The Bs →μμ\to \mu\mu decay, LFV and LFU at the LHCb experiment

<div><p>Decreased cell-substratum adhesion is crucially involved in metastasis. Previous studies demonstrated that lung cancer with floating cell clusters in histology is more likely to develop metastasis. In the present study, we investigated whether cancer cells in long-term, three-dimensional low attachment cultures acquire high metastatic potential; these cells were then used to examine the mechanisms underlying metastasis. Two KRAS-mutated adenocarcinoma cell lines (A549 and H441) were cultured and selected on ultra-low attachment culture dishes, and the resulting cells were defined as FL (for floating) sublines. Cancer cells were inoculated into NOD/SCID mice via an intracardiac injection, and metastasis was evaluated using luciferase-based imaging and histopathology. <i>In vitro</i> cell growth (in attachment or suspension cultures), migration, and invasion were assayed. A whole genomic analysis was performed to identify key molecular alterations in FL sublines. Upon detachment on low-binding dishes, parental cells initially formed rounded spheroids with limited growth activity. However, over time in cultures, cells gradually formed smaller spheroids that grew slowly, and, after 3–4 months, we obtained FL sublines that regained prominent growth potential in suspension cultures. On ordinary dishes, FL cells reattached and exhibited a more spindle-shaped morphology than parental cells. No marked differences were observed in cell growth with attachment, migration, or invasion between FL sublines and parental cell lines; however, FL cells exhibited markedly increased growth potential under suspended conditions <i>in vitro</i> and stronger metastatic abilities <i>in vivo</i>. A genomic analysis identified epithelial-mesenchymal transition (EMT) and c-Myc amplification in A549-FL and H441-FL cells, respectively, as candidate mechanisms for metastasis. The growth potential of FL cells was markedly inhibited by lentiviral ZEB1 knockdown in A549-FL cells and by the inhibition of c-Myc through lentiviral knockdown or the pharmacological inhibitor JQ1 in H441-FL cells. Long-term three-dimensional low attachment cultures may become a useful method for investigating the mechanisms underlying metastasis mediated by decreased cell-substratum adhesion.</p></div

Zinc finger E-box binding homeobox 1 (ZEB1) was induced in A549-FL cells and its knockdown (KD) led to apoptosis and the inhibition of cell growth in A549-FL cells.

<p>(A) Parental cells under detached conditions (3 days after detachment) and FL cells in a continued detachment (low attachment) culture were lysed and subjected to a Western blot analysis with antibodies to E-cadherin, vimentin, ZEB1, and β-actin. A549 cells in an attachment culture were included for comparison. Long-term low attachment (detached) cultures caused the slightly weaker expression of E-cadherin and stronger expression of vimentin and ZEB1 in A549-FL cells than in A549 cells. (B-D) A549-FL cells were transduced with lentiviral vectors (shZEB1-A, shZEB1-B, and shScramble), and after a brief selection with puromycin, used for a Western blot analysis, colony formation assay, and cell count assay, without cloning (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181342#sec002" target="_blank">Materials and methods</a> for details). (B) ZEB1 KD induced apoptosis in A549-FL cells, as indicated by the emergence of the cleaved PARP fragment. Number below gel images represent the relative protein levels of the indicated proteins after normalization to β-actin. Data shown are representative of 2 independent experiments. (C) Representative images of macroscopic colony formation assay of 4 replicates of cultures (Giemsa stain). Colony formation was inhibited more by shZEB1-A and shZEB1-B than by the control (shScramble). (D) Cell growth was inhibited more by shZEB1-A and shZEB1-B than by the control (shScramble) in attachment and detachment cultures. Data are shown as the box-and-whisker plot of 10 replicates. Statistical analysis was performed by the Kruskal-Wallis test followed by Tukey's test.</p

Transwell migration and invasion assays.

