A 549 lung carcinoma at day 28, hematoxylin-eosin stained cross section, scale bar 500 μm

<p><b>Copyright information:</b></p><p>Taken from "The selective Cox-2 inhibitor Celecoxib suppresses angiogenesis and growth of secondary bone tumors: An intravital microscopy study in mice"</p><p>BMC Cancer 2006;6():9-9.</p><p>Published online 12 Jan 2006</p><p>PMCID:PMC1360103.</p><p>Copyright © 2006 Klenke et al; licensee BioMed Central Ltd.</p> Tumor borders marked by arrows. Control (A), Celecoxib (B). : A 549 lung carcinoma at day 28, TUNEL reaction, scale bar 50 μm, apoptotic cells stained red. Control (C), Celecoxib (D)

Quantifying the spontaneous intercellular exchange rates of organelles in epithelial cells.

<p>Qtracker 605 and 525 labeled cells were co-cultured for 24 h and analyzed by flow cytometry (A). FACS analysis revealed 15.8% “double +” cells. To overcome the limitation of FACS analysis to detect and discriminate double positive cells with a low number of transfered Qdots by the tubes, we employed high-throughput fluorescence image analysis using the ImageStream™ platform (B–D). By analyzing 9335 co-cultured RPTEC, spontaneous intercellular exchange was detected in 67.5% or 6305 cells (B–C). Inhibition of tube-genesis by Lat. B resulted in marked (62%) reduction of the intercellular exchange (from 67.5% to 25.9% double positive cells). Detailed analysis revealed that the intercellular exchange resulted in three categories of double positive cells: i) transfer of 525 Qtracker to 605 Qtracker labeled cells (46.30% or 2921 cells), ii) transfer of 605 Qtracker to 525 Qtracker labeled cells (48.3% or 3046 cells) or equal content of both Qtrackers (4.57% or 288 cells) (D). Thus, the transfer of few labeled Qdots organelles contributed to the majority of spontaneous intercellular exchange among RPTEC.</p

TNT-like tubes connecting epithelial cells.

<p>Representative phase contrast images of the identified TNT-like tubes connecting HMECs. These membranous structures were able to span long distances of several micrometers between the cells (A–D). The observed “gondola”-like structures, i.e., localized changes in tube diameter (white arrows in (A)), indicated potential transfer of cargos in the lumen of the tubes. Tubes with blind ends were also found (white arrow in (B)). In contrast to TNTs the identified membranous tubes often build cross roads bridging several cells with each other (C). A frequent observation was the connection of epithelial cell “islands” via these tubes (C and D). All photomicrographs x100 view. <b>Enhanced tube-genesis followed by cellular stress</b> (E and F). Treatment of RPTEC with the glycopeptide antibiotic <i>Zeocin</i> led to a marked increase in the number of tubes in a dose dependent manner. The maximum induction of tubes was reached at 400 µg/ml dose of <i>Zeocin</i>. At higher concentrations (e.g. 1000 µg/ml) the toxicity and cell death effects of the treatment dominated the phenotype resulting in a decline of the number of tubes and vital cells per optical field. Bars represent means ± <i>SD</i> from quadruplicates measurements (p<0.01 for 50–400 µg/ml <i>Zeocin</i> treatment vs. control).</p

Intercellular exchange of Mitochondria.

<p>To detect potential exchange of other organelles such as mitochondria between RPTECs, cells were labeled with MitoTracker® reagent which specifically labels active mitochondria <i>in-vivo</i> (green). MitoTracker labeled cells were co-cultured for 24 h together with Qtracker 605 (red) labeled cells. Mitochondria could be detected in the lumen of the TNT-like tubes in RPTEC (A). The exchange of organelles between RPTEC is shown (highlighted by white arrows). Q605 labeled cells (red arrow). MitoTracker labeled cells (green arrow). Nuclear staining (blue, To-Pro3).</p

Tracing intercellular exchange by Qdot® nanocrystals.

<p>(<b>A–C</b>)<b>,</b> RPTEC were labeled with either Qtracker 605 (red) or 525 (green) and co-cultured for 24 h. To-Pro-3 was used to stain the cell nucleus (DNA, blue). Fluorescence microscopy revealed a number of “double positive” cells suggesting spontaneous intercellular exchange of cytoplasmic material between the RPTECs. Fluorescence particles were also detected in the lumen of the tubular structures (arrows) indicating endoluminal transport. Among the specific morphological features of RPTEC tubes as compared to classical TNTs are their larger caliber, their ability to bridge very long distances (>200 µm) and the more frequent appearance of bifurcations or connections of multiple cells (A and B). Occasionally To-Pro-3 positive DNA-signal (blue staining) was detected in the lumen of the tubes (C) suggesting the possibility of the exchange of genetic material between RPTEC.</p

Prospective Identification of Glioblastoma Cells Generating Dormant Tumors

<div><p>Although dormant tumors are highly prevalent within the human population, the underlying mechanisms are still mostly unknown. We have previously identified the consensus gene expression pattern of dormant tumors. Here, we show that this gene expression signature could be used for the isolation and identification of clones which generate dormant tumors. We established single cell-derived clones from the aggressive tumor-generating U-87 MG human glioblastoma cell line. Based only on the expression pattern of genes which were previously shown to be associated with tumor dormancy, we identified clones which generate dormant tumors. We show that very high expression levels of thrombospondin and high expression levels of angiomotin and insulin-like growth factor binding protein 5 (IGFBP5), together with low levels of endothelial specific marker (ESM) 1 and epithelial growth factor receptor (EGFR) characterize the clone which generates dormant U-87 MG derived glioblastomas. These tumors remained indolent both in subcutaneous and orthotopic intracranial sites, in spite of a high prevalence of proliferating cells. We further show that tumor cells which form U-87 MG derived dormant tumors have an impaired angiogenesis potential both <em>in vitro</em> and <em>in vivo</em> and have a slower invasion capacity. This work demonstrates that fast-growing tumors contain tumor cells that when isolated will form dormant tumors and serves as a proof-of-concept for the use of transcriptome profiles in the identification of such cells. Isolating the tumor cells that form dormant tumors will facilitate understanding of the underlying mechanisms of dormant micro-metastases, late recurrence, and changes in rate of tumor progression.</p> </div

