## 3 Results and Discussion
### CD44 Expression by Immunofluorescence Microscopy on U87 and LN18 Cells  
Immunofluorescent microscopy revealed a heterogeneous staining pattern, indicating that CD44 antigens might cluster on the cell surface of control cells. Intense staining was observed in polarized extensions and membrane regions that appearing to extrude from the control U87 cells often localizing to polarized areas of the cell body where actin stress fibers are commonly present (Figure 8A, arrowheads). After 48 h of treatment with 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL peel extract (Figure 8B), CD44 proteins were localized on the cell surface. However, cells treated with 400 \(\mathrm{\SIUnitSymbolMicro g}\)/mL showed a significant reduction in CD44 molecules on their surface (Figure 8C). In LN18 cells treated with 200 and 400 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of the extract for 48 h, CD44  
Figure 7: Flow cytometric analysis of CD44 expression. U87 (**A**) and LN18 (**B**) cells after treatment with 100, 200, and 400 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of peel extract at 48 and 72 h, were stained with Alexa-Fluor 488-conjugated anti-CD44 antibody. Negative controls are cells labeled with secondary goat–anti-mouse IgG Alexa-Fluor 488-conjugate. Flow cytometric analysis was performed as described in the Materials and Methods. Change in mean fluorescence intensity is depicted as mean \(\pm\) SD of three independent experiments. According to GraphPadP Prism10, unpaired _t_-test, *_p_-value < 0.05, **_p_-value 0.001 to 0.01, ***_p_-value 0.0001 to 0.001.  
antigens were generally detected on the cellular membranes without specific localization. Additionally, in Figure 9C, the reduction in CD44 molecules was more pronounced than in Figure 9B, correlating with FACS results. At the same time, in LN18 control cells, CD44 adhesion proteins were mainly detected on the cell body where actin stress fibers are commonly present (Figure 9A, arrowheads). The results of this study reveal a significant relationship between the concentration of citrus peel extract treatments and the migratory behavior of glioma cells. When the concentration of citrus peel extract increases, there is a significant decrease in the ability of tumor cells to migrate and repair wound damage; particularly in the LN18 glioblastoma cell line (Figure 7). These trends are consistent with the results obtained using fluorescence microscopy. This finding suggests that citrus peel extract could potentially serve as an inhibitor of cell migration and, consequently, the development of metastases in brain tissue. Notably, the effect is more pronounced in the LN18 cell line, where there are higher levels of P-glycoprotein (Pgp) compared to the U87 cell line. This observation aligns with previous  
Figure 8: Expression of CD44 molecules on U87 human glioma cells. Immunofluorescence observations on (**A**) (Control), (**B**) (200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), and (**C**) (400 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) of peel extract after 48 h of treatment. Green color indicates CD44 molecule, red color indicates actin filaments stained by Alexa FluorTM Plus 555 Phalloidin and blue color indicates nuclei stained by Hoechst 33258. Figure 9: Expression of CD44 molecules on LN18 human glioma cells. Immunofluorescence observations on (**A**) (control), (**B**) (200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), and (**C**) (400 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) of peel extract after 48 h of treatment. Green color indicates CD44 molecule, red color indicates actin filaments stained by Alexa FluorTM Plus 555 Phalloidin and blue color indicates nuclei stained by Hoechst 33258.  
research by [PERSON] et al. [34], which demonstrated the involvement of Pgp in migration processes in melanoma cells as well. Moreover, the major receptor for hyaluron, CD44, is involved in glioma invasion and is over-expressed in glioblastoma cancer cells [33; 35]. The results show that citrus peel extract treatment reduces CD44 expression in glioma cells (U87 and LN18, respectively).