<p>(A, B) Transwell migration assay. Representative microscopic images of cells that migrated through the transwell in the migration assay. (Giemsa stain, magnification ×200). No significant differences were observed between the FL subline and parental cell line for A549 or H441 cells. The quantitation of cells that migrated through the transwell in the migration assay. The data are shown as the box-and-whisker plot of the mean number of migrated cells per visual field (A) or 3 visual field (B) (magnification ×100) of 4 replicate wells. No significant differences were found between the FL subline and parental cell line for A549 cells or for H441 cells. (C, D) Transwell Matrigel invasion assay. Representative microscopic images of cells that invaded through the transwell in the Matrigel invasion assay. (Giemsa stain, magnification ×200). No significant differences were observed between the FL subline and parental cell line for A549 or H441 cells. Quantitation of cells that invaded through the transwell in the Matrigel invasion assay. The data are shown as the box-and-whisker plot of the mean number of cells per visual field (C) or 3 visual field (D) (magnification ×100) of 4 replicate wells. The results obtained were similar between A549 and A549-FL cells. Invasion was slightly weaker in H441-FL cells than in H441 cells. Statistical analysis was performed by the Mann-Whitney U test.</p

Metastatic tumor formation after an intracardiac injection of parental and FL cells.

<p>(A) Metastatic tumors were formed in multiple organs in all mice injected with parental or FL cells. Representative macroscopic and microscopic pictures are shown of lung (white arrow), adrenal (white arrow head), and brain metastases of luc-A549 cell lines. (B) <i>In vivo</i> luciferase imaging of luc-A549 cell lines and luc-A549-FL sublines at 5 weeks post-implantation. Representative images from ventral view are shown. Quantification of tumor-derived photons at 5 weeks post-implantation. Data are shown as the mean ± standard deviation of the photon flux of 5 animals. The Luc-A549-FL subline showed markedly greater metastatic tumor growth than luc-A549 cells. (C) <i>In vivo</i> luciferase imaging of luc-H441 cell lines and luc-H441-FL sublines at 6 weeks post-implantation. Representative images from dorsal view are shown. Quantification of tumor-derived photons at 6 weeks post-implantation. No significant differences were observed in tumor-derived photons between luc-H441 and luc-H441-FL cells. Data are shown as the box-and-whisker plot of the photon flux of 5 animals. Statistical analysis was performed by the Mann-Whitney U test.</p

Significantly amplified gene lesions with gene names in a copy number analysis of the NCI-H441-FL subline (versus the NCI-H441 cell line).

<p>Significantly amplified gene lesions with gene names in a copy number analysis of the NCI-H441-FL subline (versus the NCI-H441 cell line).</p

Levels of apoptosis and cell cycle regulators in parental and FL cells in detachment.

<p>(A) Parental and FL cells in low attachment cultures were lysed and subjected to a Western blot analysis with antibodies to poly ADP-ribose polymerase (PARP), cleaved PARP, and β-actin. Parental cells in attachment cultures were included for comparison. Under detached conditions, apoptosis was greater in FL cells than in parental cells. * in PARP indicates the cleaved fragment of PARP. (B) Parental and FL cells in low attachment cultures were lysed and subjected to a Western blot analysis with antibodies to cyclin A2, B1, D1, E1, p27, and β-actin. While the levels of cyclin A2, B1, D1, and E1 were similar between parental A549 and A549-FL cells, p27 levels were markedly lower in A549-FL cells than in parental A549 cells. Similarly, H441-FL cells showed reduced p27 levels. Number below gel images represent the relative protein levels of the indicated proteins after normalization to β-actin. Data shown are representative of 2 independent experiments.</p

FL sublines exhibit greater cell growth potential than parental cells on low-binding cultures.

<p>(A) The A549 cell line and A549-FL subline grew at a similar rate in attachment cultures. The A549-FL subline grew at a markedly higher rate than the A549 cell line in low-attachment (detached) cultures (p < 0.05). The H441-FL subline showed a slightly higher cell growth rate than the H441 cell line in attachment cultures (p < 0.05). The H441-FL subline showed a markedly higher cell growth rate than the H441 cell line in detachment cultures (p < 0.05). Data are shown as the box-and-whisker plot (minimum, lower quartile, median, upper quartile and maximum) of 5 replicates of cultures. Statistical analysis was performed by the Mann-Whitney U test. (B) The A549-FL subline showed a markedly larger fold increase in DNA than the A549 cell line in detachment cultures (p < 0.05). The H441-FL subline also showed a markedly larger increase in DNA than the H441 cell line in detachment cultures (p < 0.05). Note that H441 cells actually showed a slight decrease in DNA during the 7 days in a detachment culture. Data are shown as the box-and-whisker plot of the ratio (day 7/day 0) of the amount of DNA extracted from 4 samples of replicates of cultures. Statistical analysis was performed by the Mann-Whitney U test.</p

Score and criteria in the semi-quantitative evaluation of murine metastasis models.

<p>Score and criteria in the semi-quantitative evaluation of murine metastasis models.</p