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Standardin vitrocell culture is one of the pillars of biomedical science. However, there is increasing evidence that 2D systems provide biological responses that are often in disagreement within vivoobservations, partially due to limitations in reproducing the native cellular microenvironment. 3D materials that are able to mimic the native cellular microenvironment to a greater extent tackle these limitations. Here, we report Porous yet Dense (PyD) type I collagen materials obtained by ice-templating followed by topotactic fibrillogenesis. These materials combine extensive macroporosity, favouring the cell migration and nutrients exchange, as well as dense collagen walls, which mimic locally the Extracellular Matrix. When seeded with Normal Human Dermal Fibroblasts (NHDFs), PyD matrices allow for a faster and more extensive colonisation when compared with equivalent Non-Porous matrices. The textural properties of the PyD materials also impact cytoskeletal and nuclear 3D morphometric parameters. Due to the effectiveness in creating a biomimetic 3D environment for NHDFs and the ability to promote cell culture for more than 28 days without subculture, we anticipate that PyD materials could configure an important step towardsin vitrosystems applicable to other cell types and with higher physiological relevance.

Seeding materials with living cells has been—and still is—one of the most promising approaches to reproduce the complexity and the functionality of living matter. The strategies to associate living cells with materials are limited to cell encapsulation and colonization, however, the requirements for these two approaches have been seldom discussed systematically. Here we propose a simple two-dimensional map based on materials' pore size and the cytocompatibility of their fabrication process to draw, for the first time, a guide to building cellularized materials. We believe this approach may serve as a straightforward guideline to design new, more relevant materials, able to seize the complexity and the function of biological materials. This article is part of the theme issue ‘Bio-derived and bioinspired sustainable advanced materials for emerging technologies (part 1)’.

Liquid biopsy, based on the molecular information extracted from circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), offers the possibility to characterize the evolution of a solid tumor in real time and is highly important for diagnostic and therapeutic purposes. The aim of the present study was the development and validation of a novel liquid bead array methodology for the molecular characterization of CTCs and its application in breast cancer. In the present study we developed and evaluated a multiplex polymerase chain reaction (PCR)-coupled liquid bead array (MLBA) assay for studying simultaneously the expression of 14 genes in CTCs. The 14-gene MLBA assay is characterized by high analytical specificity, sensitivity, and reproducibility. The analytical performance of the 14-gene MLBA assay was compared with a commercially available test (AdnaTest BreastCancer, Qiagen, Germany) and our previously described multiplex quantitative reverse transcription PCR (RT-qPCR) assays. The developed assay has the potential to be further expanded in order to include up to 100 gene targets. The assay is highly specific for each target gene and is not affected by the numerous primers and probes used for multiplexing; hence, it constitutes a sample-, cost-, and time-saving analysis.

International audience; Ice templatingor freeze castinghas flourished in multiple domains as a straightforward process to shape solutions and particle suspensions into macroporous materials. Longtime used as a process to shape colloidal suspensions into lightweight ceramics, the use of ice templating has evolved to fabricate materials that mimic the architecture of biological tissues such as nacre and bone. Recently, the technique has been used to shape biopolymers for cell culture systems and tissue engineering applications and eventually to allow the fabrication of biomaterials containing living cells. Here we review how ice templating has progressed to cope with intrinsically labile biological matter and how these advances may shape the future 3D cell culture, tissue engineering and ultimately, cryobiology.

International audience; Type I collagen is the main component of the extracellular matrix (ECM). In vitro, under a narrow window of physicochemical conditions, type I collagen self-assembles to form complex supramolecular architectures reminiscent of those found in native ECM. Presently, a major challenge in collagen-based biomaterials is to couple the delicate collagen fibrillogenesis events with a controlled shaping process in non-denaturating conditions. In this work, an ice-templating approach promoting the structuration of collagen into macroporous monoliths is used. Instead of common solvent removal procedures, a new topotactic conversion approach yielding self-assembled ordered fibrous materials is implemented. These collagen-only, non-cross-linked scaffolds exhibit uncommon mechanical properties in the wet state, with a Young's modulus of 33 ± 12 kPa, an ultimate tensile stress of 33 ± 6 kPa, and a strain at failure of 105 ± 28%. With the help of the ice-patterned microridge features, normal human dermal fibroblasts and C2C12 murine myoblasts successfully migrate and form highly aligned populations within the resulting three-dimensional (3D) collagen scaffolds. These results open a new pathway to the development of new tissue engineering scaffolds ordered across various organization levels from the molecule to the macropore and are of particular interest for biomedical applications where large-scale 3D cell alignment is needed such as for muscular or nerve reconstruction.

