We report our approach to creating a microfluidic chip (namely UroLOC) that mimics the acinar/tubular structure and the luminal microenvironment of exocrine glands. The chip utilises a nanostructured membrane that is designed to provide a 3-dimensional supporting scaffold for the growth of exocrine acinus epithelial cells. The nanostructured membrane was produced using layer-by-layer assembly of polyelectrolytes, and formed into 3-dimensional hemispherical cavities and “finger-like” structures in order to mimic the natural architecture of acini found in exocrine glands. We utilised normal (PNT2) and cancerous (PC3, LNCaP) prostate epithelial cells to demonstrate the proof-of-concept of using MALDI (Matrix Assisted Laser Desorption Ionisation) profiling of secretions collected after 48 hours of cell growth, with no concentration or purification steps and without any a priori on the knowledge of targeted proteins. This MALDI profiling analysis of the crude supernatants from 3 different cell lines (PNT2, PC3 and LNCaP) demonstrated the capacity of the MALDI profiling approach to discriminate between the different secretome signatures. The UroLOC concept and secretome profiling that we describe opens new opportunities in terms of liquid-biopsy based diagnosis, particularly for the early stages of carcinogenesis.
- liquid biopsy
- 3D scaffolds
Monolayer (in 2D) cultures have been very useful models for gene discovery and early work on viral transformation, but such 2D systems do not provide a cell-model that replicates the architecture of exocrine gland tissues (1). Cell models that replicate exocrine gland architecture are important tools in the search for drugs to combat disease and cancer. For example, cancer of breast tissue is a significant cancer amongst women. Vidi et al 2013 describe methods for the growth of breast epithelial cells into physiologically relevant 3D cell culture, which demonstrate the important roles that cell polarity, cell shape and the development of proper basement membranes play in the physiology of exocrine tissues in the breast. Such physiologically relevant 3D cell culture models of breast tissue are important tools in the search for therapeutic agents to fight disease and cancer of the breast tissue. For men’s health, the exocrine prostate gland tissue is a significant cause for cancer and other disease. Prostate epithelial cells grown in micro-spheroid cultures in a Microwell-mesh 3D culture system responded appropriately to drugs used to treat prostate cancer (2) The use of organoids, as 3D culture models would provide even closer tissue-like conditions for studying drug reaction (3). Those types of 3D cell-culture systems provide information of cell responses that are measurable by imaging or spectroscopic techniques. The limitation of those 3D epithelial models for acinar structures is that it is not possible to access the luminal secretions to measure physiological responses in real-time and to identify new cancer biomarkers.
The use of microfabricated systems, or indeed novel polymer-based cell scaffolds that are coupled to microfluidics, would provide the next step toward exocrine organ-on-chip cultures that could provide real-time physiological cell responses (4). The choice of epithelial cells can assist the step toward such capability of measuring real-time physiological responses. For example, intestinal CaCo-2 epithelial cells have been cultured in a Gut-on-Chip device that included a microfluidics system to access both sides of the intestinal monolayer, which provided measurements of metabolic responses from the intestinal cells that were closer to that expected in a natural biological tissues (5, 6). That Gut-on-Chip device comprises a porous PDMS membrane to provide a permeable membrane to allow cell secretions to be collected in the microfluidic system. Nonetheless, PDMS membranes may have intrinsic transport, mechanical and structural properties that are different from those of the natural basement membrane of the tissue. Other materials were used to mimic finger-like villi topography such as biodegradable polymers (such as poly-lactic-glycolic acid and poly-lactic-acid) (7), collagen (see references in (8)). While intestine-on-chip devices (9) remain to date the closest models for finger-like acinar and tubular structure, no exocrine-gland-on a chip devices composed of hollow protuberances allowing co-cultures and direct sampling of secretions have been developed.
In this paper we describe our approach to creating a microfluidic chip (namely UroLOC) that mimics the acinar/tubular structure and the luminal microenvironment of exocrine glands. The chip utilises a nanostructured membrane that is designed to provide a 3-dimensional supporting scaffold for the growth of exocrine acinus epithelial cells. This scaffold is a porous biomaterial membrane that comprises multiple 3-dimensional and hollow protuberances on which adherent cells are grown on either side, with the acinar epithelial cells on the inside (luminal side) and stromal cells on the outside (glandular side). The acinar epithelial cells on the inside of the 3D protuberances produce small volumes of secretions, which are collected (by possibly using microfluidics) for analysis of the composition of the secreted fluid. These secretions consist of all the molecules that can be secreted in the cell culture medium such as peptides, proteins, amino acids, miRNAs, DNA, RNA. The analysis profile of these secretions makes it possible to obtain the secretome, that is to say the qualitative and quantitative profile of the secretory components.
The membrane was formed using a layer-by-layer process that utilised the polyelectrolytes poly-sodium 4-styrenesulphonate (PSS) and poly-allylamine hydrochloride (PAH) (10). The optimisation of this membrane for the UroLOC device proceeded using 2 protocols. The first protocol constructed the membrane inside 3D hemispherical cavities, with the purpose to grow the exocrine epithelial cells on these 3D hemispherical membranes and to optimize the analytical method for the small volumes of secretions from each 3D hemispherical cavity. The second protocol was to construct the membrane on a 3D acinar-mimic sacrificial mould so as to create a free-standing porous membrane, with the purpose to mimic the biological acinar architecture with epithelial cells on the inside of the membrane (luminal side) and stromal cells on the outside (basal side).
