Page 115 - SRPSKO DRUŠTVO ISTRAŽIVAČA RAKA

SDIRSACR Oncology Insights

P18

Analysis of NK-92 cytotoxicity against different colon cancer cell lines

Sonja Marinović

Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia

Keywords: NK cells, colon cancer, NKG2D, cytotoxicity

Background: Natural Killer (NK) cells play a vital role in the immune defense against colorectal cancer (CRC). Their
direct cytotoxic activity is driven through activating NK cell receptors and their ligands on target cells, leading to tumor
cell death by releasing lytic granules such as perforin. This study aimed to assess how different CRC cell lines, which
reflect varying stages of tumor progression, genetic mutations, and immune evasion strategies, affect NK cytotoxicity,
production of perforin and interferon-gamma (IFN-γ), as well as the expression of NKG2D and PD-1 receptors.
Materials and Methods: The cytotoxic activity of NK-92 cells, a commercially available NK cell line, was evaluated
against six CRC cell lines (SW480, SW620, HT-29, HCT116, DLD-1, and Caco2). NK-92 cells were co-cultured with
fluorescently pre-labeled target cells at varying effector-to-target (E:T) ratios, and target cell lysis was assessed using
flow cytometry (FACS). Additionally, FACS analysis was used to examine the expression of NKG2D and PD-1 receptors,
as well as the production of perforin and IFN-γ.
Results: At the highest E:T ratio tested (10:1), NK-92 cells exhibited enhanced cytotoxic activity against the SW480 CRC
cell line and its metastatic derivative, SW620. Notably, co-cultivation with SW620 cells led to a marked downregulation
of the activating receptor NKG2D and a reduction in perforin production in NK-92 cells, which corresponded with
decreased cytotoxic efficacy compared to that observed with the primary SW480 cells. HT-29, HCT116, and DLD-1
demonstrated greater susceptibility to NK-92-mediated cytotoxicity than Caco-2 cells, the latter exhibiting less than
10% cell lysis. This may be attributed to the close resemblance of Caco-2 cells to normal enterocytes, characterized
by the expression of epithelial ligands typically found in non-transformed tissue. Interestingly, following a 4-hour co-
culture with NK-92 cells, HT-29, HCT116, and DLD-1 cells upregulated expression of the immune checkpoint ligand PD-
L1, yet no changes in PD-1 expression were detected on NK-92 cells. Overall, NK-92 cells produced low levels of IFN-γ
in response to all CRC cell lines.
Conclusions: NK-92 cell cytotoxicity varied across CRC cell lines. Reduced NKG2D expression and perforin production
following co-culture with the metastatic SW620 suggest impaired NK-92 killing capacity. Additionally, the observed
upregulation of PD-L1 on CRC cells might contribute to NK-92 dysfunction.

Acknowledgments and funding: This research was funded by the European Union under the NextGenerationEU
Programme (MZ3-24, KP1-25).

P19

Proteomic analysis of endothelial cell derived secretomes exposed to hypoxia-induced pharyngeal cancer
cell-derived exosomes

Ozel Capik , Omer Faruk Karatas 1,2
1,2
1Molecular Biology and Genetics Department, Erzurum Technical University, Erzurum, Turkey
2Molecular Cancer Biology Laboratory, High Technology Application and Research Center, Erzurum Technical University, Erzurum,
Turkey

Keywords: hypoxic exosome, metastasis, microenvironment

Background: Head and neck squamous cell carcinomas (HNSCCs), which are associated with high mortality rates, exhibit
enhanced metastasis, invasion, angiogenesis, and immune evasion, despite advancements in diagnosis and treatment.
Recent studies indicate that exosomes, key mediators of tumor–microenvironment communication, contribute to
prognostic and pathological alterations in cancer. However, the composition of exosomes released under hypoxia in
cancers such as pharyngeal cancer and their impact on the tumor microenvironment remain poorly understood. This
study aimed to investigate the changes in the protein profiles of secretomes from endothelial cells exposed to hypoxia-

100