Consensus Micro RNAs Governing the Switch of Dormant Tumors to the Fast-Growing Angiogenic Phenotype

<div><p>Tumor dormancy refers to a critical stage in cancer development in which tumor cells remain occult for a prolonged period of time until they eventually progress and become clinically apparent. We previously showed that the switch of dormant tumors to fast-growth is angiogenesis dependent and requires a stable transcriptional reprogramming in tumor cells. Considering microRNAs (miRs) as master regulators of transcriptome, we sought to investigate their role in the control of tumor dormancy. We report here the identification of a consensus set of 19 miRs that govern the phenotypic switch of human dormant breast carcinoma, glioblastoma, osteosarcoma, and liposarcoma tumors to fast-growth. Loss of expression of dormancy-associated miRs (DmiRs, 16/19) was the prevailing regulation pattern correlating with the switch of dormant tumors to fast-growth. The expression pattern of two DmiRs (miR-580 and 190) was confirmed to correlate with disease stage in human glioma specimens. Reconstitution of a single DmiR (miR-580, 588 or 190) led to phenotypic reversal of fast-growing angiogenic tumors towards prolonged tumor dormancy. Of note, 60% of angiogenic glioblastoma and 100% of angiogenic osteosarcoma over-expressing miR190 remained dormant during the entire observation period of ∼ 120 days. Next, the ability of DmiRs to regulate angiogenesis and dormancy-associated genes was evaluated. Transcriptional reprogramming of tumors via DmiR-580, 588 or 190 over-expression resulted in downregulation of pro-angiogenic factors such as TIMP-3, bFGF and TGFalpha. In addition, a G-CSF independent downregulation of Bv8 was found as a common target of all three DmiRs and correlated with decreased tumor recruitment of bone marrow-derived CD11b+ Gr-1+ myeloid cells. In contrast, antiangiogenic and dormancy promoting pathways such as EphA5 and Angiomotin were upregulated in DmiR over-expressing tumors. This work suggests novel means to reverse the malignant tumor phenotype into an asymptomatic dormant state and may provide promising targets for early detection or prevention of cancer.</p> </div

<i>In vitro</i> cellular characteristics of U-87 MG and Clone #1 cells.

<p><b>A</b>. Representative images of cells in culture showing similar morphology. <b>B. </b><i>In vitro</i> proliferation assay: Clone #1 (black line) and U-87 MG (red line) human glioblastoma cell lines grow at a similar rate <i>in vitro. </i><b>C.</b> Clone #1 (black bars) and U-87 MG (red bars) cells migrate similarly toward serum-free media (striped bars), towards serum-containing media (solid bars) and following 12 h serum starvation (serum-free) conditions (dotted bars). <b>D.</b> Transendothelial migration<b>:</b> U-87 MG cells (red bar) migrate at a significantly higher rate (<i>p</i> = 1.9×10⁻¹¹) compared with Clone #1 cells (black bar) towards serum-free media (right panel describes the quantification of representative images on the left panel). Data represent mean± s.d. from three independent experiments. NS = non-significant, <i>*** p<0.01</i>.</p

Comparison of size-matched tumors generated from U-87 MG and Clone #1 cells.

<p><b>A.</b> Sixteen days post subcutaneous inoculation of mCherry labeled U-87 MG and Clone #1 cells, both tumor types were approximately 2 mm³ in diameter and detectable by non-invasive CRI Maestro™ imaging system (<b>upper panel</b>). Flipped skin of tumor-bearing mice revealed highly vascularized U-87 MG-generated tumors, while blood vessels were only detectable in the surrounding skin of Clone #1-generated tumors (<b>lower panel</b>). <b>B.</b> H&E, CD34 (merged image. The separate images are provided as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044395#pone.0044395.s002" target="_blank">Fig. S2</a>) and TSP-1 staining of U-87 MG and Clone #1 tumor sections. TSP-1 staining was done on size-matched tumors from day 16 (2 mm³) and on large tumors (U-87 MG tumors at end point of experiment and Clone #1 tumors after escape from dormancy) (1800 mm³). <b>C.</b> Contrast-enhanced US imaging of U-87 MG and Clone #1 subcutaneous tumors show high vascularization of the U-87 MG fast-growing tumor (red bar, n = 5) compared with Clone #1 dormant tumors (black bar, n = 3) (<i>p</i> = 0.008). Data represent mean ± s.d. <i>*** p<0.01</i>.</p

MiR-190 expression in osteosarcoma model.

<p>Tumor growth kinetic of vector control (GFP; n = 5) vs. miR-190 (n = 5) expressing angiogenic fast-growing “A-Osteosarcoma” (A). MiR-190 expression led to complete (i.e., 100% inhibition, 5/5) phenotypic reversal of fast-growing osteosarcoma resulting in significant increase of overall survival (p<0.005 by log-rank test) (B). Death event: mouse sacrificed based on large tumor size.</p