A short-term colorectal cancer sphere culture as a relevant tool for human cancer biology investigation.

BACKGROUND: Ex vivo colospheres have been previously characterised as a colorectal cancer (CRC) well-rounded multicellular model, exclusively formed by carcinoma cells, and derived from fresh CRC tissue after mechanical dissociation. The ability to form colospheres was correlated with tumour aggressiveness. Their three-dimensional conformation prompted us to further investigate their potential interest as a preclinical cancer tool. METHODS: Patient-derived CRC xenografts were used to produce numerous colospheres. Mechanism of formation was elucidated by confocal microscopy. Expression analysis of a panel of 64 selected cancer-related genes by real-time qRT-PCR and hierarchical clustering allowed comparison of colospheres with parent xenografts. In vitro and in vivo assays were performed for migration and chemosensitivity studies. RESULTS: Colospheres, formed by tissue remodelling and compaction, remained viable several weeks in floating conditions, escaping anoikis through their strong cell-cell interactions. Colospheres matched the gene expression profile of the parent xenograft tissue. Colosphere-forming cells migrated in collagen I matrix and metastasised when subrenally implanted in nude mice. Besides, the colosphere responses to 5-fluorouracil and irinotecan, two standard drugs in CRC, reproduced those of the in vivo original xenografts. CONCLUSION: Colospheres closely mimic biological characteristics of in vivo CRC tumours. Consequently, they would be relevant ex vivo CRC models.

Newly characterised ex vivo colospheres as a three-dimensional colon cancer cell model of tumour aggressiveness.

BACKGROUND: New models continue to be required to improve our understanding of colorectal cancer progression. To this aim, we characterised in this study a three-dimensional multicellular tumour model that we named colospheres, directly obtained from mechanically dissociated colonic primary tumours and correlated with metastatic potential. METHODS: Colorectal primary tumours (n=203) and 120 paired non-tumoral colon mucosa were mechanically disaggregated into small fragments for short-term cultures. Features of tumours producing colospheres were analysed. Further characterisation was performed using colospheres, generated from a human colon cancer xenograft, and spheroids, formed on agarose by the paired cancer cell lines. RESULTS: Colospheres, exclusively formed by viable cancer cells, were obtained in only 1 day from 98 tumours (47%). Inversely, non-tumoral colonic mucosa never generated colospheres. Colosphere-forming capacity was statistically significantly associated with tumour aggressiveness, according to AJCC stage analysis. Despite a close morphology, colospheres displayed higher invasivity than did spheroids. Spheroids and colospheres migrated into Matrigel but matrix metalloproteinase (MMP)-2 and MMP-9 activity was detected only in colospheres. Mouse subrenal capsule assay revealed the unique tumorigenic and metastatic phenotype of colospheres. Moreover, colospheres and parental xenograft reproduced similar CD44 and CD133 expressions in which CD44+ cells represented a minority subset of the CD133+ population. CONCLUSION: The present colospheres provide an ex vivo three-dimensional model, potentially useful for studying metastatic process.

Nicephor[e]: a web-based solution for teaching forensic and scientific photography.

Nicephor[e] is a project funded by "Swiss Virtual Campus" and aims at creating a distant or mixed web-based learning system in forensic and scientific photography and microscopy. The practical goal is to organize series of on-line modular courses corresponding to the educational requirements of undergraduate academic programs. Additionally, this program could be used in the context of continuing educational programs. The architecture of the project is designed to guarantee a high level of knowledge in forensic and scientific photographic techniques, and to have an easy content production and the ability to create a number of different courses sharing the same content. The e-learning system Nicephor[e] consists of three different parts. The first one is a repository of learning objects that gathers all theoretical subject matter of the project such as texts, animations, images, and films. This repository is a web content management system (Typo3) that permits creating, publishing, and administrating dynamic content via a web browser as well as storing it into a database. The flexibility of the system's architecture allows for an easy updating of the content to follow the development of photographic technology. The instructor of a course can decide which modular contents need to be included in the course, and in which order they will be accessed by students. All the modular courses are developed in a learning management system (WebCT or Moodle) that can deal with complex learning scenarios, content distribution, students, tests, and interaction with instructor. Each course has its own learning scenario based on the goals of the course and the student's profile. The content of each course is taken from the content management system. It is then structured in the learning management system according to the pedagogical goals defined by the instructor. The modular courses are created in a highly interactive setting and offer autoevaluating tests to the students. The last part of the system is a digital assets management system (Extensis Portfolio). The practical portion of each course is to produce images of different marks or objects. The collection of all this material produced, indexed by the students and corrected by the instructor is essential to the development of a knowledge base of photographic techniques applied to a specific forensic subject. It represents also an extensible collection of different marks from known sources obtained under various conditions. It allows to reuse these images for creating image-based case files.

A comparison of efficiency of manual and automatic fibres search with the Maxcan fibre finder.

