LACSLCs derived from A549 cells exhibit highly invasive and migratory capabilities.

<p>(<b>A</b>) Morphology of sphere-LACSLCs derived from lung cancer A549 cell line. Representative images from light microscopy (monolayer cells 10×, LACSLCs 20×) are shown. (B) Wound healing assay for cell migration of A549 LACSLCs and A549 monolayer cells. Representative images of cell migration into the wounded area at 0 and 16 hours post-injury (<i>left panel</i>) are shown. Quantitative analysis shows wound repair capability of migrating cells at 16 h post-injury (<i>right panel</i>). (<b>C</b>) Comparison of invasive capability in A549 LACSLCs and monolayer cells detected by transwell assay. Representative images of invading cells visualized by crystal violet staining (20×) (<i>left panel</i>). Quantitative analysis of cell invading capacity at 24 h after seeding (<i>right panel</i>). (<b>D</b>) Histological images by H&E staining under light microscopy. Tumors formed by A549 LACSLCs were highly invasive and the tumor cells invaded neighboring muscle layer (red arrows; 20×). All experiments were carried out at least in triplicates and the data are presented as the mean ± SEM. Student <i>t</i> test was performed to evaluate the difference.</p

Examination of POU5F1 and MMP-2 by immunostaining and Western blot analyses.

<p>(<b>A</b>) Immunostaining of POU5F1 and MMP-2 observed by confocal scanning microscopy. (<b>B</b>) Expression of stem cell transcription factor POU5F1 was detected by Western blot. (<b>C</b>) Immunohistochemical analysis of MMP-2 protein in tumor tissues formed by A549 LACSLCs and monolayer cells. Representative images of MMP-2 expression by IHC staining (20×) (<i>left panel</i>). Quantitative analysis of MMP-2 protein levels in tumor tissues formed by A549 LACSLCs and monolayer cells. All experiments were carried out at least in triplicates and the data are presented as the mean ± SEM. Student <i>t</i> test was performed to evaluate the difference.</p

The expression of POU5F1 and MMP-2 in 55 cases of lung adenocarcinoma.

<p>^a Mean age.</p

Invasive U87 sphere cells express CD133.

<p>A. U87 sphere cells with various invasion capability within zebrafish embryos. The extent of invasion was classified in three degrees: Low: less than 5 migrated cells; Medium: 5–20 migrated cells; High: more than 20 migrated cells. Representative images at higher magnification show the invasive RFP-labeled U87 sphere cell masses (red) in the tail region of the embryos <i>via</i> EGFP-labeled host vessels (green). B. Detection of CD133 expression on non-invasive and invasive U87 sphere cells at 2 dpi by immunofluorecent staining. All of U87 sphere cells within injected embryos were stained with monoclonal anti-CD133 antibody (1∶300) and examined by confocal microscopy. Green: Tg (<i>fli1</i>:EGFP)^y1 microvessels; red: RFP-labeled U87 sphere cells; blue: CD133 positive U87 cells. C. Quantitative analysis of CD133-expressing cells in non-invasive cell group (n = 713) and high-invasive cell group (n = 175) at 2 dpi. (<i>p</i><0.001).</p

The establishment of U87 glioma sphere cell invasion model in zebrafish embryos.

<p>A. Dual color confocal image shows that U87 sphere cells (RFP labeled, red) were microinjected into the middle of yolk <i>sac</i> within Tg (<i>fli1</i>:EGFP)^y1 transgenic zebrafish embryos (EGFP labeled, green). B. Different numbers of U87-RFP glioma sphere cells were microinjected into Tg (<i>fli1</i>:EGFP)^y1 embryos (n = 300 in each group), and the percentage of embryos with invasive tumor cells was quantitated. C. The survival rate of Tg (<i>fli1</i>:EGFP)^y1 zebrafish embryos microinjected with different numbers of U87-RFP glioma sphere cells (n = 300 in each group). D. Representative dual color confocal images of RFP-labeled U87 sphere cells within Tg (<i>fli1</i>:EGFP)^y1 zebrafish embryos at the different invasive stages. Red: RFP-labeled U87 sphere cells; Green: Tg (<i>fli1</i>:EGFP)^y1 microvessels.</p

MMP-9 mediates invasion and spread of CD133⁺ U87 GSCs in zebrafish embryos.