Background Liquid biopsy is based on minimally invasive blood tests and has the potential to characterize the evolution of a solid tumor in real time, by extracting molecular information from circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Epigenetic silencing of tumor and metastasis suppressor genes plays a key role in survival and metastatic potential of cancer cells. Our group was the first to show the presence of epigenetic alterations in CTCs. Methods We present the development and analytical validation of a highly specific and sensitive Multiplex Methylation Specific PCR-coupled liquid bead array (MMSPA) for the simultaneous detection of the methylation status of three tumor and metastasis suppressor genes (CST6, SOX17 and BRMS1 ) in liquid biopsy material (CTCs, corresponding ctDNA) and paired primary breast tumors. Results In the EpCAM-positive CTCs fraction we observed methylation of: a) CST6, in 11/30(37%) and 11/30(37%), b) BRMS1 in 8/30(27%) and 11/30(37%) c) SOX17 in 8/30(27%) and 13/30(43%) early breast cancer patients and patients with verified metastasis respectively. In ctDNA we observed methylation of: a) CST6, in 5/30(17%) and 10/31(32%), b) BRMS1 in 8/30 (27%) and 8/31 (26%) c) SOX17 in 5/30(17%) and 13/31(42%) early breast cancer patients and patients with verified metastasis respectively. Conclusions Our results indicate a high cancerous load at the epigenetic level in EpCAM-positive CTCs fractions and corresponding ctDNA in breast cancer. The main principle of the developed methodology has the potential to be extended in a large number of gene-targets and be applied in many types of cancer.

Molecular characterization of CTCs holds considerable promise for the identification of therapeutic targets and resistance mechanisms and for real-time monitoring of the efficacy of systemic therapies in patients with metastatic breast cancer. A variety of state-of-the-art systems are available for CTC isolation, detection, and molecular characterization. Analysis of CTCs at the gene expression, DNA methylation, and DNA mutation level holds considerable promise for an individualized treatment approach for patients with metastatic breast cancer.

article i nfo VEGF splice variants Liquid bead array Multiplex PCR Non-small cell lung cancer Personalized medicine Objectives: To develop a multiplex PCR-coupled liquid bead array assay for the expression of VEGF splice variants. Design and methods: The assay was based on the combination of multiplex PCR with liquid bead array tech- nology, and optimized and evaluated in terms of analytical sensitivity, specificity, and reproducibility using the MCF-7 cell line.Clinicalperformance wasevaluatedin16pairsoffresh frozencancerousandcorrespondingnon- cancerous adjacent tissues from NSCLC patients. Results: The assay is highly sensitive, reproducible and can detect specifically VEGF splice variants in clinical samples. When applied in 32 clinical samples it gave comparable results to RT-qPCR (concordance of 81%, 75%, 88% and 81% for PBGD, VEGF121, VEGF165, and VEGF189 respectively). Conclusions:ThedevelopedassaycansimultaneouslydetectthreeVEGFsplicevariantswithhighspecificityand sensitivity and can be further used to evaluate the role of each individual VEGF splice variant in molecular ther- apies targeted against VEGF.

Background Circulating tumor cells (CTCs) have been associated with prognosis especially in breast cancer and have been proposed as a liquid biopsy for repeated follow up examinations. Molecular characterization of CTCs is difficult to address since they are very rare and the amount of available sample is very limited. Methods We quantified by RT-qPCR CK-19, MAGE-A3, HER-2, TWIST1, hTERT α+β+, and mammaglobin gene transcripts in immunomagnetically positively selected CTCs from 92 breast cancer patients, and 28 healthy individuals. We also compared our results with the CellSearch system in 33 of these patients with early breast cancer. Results RT-qPCR is highly sensitive and specific and can detect the expression of each individual gene at the one cell level. None of the genes tested was detected in the group of healthy donors. In 66 operable breast cancer patients, CK-19 was detected in 42.4%, HER-2 in 13.6%, MAGE-A3 in 21.2%, hMAM in 13.6%, TWIST-1 in 42.4%, and hTERT α+β+ in 10.2%. In 26 patients with verified metastasis, CK-19 was detected in 53.8%, HER-2 in 19.2%, MAGE-A3 in 15.4%, hMAM in 30.8%, TWIST-1 in 38.5% and hTERT α+β+in 19.2%. Our preliminary data on the comparison between RT-qPCR and CellSearch in 33 early breast cancer patients showed that RT-qPCR gives more positive results in respect to CellSearch. Conclusions Molecular characterization of CTCs has revealed a remarkable heterogeneity of gene expression between breast cancer patients. In a small percentage of patients, CTCs were positive for all six genes tested, while in some patients only one of these genes was expressed. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome for these patients is known.