The aim of this work was to study the efficiency of automatic fibre searching with the Maxcan fibre finder (Cox Analytical Systems, Sweden) in comparison to manual searching. The influence of some parameters (color, thickness, background noise) on the results of a fibre search was considered. Eighteen experimental tapes with different target fibres and different background noises were prepared in the laboratory. Searching of fibres was performed manually and with the Maxcan fibre finder by different operators from four European laboratories. Two laboratories have the Maxcan fibre finder system and the two instruments were used and compared in this study. The results show that searching with the Maxcan is generally as efficient as manual searching, except for very pale or very dark fibres. Note that the tapes used for these experiments were prepared in laboratory, and are not completely representative of the tape that could be obtained in real cases. To generalize the results obtained, further research on real case samples would be necessary.

Raman spectroscopy and the forensic analysis of black/grey and blue cotton fibres Part 1: investigation of the effects of varying laser wavelength.

Raman spectroscopy was investigated to determine the optimal conditions, mainly laser wavelength/s, for the analysis of the commonly encountered black/grey and blue cotton fibres dyed with reactive dyes. In this first part, a single blue cotton fibre, its three dye components, and an undyed cotton fibre were analysed with five different laser wavelengths from two different Raman microprobe spectrometers. The quality of the spectra, fibre degradation and speed of acquisition were used to determine that, under the conditions used, the 785 and 830 nm lasers gave superior results. The 632.8 nm laser wavelengths provided good results with little acquisition time and no spectral degradation. Results indicate that, at least, the major dye component could be identified using Raman spectroscopy.

A market study of green spray paints by Fourier transform infrared (FTIR) and Raman spectroscopy.

A market study of 40 different green spray paints was carried out using infrared (FTIR) and Raman spectroscopy. The infrared technique distinguished between the 12 main groups based on their binder and extender composition. After visual comparison of the spectra 22 subgroups were observed. Raman spectroscopy was also carried out on the 40 reference paints in order to determine the pigment content. Analyses were undertaken using two different excitation sources: Argon ion (514.5 nm) and Helium-Neon (632.8 nm). The first generated strong fluorescence for most of the samples and created eight groups. Using the red laser, 15 classes were observed. Finally, using an analytical sequence starting with infrared spectroscopy followed by Raman Helium-Neon and then by Raman Argon laser, most of the paints were differentiated. In this study infrared and Raman spectroscopy complemented each other. FTIR supplied information about the binder and some extenders, and Raman provided information on the main organic pigments present.

Raman spectroscopy and microspectrophotometry of reactive dyes on cotton fibres: analysis and detection limits.

A collaborative study on Raman spectroscopy and microspectrophotometry (MSP) was carried out by members of the ENFSI (European Network of Forensic Science Institutes) European Fibres Group (EFG) on different dyed cotton fabrics. The detection limits of the two methods were tested on two cotton sets with a dye concentration ranging from 0.5 to 0.005% (w/w). This survey shows that it is possible to detect the presence of dye in fibres with concentrations below that detectable by the traditional methods of light microscopy and microspectrophotometry (MSP). The MSP detection limit for the dyes used in this study was found to be a concentration of 0.5% (w/w). At this concentration, the fibres appear colourless with light microscopy. Raman spectroscopy clearly shows a higher potential to detect concentrations of dyes as low as 0.05% for the yellow dye RY145 and 0.005% for the blue dye RB221. This detection limit was found to depend both on the chemical composition of the dye itself and on the analytical conditions, particularly the laser wavelength. Furthermore, analysis of binary mixtures of dyes showed that while the minor dye was detected at 1.5% (w/w) (30% of the total dye concentration) using microspectrophotometry, it was detected at a level as low as 0.05% (w/w) (10% of the total dye concentration) using Raman spectroscopy. This work also highlights the importance of a flexible Raman instrument equipped with several lasers at different wavelengths for the analysis of dyed fibres. The operator and the set up of the analytical conditions are also of prime importance in order to obtain high quality spectra. Changing the laser wavelength is important to detect different dyes in a mixture.

Applications of a transportable Raman spectrometer for the in situ detection of controlled substances at border controls.

A transportable Raman spectrometer was tested for the detection of illicit drugs seized during border controls. In a first step, the analysis methodology was optimized using reference substances such as diacetylmorphine (heroin), cocaine and amphetamine (as powder or liquid forms). Adequate focalisation distance and times of analysis, influence of daylight and artificial light sources, repeatability and limits of detection were studied. In a second step, the applications and limitations of the technique to detect the illicit substances in different mixtures and containers were evaluated. Transportable Raman spectroscopy was found to be adequate for a rapid screen of liquids and powders for the detection and identification of controlled substances. Additionally, it had the advantage over other portable techniques, such as ion mobility spectrometry, of being non-destructive and capable of rapid analysis of large quantities of substances through containers such as plastic bags and glass bottles.