To screen and identify CD98 light chain L-type amino acid transporter 1(LAT1) as a new glioma stem cell marker.

The flow cytometric single-color labeling method was used to detect the expression of CD98 heavy chain protein CD98hc, CD98 light chain protein LAT1 and expression of CD133 in U87 and U251 glioma cells to select possible and potential stem cell markers in glioma cells. LAT1⁺ and LAT1^- glioma cells were sorted by flow cytometry. Cellular immunofluorescence, sphere-formation assay, and in vitro limiting dilution assay were used to identify cell stemness. Differentiated glioma stem cells were cultured, and the expressions of β-tubulin Ⅲ, GFAP, and LAT1 were detected by Western blot. By constructing a nude mouse subcutaneous tumor model, we observed the tumor formation of LAT1 ⁺ and LAT1^- glioma cells under the skin of nude mice (LAT1⁺ group and LAT1^- group respectively).

Flow cytometry results showed that the positive rate of LAT1 (SLC7A5) in U87 cells was (1.99±0.18)%, the positive rate of CD133 in U87 cells was (2.18±0.29)%; the positive rate of LAT1 (SLC7A5) in U251 cells was (6.24±0.69)%, and the positive rate of CD133 in U251 cells was (4.79±0.64)%. Immunofluorescence detection results showed that the positive expression rates of Sox2, Olig2 and CD133 in LAT1⁺ -U251 and LAT1⁺ -U87 cells were higher; the positive expression rates of Sox2, Olig2 and CD133 in LAT1^--U251 and LAT1^--U87 cells were all lower. The sphere-formation assay and the limiting dilution assay showed that the sphere-formation rate of LAT1⁺ -U251 cells was better than that of LAT1⁺ -U87 cells (P=0.005); LAT1^--U251 and LAT1^--U87 cells did not form spheroids. Western blotting results showed that LAT1^--U251 cells had low expression of β-tubulin Ⅲ and GFAP when they were undifferentiated and cultured without induction; LAT1 ⁺ -U251 cells had low expression of β-tubulin Ⅲ and GFAP before differentiation, but after induced differentiation the expression of β-tubulin Ⅲ and GFAP increased significantly (all P<0.05), and the expression of LAT1 decreased significantly (P=0.007). The results of the nude mouse tumorigenesis model showed that the tumor volume of the LAT1⁺ group was larger than that of the LAT1^- group at 21 d and 28 d, and the differences were statistically significant (all P<0.05); at 28 d, the tumor weight in the LAT1⁺ group was greater than that of LAT1^- group, and the difference in tumor weight between the two groups was statistically significant (P=0.007).

CD98 light chain protein LAT1 is a potential glioma stem cell marker.

Glioblastoma; Tumor stem cell; Immunohistochemistry; Biological markers; L-type amino acid transporter 1