<p>A. The MMP-2 and MMP-9 RNA in CD133⁻ U87 cells and CD133⁺ U87 GSCs were examined by qRT-PCR. B. The MMP-2 and MMP-9 proteins in CD133⁻ U87 cells and CD133⁺ U87 GSCs examined by Western blot. C. The inhibitory effect of MMP-9 inhibitor (AG-L-66085) on the invasion of CD133⁺ U87 GSCs within zebrafish embryos. The embryos xenografted with CD133⁺ U87 GSCs were treated with 2 µM AG-L-66085 or DMSO control. The percentages of invasive cells in injected embryos (low, medium, or high-invasion) were measured at 2 dpi. The data were obtained from three replicate experiments with the number of embryos: n = 123 for DMSO control group, n = 119 for MMP-9 inhibitor group, and n = 144 for negative control group (<i>p</i><0.001).</p

Quantitation of invading tumor cells within zebrafish embryos injected with U87 cells.

<p>U87 sphere cells and U87 CD133⁺ GSCs. The data were obtained from three replicate experiments of 50 injected embryos for each experiment.</p

CD133+ U87 GSCs are highly invasive within zebrafish embryos.

<p>A. Representative images of the invasion of differentiated U87 cells, U87 sphere cells, and CD133+ U87 GSCs within the injected embryos at 2 dpi. The images at higher magnification show the invasive RFP-labeled cell masses at tail region of embryos <i>via</i> host vessels. B. The percentage of the embryos with invasive cells injected with RFP-labeled differentiated U87 cells, U87 sphere cells, and CD133⁺ U87 GSCs. The data were obtained from three replicate experiments of 50 injected embryos in each experiment: n = 124 for live embryos injected with differentiated U87 cells, n = 121 for embryos injected with U87 sphere cells, and n = 120 for embryos injected with CD133⁺ cells C. The percentage of invasive cells within total injected cells (Invasion Index) in the embryos. All injected cells including invasive or non-invasive cells within zebrafish embryos were evaluated by ImageJ software through fluorescence intensity. n = 37200 (300 injected cells per embryo among 124 live embryos) for differentiated U87 cell group, n = 36300 (300 injected cells per embryo among 121 live embryos) for U87 sphere cell group, and n = 36000 (300 cells per embryo among 120 live embryos) for CD133⁺ U87 GSCs group (<i>p</i><0.001).</p

A Novel Zebrafish Xenotransplantation Model for Study of Glioma Stem Cell Invasion

<div><p>Invasion and metastasis of solid tumors are the major causes of death in cancer patients. Cancer stem cells (CSCs) constitute a small fraction of tumor cell population, but play a critical role in tumor invasion and metastasis. The xenograft of tumor cells in immunodeficient mice is one of commonly used <i>in vivo</i> models to study the invasion and metastasis of cancer cells. However, this model is time-consuming and labor intensive. Zebrafish (<i>Danio rerio</i>) and their transparent embryos are emerging as a promising xenograft tumor model system for studies of tumor invasion. In this study, we established a tumor invasion model by using zebrafish embryo xenografted with human glioblastoma cell line U87 and its derived cancer stem cells (CSCs). We found that CSCs-enriched from U87 cells spreaded <i>via</i> the vessels within zebrafish embryos and such cells displayed an extremely high level of invasiveness which was associated with the up-regulated MMP-9 by CSCs. The invasion of glioma CSCs (GSCs) in zebrafish embryos was markedly inhibited by an MMP-9 inhibitor. Thus, our zebrafish embryo model is considered a cost-effective approach tostudies of the mechanisms underlying the invasion of CSCs and suitable for high-throughput screening of novel anti-tumor invasion/metastasis agents.</p></div

Additional file 1: of Cripto-1 acts as a functional marker of cancer stem-like cells and predicts prognosis of the patients in esophageal squamous cell carcinoma

Supplementary materials and methods, including Quantitative RT-PCR, Western blotting, Flow cytometry, Silencing CR-1 with shRNA, and Immunofluorescence staining. Table S1. Primer sequences for qRT-PCR assay. Table S2. shRNA sequences targeting CR-1. Table S3. Incidence of tumor formation in nude mice injected with ESCC cells. Table S4. The frequency of lung metastasis (1x104 cells/mouse). Table S5. Significant difference of CR-1 expression between carcinoma and adjacent normal tissues. Figure S1. The levels of CR-1expression and the silencing efficiency of CR-1 shRNA in ESCC cells. Figure S2. Silencing CR-1 expression significantly represses the self-renewal and tumorigenicity inTE-1 cells. Figure S3. Suppression of CR-1 expression inhibits the invasive and metastatic capabilities of TE-1 cells in vitro and in vivo. Figure S4. The effect of silencing CR-1 on the expression of MMPs in EC109 cells. (DOC 1602 kb