The effect of detoxified Shigella sonnei lipopolysaccharide on the metastasis of B16 melanoma cells

• Elena M. Novikova
• Elena S. Chukhina
• Nadezhda A. Razvalyayeva
• Olga V. Kozyreva
• Rodion N. Stepanenko

Abstract

Introduction. According to current concepts, metastasis is the result of a complex multi-stage process determined by the interaction between metastatic cells, tumor microenvironment and homeostatic mechanisms. Surface receptors present on the cells of the tumor itself play a major role in the progression and metastasis of melanoma. First of all, this applies to toll-like receptors (TLRs). Among the TLRs expressed by malignant melanocytes, there is TLR4. This is one of the most important representatives of the TLRs family, high expression of which on tumor cells and cells of the tumor microenvironment in various types of cancer closely correlates with the onset of oncogenesis, tumor progression and drug resistance. On the other hand, there is data on the suppression of tumor growth after stimulation of melanoma cells expressing TLR4 with lipopolysaccharide (LPS). At the same time as shown in a number of studies, when culturing tumor cells in vitro with detoxified TLR agonists, the number of cells expressing TLR4 increases, which can have a significant impact on tumor progression and metastasis upon subsequent administration of these cells to experimental animals.

Along with TLR4, there are a number of other antigens (Ag) on the surface of melanoma cells, changes in the expression of which can have a significant impact on the ability of tumor cells to metastasize. These include the chemokine CXCR4 (CD184), integrins, the receptor for hyaluronic acid (CD44) and the tumor stem cell marker, the CD24 protein.

Aim – to study the in vitro effect of detoxified lipopolysaccharide (Ac3-S-LPS) Shigella sonnei, phase 1, on metastasis and expression by melanoma cells B16 TLR4, chemokine receptor CXCR4 (CD184), integrin avß5, receptor for hyaluronic acid (CD44), tumor stem cell marker (CD24) and PD-L1 (CD274) protein (the ligand of the programmed cell death receptor PD-1) and MHC I.

Material and methods. B16 melanoma cells were cultured with different concentrations of Ac3-S-LPS for 48 hours at 37 °C in a CO₂ incubator and after the end of cultivation were injected intravenously into C57Bl/6 mice. On the 21^st day after tumor cell inoculation, the animals were sacrificed and the number of lung metastases was counted. To determine the effect of Ac3-S-LPS on the expression of surface Ag chemokine CXCR4, hyaluronic acid receptor, tumor stem cell marker, MHC I and PD-L1 protein, the cells were cultured and then stained with monoclonal antibodies (Ab) conjugated with fluorescent dyes. The samples were analyzed on a flow cytometer. To assess the expression of TLR4 and integrin avß5, tumor cells after cultivation with Ac3-S-LPS were stained with primary recombinant polyclonal At to TLR4, then secondary At – anti-rabbit IgG (H+L) fragment F(ab')2 conjugated with Alexa Fluor 488 and analyzed under a fluorescent microscope.

Results. The obtained results indicate that culturing tumor cells with a TLR4 agonist (Ac3-S-LPS) reduces their metastatic potential, increases the number of cells expressing TLR4, reduces the number of cells carrying CD184, and increases the number of cells carrying a receptor for hyaluronic acid (CD44). At the same time, the number of cells expressing tumor stem cell marker (CD24) and integrin αvβ5 remained virtually unchanged. The presence of the CXCR4 receptor on tumor cells is of great importance for the formation of metastases in the lungs. The effect of culturing tumor cells with Ac3-S-LPS on the expression of CXCR4 on cells carrying CD44, CD24 and PD-L1 (CD274) was studied. It was found that culturing melanoma B16 cells with Ac3-S-LPS for 48 hours led to a decrease in the number of cells expressing CD184⁺/CD44⁺, and had virtually no effect on the number of cells carrying CD184⁺/CD24⁺ and CD184⁺/CD274⁺. At the same time, culturing tumor cells with Ac3-S-LPS led to an increase in the number of malignant melanocytes in the population expressing MHC I and PD-L1 proteins.

Conclusion. Cultivation of B16 melanoma cells with detoxified Shigella sonnei, phase 1, LPS leads to a decrease in the metastatic potential of tumor cells. This effect is due to a decrease in the number of cells carrying CXCR4 under the influence of Ac3-S-LPS. And although after cultivation with an agonist, the number of cells carrying CD44⁺ increases, which greatly facilitates the emigration of cells from primary tumor nodes, the absence of the CXCR4 receptor on them does not allow them to be localized in lung tissue.

Keywords: melanoma B16; malignant cells; detoxified lipopolysaccharide; TLR4; CXCR4; CD44

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