1st Studio Siberian Mouse 182 !EXCLUSIVE!

1st Studio Siberian Mouse 182 !EXCLUSIVE!

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To investigate the possible action of 5HT on HTR3A in the progression of tumors originating from sympathoadrenal cells, we analyzed several clones of human-derived neuroblastoma for HTR3A expression and tumorigenicity using an immunodeficient mouse model. Based on mRNA (Fig. 6a) and protein expression levels (Fig. 6b, c), the examined neuroblastoma cell lines could be characterized as either HTR3Ahigh (SH-SY5Y, CHLA-15, and CHLA-20), expressing markedly high levels of HTR3A, or HTR3Alow (NBL-28, NBL-38, and NBL-40), with only weak HTR3A expression. While all cell lines were negative for MYCN amplification (two copies of gene in the genome), HTR3A protein expression was associated with expression of major drivers of aggressive neuroblastomas, N-MYC and c-MYC44,45, or one of the core stemness factors SOX2 (Fig. 6b). Intriguingly, the same association was observed in NTERA-2 embryonal pluripotent carcinoma cells, which served as a positive control for HTR3A expression, and which are known to express high levels of N-MYC and share characteristics with early neural progenitors. To investigate the possible role of HTR3A receptor in regulation of proliferation in tumor cells, we treated the cells with the HTR3A agonists, N-methylquipazine dimaleate (NMQ) and SR57277, as well as antagonists, VUF10166 and granisetron HCl, in the presence of 5HT. HTR3A agonists dramatically limited proliferation of HTR3Ahigh cell lines, whereas they did not affect HTR3Alow cell lines, or the effects were seen only at much higher doses (Fig. 6d, e). No cleaved caspase-3 was detected after the treatment with NMQ, indicating that such treatment does not induce apoptosis (Supplementary Fig. 9). In contrast, there was no significant effect of HTR3A antagonists on cell growth of both HTR3Ahigh SH-SY5Y and HTR3Alow NBL-28 cell lines (Fig. 6f, g).

NOD/ShiLtSz-scid/Il2rγnull mice were used as a NSG model. All NSG mouse experiments were conducted in accordance with a study (21379/2011-30) approved by the Institutional Animal Care and Use Committee of Masaryk University and registered by the Ministry of Agriculture of the Czech Republic as required by national legislation.

We re-analyzed single-cell RNA-seq data of mouse adrenal gland from Furlan et al.6. Gene counts were obtained from GEO database (GSE99933). Gene count matrix was analyzed with a standard Seurat (version 3.0.2) workflow88. We used the original embeddings and clustering from ref. 6 (Figs. 5B and 5G), downloaded from the published pagoda apps: -bin/R/rook/nc.SS2_16_249-2/pathcl.json and -bin/R/rook/nc.SS2_16_250-2/pathcl.json (json slots embedding/data for the embedding and colcols/clusters/data for the cluster labels).

The raw and processed data of single-cell transcriptomic experiments generated in this study have been deposited in the GEO database under accession codes: GSE180861 (mouse), GSE195929 (human). The single-cell RNA-seq data of mouse adrenal gland from Furlan et al. (2017) used in this study are available in the GEO database under accession code GSE99933, mm10_ERCC genome used in this study is available in the RefSeq database under accession code GCF_000001635.20. The data other than RNA-seq data generated in this study are provided in the Source data file. 2b1af7f3a8