patient, based on its stage, condition, and patient’s decision. Surgery is the first-line treatment for ovarian cancer which followed by adjuvant therapy [4]. Adjuvant therapies like chemotherapy or radiotherapy oftentimes caused cancer resistance. This increased the risk of cancer recurrence and decreased long-term survival in patients [5]. To overcome this, immunotherapy using natural killer (NK) cells has been reported in phase 1-2 clinical trial in hematology malignancy. NK cells as cellular-based immunotherapy relies on the capacity to kill target cells without prior sensitization also on the capacity to acquire immunology memory [6].
NK cells as cellular-based immunotherapy have low capacity and efficacy on infiltrating ovarian cancer’s tumor microenvironment [7]. Ovarian cancer’s tumor microenvironment (TME) is capable of suppressing cytolytic cells including NK cells. Immunosuppressive TME exerts multiple ligands that interacted with NK cells receptor thus inhibiting NK cells' function [8]. The strategy to enhance the impaired NK cells' function is to induce them with certain activating cytokine/s or chemokines such as interleukin [9]. Studies have shown that antigen-induced NK cells have
enhanced function, proliferation, and cytotoxic activity including cytokine production and in in vitro NK-cell mediated killing of ovarian cancer. The cytotoxic activity is seen by co-culturing ovarian cancer cell lines with induced and not induced NK cells [10]. The co-culturing method causes physical contact between NK cells and ovarian cancer cell lines resulting in cell-cell communication that is related to the cancer cell invasion mechanism. This cell-cell communication is quite similar to in vivo condition however can be analyzed in vitro [11].
There are over 100 commercially available ovarian cancer cell lines in the world. Immunotherapy study screening, which relies heavily on cell lines must use the most suitable, effective, good quality cell lines to achieve the desired result [12]. Therefore it is important to compare enhanced NK cells cytotoxicity against commonly used cancer ovarian cell lines also the potential risk of using cell lines for in vitro study and how to decrease the risk.
MATERIALS AND METHODS
This systematic literature search was performed in June 2020 in numerous databases. NCBI, ScienceDirect, and Google Scholar are the three main source database. The combinations of terms used for the search are, “ovarian cancer cell lines”, “NK cells in vitro co-culture”, and “NK cells cytotoxicity assay”. The inclusion criteria being used in this systematic review including:
(i) Article published in the last 10 years (ii) Review article and research article (iii) Full-text article open access
RESULTS AND DISCUSSIONS
The result of the combined keywords is 3.942 articles in NCBI; 1.415 articles in ScienceDirect; 6.260 articles in Google Scholar. Three studies that correlated with ovary cancer cell lines used for induced NK cell in vitro co-culture cytotoxic assay were found. From these studies, seven ovarian cancer cell from different epithelial origin were used.
They are shown in Table 1.
TABLE 1. Types of ovarian cancer cell lines and histopathology origin Histopathology Type of ovarian cancer cell line Serous tumors
Malignant (serous adenocarcinoma), high grade serous
OVCAR-3, CAOV-3, OVCARS-3, MA148, A1847
Clear Cell SKOV-3
Endometrioid tumors A2780
90% of ovary tumors are of epithelial origin with four major histotypes, which are serous, endometrioid, mucinous, and clear cell. Serous ovarian carcinoma is accountable for almost 70% of epithelial ovarian cancer (EOC). High- grade serous ovarian cancer (HGSC) is responsible for 90% of serous carcinoma and the origin of most commonly used ovarian cancer cell lines, such as OVCAR-3 and CAOV3 [13]. Other cell lines that are commonly used are clear ovarian cancer cell line, SKOV-3, and ovarian endometrioid carcinoma cell line A2780 [14].
OVCAR-3, CAOV3, SKOV-3, and A2780 are used by Sun et al. to demonstrate enhanced NK cell with interleukin-15 (500 IU/ml) and autoplasma (1%) cytotoxic activity by co-culturing them together on culture medium.
