Protocols and characterization data for 2D, 3D, and slice-based tumor models from the PREDECT project

Two-dimensional (2D) culture of cancer cells in vitro does not recapitulate the three-dimensional (3D) architecture, heterogeneity and complexity of human tumors. More representative models are required that better reflect key aspects of tumor biology. These are essential studies of cancer biology and immunology as well as for target validation and drug discovery. The Innovative Medicines Initiative (IMI) consortium PREDECT (www.predect.eu) characterized in vitro models of three solid tumor types with the goal to capture elements of tumor complexity and heterogeneity. 2D culture and 3D mono-and stromal cocultures of increasing complexity, and precision-cut tumor slice models were established. Robust protocols for the generation of these platforms are described. Tissue microarrays were prepared from all the models, permitting immunohistochemical analysis of individual cells, capturing heterogeneity. 3D cultures were also characterized using image analysis. Detailed step-by-step protocols, exemplary datasets from the 2D, 3D, and slice models, and refined analytical methods were established and are presented.Peer reviewe

Capturing tumor complexity in vitro : Comparative analysis of 2D and 3D tumor models for drug discovery

Two-dimensional (2D) cell cultures growing on plastic do not recapitulate the three dimensional (3D) architecture and complexity of human tumors. More representative models are required for drug discovery and validation. Here, 2D culture and 3D mono-and stromal co-culture models of increasing complexity have been established and cross-comparisons made using three standard cell carcinoma lines: MCF7, LNCaP, NCI-H1437. Fluorescence-based growth curves, 3D image analysis, immunohistochemistry and treatment responses showed that end points differed according to cell type, stromal co-culture and culture format. The adaptable methodologies described here should guide the choice of appropriate simple and complex in vitro models.Peer reviewe

A Phytase-Based Reporter System for Identification of Functional Secretion Signals in Bifidobacteria

<div><p>Health-promoting effects have been attributed to a number of <i>Bifidobacterium</i> sp. strains. These effects as well as the ability to colonise the host depend on secreted proteins. Moreover, rational design of protein secretion systems bears the potential for the generation of novel probiotic bifidobacteria with improved health-promoting or therapeutic properties. To date, there is only very limited data on secretion signals of bifidobacteria available. Using <i>in silico</i> analysis, we demonstrate that all bifidobacteria encode the major components of Sec-dependent secretion machineries but only <i>B</i>. <i>longum</i> strains harbour Tat protein translocation systems. A reporter plasmid for secretion signals in bifidobacteria was established by fusing the coding sequence of the signal peptide of a sialidase of <i>Bifidobacterium bifidum</i> S17 to the phytase gene <i>appA</i> of <i>E</i>. <i>coli</i>. The recombinant strain showed increased phytase activity in spent culture supernatants and reduced phytase levels in crude extracts compared to the control indicating efficient phytase secretion. The reporter plasmid was used to screen seven predicted signal peptides in <i>B</i>. <i>bifidum</i> S17 and <i>B</i>. <i>longum</i> E18. The tested signal peptides differed substantially in their efficacy to mediate protein secretion in different host strains. An efficient signal peptide was used for expression and secretion of a therapeutically relevant protein in <i>B</i>. <i>bifidum</i> S17. Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of <i>B</i>. <i>bifidum</i> S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell. Selection of appropriate signal peptides for defined protein secretion might improve therapeutic efficacy as well as probiotic properties of bifidobacteria.</p></div

Signal peptides used to establish or tested in the phytase reporter assay and corresponding information on the predicted localization.

<p>^a E-score, extracellular score, calculated by cPSORTdb (version 3)</p><p>^b D-score, discrimination score, calculated by SignalP 4.1.</p><p>^c amino acid positions (aa), the sequence and the exact cleavage site (*) of the predicted SPs are indicated</p><p>^d This SP belong to a protein which is predicted to be located in the cell wall. The respective C-score for cell wall localization calculated by PSORTdb was 9.26.</p><p>Signal peptides used to establish or tested in the phytase reporter assay and corresponding information on the predicted localization.</p

Components of the major protein secretion machineries encoded on the genomes of representative <i>Bifidobacterium sp</i>.

