Additional file 6: of Metabolic signatures differentiate ovarian from colon cancer cell lines

Supplemental Figure 3. Dipeptides were significantly upregulated in ovarian cancer cells compared with colon cancer cells. The log-scaled metabolite intensities are presented as box plots representing the median values of experiments performed in 10 (HCT15, HTC116, OVCAR3) and 8 (SKOV3) replicates. Values were obtained after statistical data analysis using the metaP server

Additional file 1: of Metabolic signatures differentiate ovarian from colon cancer cell lines

Supplemental Figure 1. Identification of outliers using PCA. PCA score plots generated by the metaP server reveal distinct clustering of HCT15 (dark blue), HCT116 (light blue), OVCAR3 (red), and SKOV3 (orange). Two sample outliers were identified in the SKOV3 cell line and were removed from the analysis

Additional file 5: of Metabolic signatures differentiate ovarian from colon cancer cell lines

Supplemental Table 3. Metabolites that significantly distinguish HCT116 from HCT15

Using Runtime Traces to Improve Documentation and Unit Test Authoring for Dynamic Languages

Supplemental Figure 3. Dipeptides were significantly upregulated in ovarian cancer cells compared with colon cancer cells. The log-scaled metabolite intensities are presented as box plots representing the median values of experiments performed in 10 (HCT15, HTC116, OVCAR3) and 8 (SKOV3) replicates. Values were obtained after statistical data analysis using the metaP server

Additional file 2: of Metabolic signatures differentiate ovarian from colon cancer cell lines

Supplemental Table 1. Metabolites present in all examined cell lines. Star (*) indicates compounds that have not been officially “plexed” (based on a standard), although we are confident in their identity

Endothelial Cells Provide a Notch-Dependent Pro-Tumoral Niche for Enhancing Breast Cancer Survival, Stemness and Pro-Metastatic Properties

<div><p>Treating metastasis has been challenging due to tumors complexity and heterogeneity. This complexity is partly related to the crosstalk between tumor and its microenvironment. Endothelial cells -the building blocks of tumor vasculature- have been shown to have additional roles in cancer progression than angiogenesis and supplying oxygen and nutrients. Here, we show an alternative role for endothelial cells in supporting breast cancer growth and spreading independent of their vascular functions. Using endothelial cells and breast cancer cell lines MDA-MB231 and MCF-7, we developed co-culture systems to study the influence of tumor endothelium on breast tumor development by both <i>in vitro</i> and <i>in vivo</i> approaches. Our results demonstrated that endothelial cells conferred survival advantage to tumor cells under complete starvation and enriched the CD44^HighCD24^Low/- stem cell population in tumor cells. Moreover, endothelial cells enhanced the pro-metastatic potential of breast cancer cells. The <i>in vitro</i> and <i>in vivo</i> results concordantly confirmed a role for endothelial Jagged1 to promote breast tumor through notch activation. Here, we propose a role for endothelial cells in enhancing breast cancer progression, stemness, and pro-metastatic traits through a perfusion-independent manner. Our findings may be beneficial in developing novel therapeutic approaches.</p></div

E4-ECs promote BCCs self-renewal and survival in a contact-dependent manner.

<p><b>A</b>) Schematic representation of the co-culture system developed for assessing tumor cell proliferation in direct contact with GFP⁺E4-ECs. BCCs and GFP⁺E4-ECs were co-cultivated at 1∶5 ratio without serum and cytokine supplementation and proliferation and survival of BCCs was evaluated 2, 4, and 7 days post co-culture by counting the GFP^-BCCs using a fluorescent microscope. <b>B</b>) Phase contrast and fluorescent microscopy images showing higher BCC proliferation (dark gray cells) in co-culture with GFP⁺E4-ECs as compared with BCCs grown without ECs. <b>C</b>) Quantitative analysis of breast cancer cells MDA-231 and MCF-7 proliferation cultured with or without E4-ECs (***<i>p</i><0.001, mean ± SEM). <b>D</b>) Schematic representation of a transwell system used for co-culturing BCCs and E4-EC without any direct contact. BCCs and E4-ECs were separately grown as monolayers in multi-well culture plates and transwell inserts respectively. Then, inserts were positioned in the multi-well plates and both cell types were continued to grow in a serum- and cytokine-free medium (conditioned medium) and the proliferation of BBCs was evaluated at 2, 4, and 7day intervals by manual counting. <b>E</b>) Phase contrast and fluorescent microscopy images demonstrating BCCs grown under starvation either alone or in E4-EC conditioned media (CM). <b>F</b>) Quantitative analysis of proliferation of breast cancer cells MDA-231 and MCF-7 with or without direct physical contact with E4-ECs demonstrating the importance of contact (***<i>p</i><0.001, mean ± SEM).</p

E4-ECs promote mammosphere enrichment.