The results are the enhanced NK cells effectively lysed those cancer cell lines differently depending on the dose and time. On 24, 48, and 72 hours post-treatment with different effector: target doses (1:1; 5:1; 15;1) SKOV-3 is the most affected one and CAOV-3 is the least affected one. The enhanced NK cells also reduce the ovarian cancer cell line migratory activities [5].
Klapdor et al. successfully engineered NK-cell line NK-92 with anti-CD24 CAR and anti-CD19 control CAR using lentiviral transduction. Engineering the NK-92 is a novel immunotherapy approach toward ovarian cancer. The engineered NK-92 cells showed high cytotoxic activity against CD24-positive ovarian cancer cell lines (SKOV-3 and
OVCAR3). This effect was restricted to CD24-expressing cells. The experiment also showed that the SKOV3 cell is more resistant to unspecific killing by control cells compared with low expressing CD24, A2780 cells [15].
NK cells incubated with interleukin (IL)-12, IL-15, and IL-18 produces cytokine-induced memory-like (CIML) NK cells. CIML NK displayed increasing interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) production against MA148, A1847, OVCAR5, and SKOV3 ovarian cancer cell line. Moreover, CIML NK cultures show a drastic reduction in tumor cell count, demonstrating an enhanced killing by CIML NK [10].
In immunotherapy studies, cancer cell lines became target cells against immune cells like NK cells as the effector cell. However, limited understanding of the NK cells cytotoxicity towards different ovarian cancer cell lines activity becomes an obstacle in the experimental design. Each ovarian cancer cell lines are different because of its origin as there are many types of ovary cancer.
The potential risk of using cell lines for in vitro study started with the diverse origin for in vitro therapy screening which is caused by different genetic makeup between each ovarian cancer cell line. This may indicate different ligands expressed by the ovarian cancer cell lines. Eventually will lead to a misanalysis of the in vitro result, resulting as a wrong interpretation of the result. More research is required to be done to understand the interaction between ligands expressed by ovarian cancer cell lines with NK cell receptors. In addition, the standard for commercially available cell lines with legit identification will secure cell lines' quality [12,13].
CONCLUSION
Seven ovarian cancer cell lines as NK cell’s target cell were used (SKOV-3, OVCAR-3, MA148, CAOV3, A1847, OVCAR5, A2780) in three studies correlated to NK cell cytotoxicity assay. The ovarian cancer cell lines could involve either from the different epithelial origin or derived from the same origin which is high-grade serous carcinoma. The SKOV-3 and OVCAR3 cells had a high expression of CD24, while A2780 which lacked CD24 expression.
TABLE 1. Comparison Between Co-Cultured Different Enhanced NK Cells and Ovarian Cancer Cell Lines Cytotoxic
Inducer/
Antigen[Ref] Culture system
NK cell expansion
rate
Target cells (ovarian cancer cell
lines)
NK cell function
Autoplasma (1%) + interleukin-2 (500
IU/ml)[5]
Ovarian cancer patient NK cells +
inducer in MEM Medium using
VarioMACS
14 days
OVCAR-3, CAOV3, SKOV-3, and A2780
- Lyse target cells - Reducing target cells
migratory activities by analyzing the inhibitory effects
Anti-CD24 CAR and anti-CD19 control CAR[15]
NK-cell line NK-92 + inducer using
lentiviral transduction N/A
SKOV3, OVCAR3, and A2780
- High cytotoxic activity against CD24-positive ovarian cancer cell lines (SKOV-3 and OVCAR3 - SKOV3 cells are more
resistant to unspecific killing by control cells compared with low expressing CD24, A2780 cells
IL-12, IL-15, and IL-18[10]
Healthy NK Cells + inducer + in RPMI-10
supplemented with rhIL-15 (1ng/mL)
7 to 14 days
MA148, A1847, OVCAR5, and SKOV3
- Enhanced interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) production
- NK cultures show a drastic reduction in target cell count
ACKNOWLEDGEMENT
This study is funded by research grant PDUPT from RISTEKDIKTI/BRIN 2020 year 2 research with the title Induction of peptide-specific NK cells as adoptive immunotherapy for advance stage Ovary Cancer. The principal investigator is Radiana Dhewayani Antarianto.
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