Cancer stem cells resemble normal tissue-specific stem cells in many aspects, such as self-renewal and plasticity. Like their non-malignant counterparts, cancer stem cells are suggested to exhibit a relative quiescence. The established cancer cell lines reportedly harbor slow-proliferating cells that are positive for some cancer stem cells markers. However, the fate of these cells and their progeny remains unknown. We used time-lapse microscopy and the contrast-based segmentation algorithm to identify and monitor actively dividing and non-dividing cells in human osteosarcoma MG-63 cell line. Within the monitored field of view the non-dividing cells were represented by three cells that never divided, and one cell that attempted to divide, but failed cytokinesis, and later, after significantly prolonged division, produced the progeny with enlarged segmented nuclei, thus pointing to a possible mitotic catastrophe. Together, these cells initially constituted about 6.2% of the total number of seeded cells, yet only 0.02% of all cells at the end of the observation period when cells became confluent. Non-dividing cells were characterized by rounded shape, dark nuclei, random cytoplasmic streaming and subtle oscillatory movement, however, they did not migrate and rarely formed cell-cell contacts as compared to actively dividing cells. Our data indicate that the observed non-dividing MG-63 cells do not have a growth advantage over other cells and, therefore, they do not contribute to the cancer stem cells pool.

* equally contributed Grant #5P20GM103443 (NIGMS). Single nucleotide polymorphisms (SNPs) can either create or destroy microRNA binding sites. We analyzed 200 previously reported cancer associated SNPs within microRNA binding sites and found that more than 90% of them were surrounded by single and multiple low-frequency SNPs. The low-frequency SNPs were positioned within the expected microRNAs seed matching areas (58%), within the whole microRNA matching regions (71% incidence), and within the distance where they potentially can affect microRNA-mRNA interaction (36% incidence). We further analyzed the presence of SNPs within microRNA-binding sites in the 3'UTRs of mRNAs encoding the human V-set domain containing T-cell activation inhibitor 1 (VTCN1) and an AT-rich interaction domain 5B (ARID5B). In VTCN1 single SNPs were present in 36% of microRNAs seed matching areas, and two and more SNPs in 8% of microRNA seed matching sites. For ARID5B, single SNPs were present in 42%, and two and more SNPs in 26% of microRNA seed matching sites. In both VTCN1 and ARID5B, some microRNA seed matching areas harbored as many as 4 SNPs. The predicted binding site for microRNA-6870-5p (miR-6870) within the VTCN1 3'UTR consists of 11 uninterrupted and 2 additional Watson-Crick pairs. The length of mature miR-6870 is 19 bases, and the corresponding VTCN1 mRNA fragment harbors 6 nucleotide variations: rs758251859, rs1001277215, rs551576201, rs539444165, rs949692788, and indel rs9055595515. Hypothetically, the rs1001277215 minor allele (MA) eliminates the miR-6870-mRNA complementarity between the corresponding nucleotides. The rs551576201 MA weakens miR-6870-mRNA complementarity by creating a wobble G-U pair, the rs949692788 MA enhances miR-6870-mRNA complementarity by switching from a non-canonical G-U to canonical Watson-Crick A-U pair, and for indel rs9055595515, the absence of deletion preserves miR-6870-mRNA complementarity between CA and GU nucleotides. The probabilities of the “best” and “worst” matches between miR-6870 and VTCN1 mRNA are 0.009995% and 0.000006% correspondingly. The binding between miR-6870 and VTCN1mRNA may also be affected by variations between A and G within rs758251859 (MA frequency is unknown). As microRNA-binding efficacy depends on the mRNA sequences outside the target region, the indels rs35182629 (located 5 nucleotides upstream and covers 2 bases) and rs896747700 (located 1 nucleotide downstream and covers 1 base) may also impact miR-6870-mediated regulation of VTCN1 expression. The possible coincidence of hyper-functional or completely disabled microRNA-binding sites may result in significant phenotypic variations and predisposition to cancers. Citation Format: Amber Budmark*, Michael Catalano*, Tyrel Haley*, Brady Hicks*, Maria Koenen*, Thea Patrick*, Tyler Larson*, Tyler Wagner*, Clark Butler, Joshua Feiner, Rebecca Frick, Sierra Haage, James Miller, Mackayla Nohr, Dillon Stadlman, Dillon Turner, Sara Husher, Nicholas Woslum, Nathan Stadem, John Dosch, Tyler Fortuna, Chandler Fredrich, Elise Hadley, Brooklynn Oehlerking, Delayna Paulson, Cal Wiese, Paula Mazzer, Tim Mullican, Cynthia Anderson, Mark Larson, Elena Paryiskaya, Alexandra Kharazova, Paola Vermeer, Samuel Milanovich, Alexei Savinov, John Collins, Alexander Kofman. Single nucleotide variations within and around microRNA-binding sites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 490.