Fig. 6: HTR3A^high neuroblastoma cells are highly tumorigenic and reduce their proliferation under excessive HTR3A stimulation.

a Gene expression analysis by qRT-PCR revealed marked differences in relative expression of HTR3A among individual human neuroblastoma cell lines. NTERA-2 pluripotent embryonal carcinoma cells served as a positive expression control (Ctrl). Data presented as mean ± SD, biological n = 3, technical n = 3; *p < 0.05, **p < 0.01 using one-way ANOVA followed by Tukey’s post hoc test. b Western blotting showed differences in HTR3A protein levels, which also corresponded to the differences in expression of N-MYC, c-MYC, and SOX2 proteins between HTR3A^high and HTR3A^low groups. Blots are representative of three experiments. c Densitometric quantitation of the HTR3A protein expression confirms the defined HTR3A^high and HTR3A^low groups. Data presented as mean ± SD, biological n = 3; ***p < 0.01 using one-way ANOVA followed by Tukey’s post hoc test. d–g MTT assay on cells treated with agonists of HTR3A receptor (d, e) revealed significant dose-response decrease in proliferation of HTR3A^high neuroblastoma cells after 5 days of treatment with either N-methylquipazine dimaleate (NMQ, d) or SR57227 (e); treatments with HTR3A antagonists (f, g) did not exert a significant effect on cell proliferation. Data presented as mean ± SD, biological n = 3–5, technical n = 5. h Schematic depiction of limiting dilution sphere formation assay: neuroblastoma cells were pretreated with 75 µM NMQ or vehicle (DMSO) for 5 days and serially diluted in fresh serum-free media w/o the drugs at indicated numbers per well. i The frequencies of sphere-forming cells significantly differed between HTR3A^high and HTR3A^low cell lines, while NMQ pretreatment did not reduced sphere formation capacity of the tested cells. Data are shown as mean ± 95% confidence interval, frequencies, and probability were computed using ELDA software⁷⁹. ***p < 0.01, χ² pairwise test. j–l Only HTR3A^high neuroblastoma cell lines formed xenograft tumors in NOD/SCID gamma (NSG) mice (k). The higher levels of HTR3A expression in SH-SY5Y cells corresponded to the earlier onset of tumor formation (j) and increased tumor growth (l) when compared with CHLA-20 cells; ***p < 0.01 using one-way ANOVA followed by Tukey’s post hoc test.