Introduction. The presence of circulating tumor cells (CTCs) in patients’ bloodstream is responsible for hematogenous metastases and as a “liquid biopsy” these cells should be considered as a useful tool for the detection and the characterization of micrometastasis. Molecular characterization of CTCs is really helpful for diagnostic and therapeutic purposes. We recently developed a liquid bead array hybridization assay for studying the expression of 6 genes in CTCs in breast cancer (Markou et al, Clin Chem 2011). The aim of the present study was to expand the potential of this assay by adding more genes of clinical interest in the genes panel. Materials and Methods. We designed and evaluated a multiplexed PCR-coupled liquid bead array for studying simultaneously the expression of 14 genes. We firstly designed in silico primers and capture probes for: a) breast cancer stem cell markers: ALDH1, CD24, CD44, b) EMT markers: SNAIL, TWIST-1 and VIM, c) breast cancer markers: ERBB2, mammaglobin A, MAGEA3, PTEN, d) epithelial markers: CK-19, e) housekeeping genes: PBGD, HPRT1, and PDCD4 as a gene related with apoptosis. The assay is based on the following steps: total RNA isolation, cDNA synthesis, multiplex PCR for 14 genes, biotinylation of multiplex PCR products, hybridization of the biotinylated multiplex PCR products to xMAP microspheres, addition of streptavidin-phycoerythrin and measurement in the Luminex® 100 analyzer. Every single step was optimized in terms of analytical specificity, repeatability and reproducibility using the breast cancer cell lines MCF-7, MDA-MB-231 and SK-BR-3. To evaluate the performance of this assay we used cancer cell lines originated from the following tissues: a. human colon (COLO-205), b. human cervix (HeLa) and c. human mammary gland (T-47D). Results. The total duration of the assay, including hands on work, is approximately 6h. The assay is highly specific for each gene in complex multiplexed formats. Intra-assay CVs (n=3) ranged from 0.2% to 15.3% and inter-assay CVs (n=3) ranged from 1.0% to 18.9%. The developed assay was successfully applied for studying gene expression in COLO-205, HeLa, MCF-7, MDA-MB-231, SK-BR-3 and T-47D cancer cell lines. Conclusions. The previously developed liquid bead array has been successfully expanded including 8 additional gene targets of clinical interest. The developed assay could be applied for the molecular characterization of CTCs, since the expression of 14 genes can be measured simultaneously and reliably, thereby saving precious sample and reducing the costs and time of analysis. The developed assay will be further extended to include additional gene targets. Citation Format: Cleo Parisi, Athina Markou, Areti Strati, Evi Lianidou. Development of a multiplexed RT-PCR-coupled liquid bead array assay for the molecular characterization of CTCs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1462. doi:10.1158/1538-7445.AM2013-1462

Introduction: Comparison studies between different analytical methodologies for circulating tumor cells (CTC) detection and molecular characterization are urgently needed, since standardization of assays is essential before their use in clinical practice. Methods: We compared three different CTC molecular assays. To avoid discrepancies due to pre-analytical errors we used the same cDNAs throughout our study. CTC were isolated using anti-EpCAM and anti-MUC1 coated magnetic beads from 2 × 5 ml of peripheral blood of 254 early and 51 metastatic breast cancer patients and 30 healthy individuals. The same cDNAs were analyzed by: a) singleplex RT-qPCR assay for CK-19; b) multiplex RT-qPCR for CK-19, HER-2, MAGE- A3, and PBGD; and c) a commercially available molecular assay (AdnaTest BreastCancer) for GA733-2, MUC-1, HER-2 and beta-actin. Results: In early breast cancer, CK-19 RT-qPCR, multiplex RT-qPCR and the AdnaTest, were positive for the presence of CTC in 14.2%, 22.8% and 16.5% subjects, respectively. The concordance between the AdnaTest and CK-19 RT-qPCR was 72.4% while between the AdnaTest and multiplex RT-qPCR was 64.6%. In patients with overt metastasis, CK-19 RT-qPCR, multiplex RT-qPCR and the AdnaTest were positive in 41.2%, 39.2% and 54.9% patients, respectively. The concordance between the AdnaTest and CK-19 RT-qPCR was 70.6% while between the AdnaTest and multiplex RT-qPCR was 68.6%. Conclusions: All CTC assays gave similar results in about 70% of cases. Better agreement was found in the metastatic setting, possibly explained by the higher tumor load in this group. Discordances could be attributed to the different gene transcripts used to evaluate CTC positivity. Our results indicate the importance of CTC heterogeneity for their detection by different analytical methodologies. © 2013 Strati et al.; licensee BioMed Central Ltd. CA extern