<p><b>A</b>) Schematic representation of sphere forming assay for enriching CSCs. PKH26-BCCs were cultured with and without GFP⁺E4-ECs (1∶10 ratio) under low adherent condition for 5 days and the rate of mammo-angiosphere or mammosphere enrichment was evaluated to determine the role of endothelial cells in CSC propagation. <b>B</b>) Phase contrast microscopy images of sphere forming assay illustrating the effect of E4-ECs on mammary stem cell development. <b>C</b>) Quantitative analysis of mammosphere formation grown with and without E4-ECs. When E4-ECs mingled with BCCs, the rate of mammosphere formation increased by 3 to 4-fold (***<i>p</i><0.001, mean ± SEM). <b>D</b>) Quantitative analysis of secondary mammosphere formation. Initially, primary mammospheres or mammo-angiospheres were cultivated for 5 days, then dissociated and flow cytometry-sorted mammospheroids were plated under low attachment condition to obtain secondary mammosphere (***<i>p</i><0.001, mean ± SEM). <b>E</b>) Phase contrast microscopy showing significant increase in secondary mammosphere growth in spheroids pre-exposed to E4-ECs. <b>F</b>) Immunofluorescent live confocal imaging of the daily process of mammo-angiosphere formation. GFP⁺E4-ECs serve as a core for the accumulation and enrichment of mammary stem cells (red). PKH26^High CSCs remain in close vicinity of E4-ECs (white arrowheads).</p

Human Embryonic Stem Cell Derived Mesenchymal Progenitors Express Cardiac Markers but Do Not Form Contractile Cardiomyocytes

<div><p>Mesenchymal progenitors or stromal cells have shown promise as a therapeutic strategy for a range of diseases including heart failure. In this context, we explored the growth and differentiation potential of mesenchymal progenitors (MPs) derived in vitro from human embryonic stem cells (hESCs). Similar to MPs isolated from bone marrow, hESC derived MPs (hESC-MPs) efficiently differentiated into archetypical mesenchymal derivatives such as chondrocytes and adipocytes. Upon treatment with 5-Azacytidine or TGF-β1, hESC-MPs modified their morphology and up-regulated expression of key cardiac transcription factors such as <em>NKX2-5</em>, <em>MEF2C</em>, <em>HAND2</em> and <em>MYOCD</em>. Nevertheless, NKX2-5⁺ hESC-MP derivatives did not form contractile cardiomyocytes, raising questions concerning the suitability of these cells as a platform for cardiomyocyte replacement therapy. Gene profiling experiments revealed that, although hESC-MP derived cells expressed a suite of cardiac related genes, they lacked the complete repertoire of genes associated with bona fide cardiomyocytes. Our results suggest that whilst agents such as TGF-β1 and 5-Azacytidine can induce expression of cardiac related genes, but treated cells retain a mesenchymal like phenotype.</p> </div

Functional characterization of E4-EC enriched mammospheres.

<p><b>A</b>) Flow cytometry analysis of CD44^HighCD24^Low/- BCSC population in mammospheres grown with or without GFP⁺E4-ECs. Mammospheres or mammo-angiospheres were dissociated by enzymatic processing and labeled with PE-CD44 and APC-CD24 antibodies after sorting from GFP⁺E4-ECs. Then, the percentage of CD44^HighCD24^Low/- mammospheres was evaluated. <b>B</b>) Sphere forming assay was performed to compare the stemness capacity of CD44^HighCD24^Low/- mammospheres. Primary mammospheres were grown and labeled with PE-CD44 and APC-CD24 antibodies followed by sorting this subpopulation. Consequently, the secondary sphere forming ability of CD44^HighCD24^Low/- spheroids was compared with the bulk of primary spheroids (***<i>p</i><0.001, mean ± SEM). <b>C</b>) Confocal images illustrating PKH dye retention in mammospheres cultured with or without E4-ECs. PKH26-stained breast cancer cells (red) while mingled with E4-ECs showed slower cell division and tend to retain the PKH dye. In the absence of E4-ECs, the dye is significantly diluted within the same culture period. <b>D</b>) Flow cytometry scatter plot demonstrating gating strategies used to define PKH26^High and PKH26^Low/- populations for cell sorting. <b>E</b>) Phase contrast microscopy of the secondary sphere formation of PKH26^High versus PKH26^Low/- spheroids. PKH26-stained primary mammo-angiospheres were dissociated and PKH26^High and PKH26^Low/- populations were sorted and their secondary mammosphere capacity was compared. <b>F</b>) Quantification of secondary sphere forming ability of PKH26^High versus PKH26^Low/- primary spheres demonstrates an almost 2-fold increase in secondary sphere formation by PKH26^High cells (**<i>p</i><0.01, mean ± SEM). <b>G</b>) qPCR analysis of the expression of pluripotency markers in primary mammo-angiospheres made by MDA-231 cells (Left panel) or MCF-7 cells (right panel) as compared with mammospheres of each cell type after sorting from GFP⁺E4-ECs.</p