<p>^a protein size in amino acid residues (aa).</p><p>^b percent identity on amino acid sequence level to the respective homologue of <i>E</i>. <i>coli</i> K12-W3110.</p><p>Components of the major protein secretion machineries encoded on the genomes of representative <i>Bifidobacterium sp</i>.</p

List of <i>B</i>. <i>bifidum</i> S17 proteins with predicted extracellular localization and information on the signal peptides identified in their amino acid sequences.

<p>List of <i>B</i>. <i>bifidum</i> S17 proteins with predicted extracellular localization and information on the signal peptides identified in their amino acid sequences.</p

Phytase reporter system to monitor protein secretion by bifidobacteria.

<p>(A) Schematic representation of the secretion reporter plasmid pMgapS0P. The vector was constructed by fusing the SP of BBIF_1734 (S0) to the phytase gene <i>appA</i> of <i>E</i>. <i>coli</i> K12 and cloning of the construct under the control of the <i>gap</i> promoter (P_<i>gap</i>) of <i>B</i>. <i>bifidum</i> S17 in the vector backbone of pMDY23-P_<i>gap</i> using <i>Xho</i>I and <i>Hind</i>III. Relevant other features are: <i>rep</i> (origin of replication for <i>E</i>. <i>coli</i>), <i>repAB</i> (origin of replication for bifidobacteria), <i>spc</i> (spectinomycin resistance gene). (B)+(C) Phytase activity in supernatants (B) or crude extracts (C) of <i>B</i>. <i>bifidum</i> S17/pMgapP (S17 pMgapP) and <i>B</i>. <i>bifidum</i> S17/pMgapS0P (S17 pMgaS0P) during growth in RCM batch cultures. Values are relative phytase units (RPU) per ml supernatant (B) or mg protein in crude extracts (C) and are mean +/- standard deviation of three independent cultures measured in technical triplicates.</p

Phytase activity in supernatants recombinant bifidobacteria strains expressing phytase with different signal peptides.

<p>Phytase activity was measured in culture supernatants of <i>B</i>. <i>bifidum</i> S17 (A) or <i>B</i>. <i>longum</i> E18 (B) harbouring pMgapP-derived plasmids containing different SPs (S0-S6) during growth in RCM batch cultures. The control plasmid pMgapP (-) contains no SP and serves as a background control for expression of a non-secreted phytase. Values are relative phytase units (RPU) per ml supernatant (B) and are mean +/- standard deviation of three independent cultures measured in technical triplicates. Statistical analysis was performed by one-way ANOVA with Bonferroni post-tests for multiple comparisons. Phytase activity in the supernatants of each strain was compared to all other strains. Letters indicate statistical significance of the difference (a: <i>p</i> < 0.001 for all comparisons; b: <i>p</i> < 0.001 for all comparisons except S0 vs. S1; differences for all other signal peptides were significant at p<0001 for less than 5 other signal peptides). For complete results of the analysis see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128802#pone.0128802.s004" target="_blank">S3 Table</a>.</p

Ca-phytate degradation of recombinant bifidobacteria expressing phytase with different signal peptides.

<p>Calcium phytate degradation by recombinant strains of <i>B</i>. <i>bifidum</i> S17 (A) and <i>B</i>. <i>longum</i> E18 (B) harbouring pMgapP-derived plasmids containing different SPs (S0-S6). The control plasmid pMgapP contains no SP and serves as a background control for expression of a non-secreted phytase. Overnight cultures of all strains were spotted in triplicate on RCM agar supplemented with 0.15% calcium phytate and imaged after anaerobic incubation for 48 h at 37°C. One representative spot of three independent cultures is shown.</p