The 3D hemispherical cavities were produced from a master mould.
The derived mould was used to form 3D hemispherical cavities in a PDMS chip. Multi-layered polyelectrolyte films were assembled inside these 3D hemispherical cavities by alternate coating of PSS (poly (sodium 4-styrenesulphonate; Sigma 243051)) and PAH (poly (allylamine hydrochloride; Sigma 283223)) following the LbL (Layer-by-Layer) process we have previously described (10, 11) (see also
RWPE1 and WPE1-int lines were used to validate the structural and functional phenotype of prostatic cells grown on the PE films inside the 3D hemispherical cavities. The RWPE1 (ATCC Ref. CRL11609) cell-line was derived from non-neoplastic human prostate epithelial cells via immortalization with human papillomavirus. They were used as model for normal prostate epithelial cell behaviour, as characterised by a well-established polarised morphology (12) and clear cell-cell junctions when grown on a flat or curve surface (13). The WPE1-int cells (ATCC ref CRL2888) were derived from the RWPE-1 cell-line after two consecutive cycles of single cell cloning and were used as luminal prostatic cells that have the property to secrete Prostate Specific Antigen (PSA) while conserving a normal epithelial morphology.
Cells were cultured on the PE films inside the 3D hemispherical cavities from 5 to 8 day days after seeding. Vinculin was chosen to visualize the cell focal adhesions on the PE films inside the 3D hemispherical cavities. Cells were permeabilized for 40 seconds with Triton X-100 and fixed with 4% (v/v) PFA in PBS for 20 minutes and washed once with PBS. To block nonspecific antibody binding, cells were incubated with 0.1 BSA and 10% goat serum for 1 hour. Primary antibody against vinculin (Sigma Aldrich) was used at 1:700 dilution (in PBS+Tween 20 0.05%, and 5% goat serum) and incubated 1 hour at RT followed by 4 PBS washes (45 minutes in total). Cultures were then incubated with anti-mouse Cy5 (Jackson, dilution 1/500 in PBS+, Tween 20 0.05%, and 5% goat serum). The cells were subsequently washed 4 times for 15 minutes with PBS. Nuclei were stained with SYBR green (2μg/ml in PBS). Confocal acquisition was achieved by exciting with 2 lasers, 488 (SYBR green) and 543 (Cyanine 5), which allowed for 3D reconstruction of 50 optical slices (1 μm step) which were acquired in the Z-axis from the top of the mould (flat region, red) to the bottom of the 3D cavity (blue).
The volume of secretions from cells grown on the PE-scaffold inside the hemispherical cavities was estimated as followed (
PDMS moulds were placed in 9 well plates for cell culture. The cells were seeded over the PDMS mould at high concentration (diluted by one-third from T25 cm2) and culture medium without FCS was placed over the PDMS mould to form a large convex meniscus. 24h later the meniscus had decreased so that the culture medium was filling only the hemispherical cavities present on the moulds. Cells were then stimulated with dihydrotestosterone (DHT, 100 nM) for 24 hours inside the incubator in order to stimulate PSA secretion. Particular attention was paid to only adding DHT to the hemispherical cavities of the PDMS moulds and not across the entire surface of each mould. For each condition, the medium from 3 moulds was pooled into 1 sample for ELISPOT-based analysis, in each of the 4 following conditions: WPE1-int – DHT, WPE1-int +DHT, RPTEC and 3ng of pure PSA. Volumes of secretions collected from moulds were spotted on a 1.5 cm2 nitrocellulose membrane. Each spot corresponds to 10 μl of supernatant and all dilutions have been made with milli-Q water. ELISPOT analysis was performed as described in section 4
he concept was based on the modular assembly of 3D printed pieces that provided a system to shape the self-supporting “acinar-like” PE scaffolds. The methodology relied on using alginate as a sacrificial core for PE coating from a shape of either alginate “finger-like” structures (
For the first approach (
In the second approach (
Some technical issues were addressed during the assembly procedure. First, it was required to have a perfect alignment between the wells created on the alginate mould and the holes in the second piece. This issue was solved by designing the blue and green piece with the pillars/holes respectively in the exact same position. Secondly, the addition of liquids to the system was necessary for alginate gelation, polyelectrolyte coating and cell culture. Alginate needs to be contained in a support and a non-leaky connection needs to be created between the black and blue piece. Therefore, both pieces contain walls to avoid these drawbacks. Finally, the green piece was originally created with symmetrical walls on both sides of the horizontal plane. However, this design prevented liquid from entering the bottom chamber, and placed the horizontal plane outside the normal working distance for microscope objectives. This design was not appropriate for cell culture nor for imaging by inverted microscopy. For these reasons, a new (grey) piece was created with a castellation shape to enable the passage of liquid to the bottom chamber as well as to provide the opportunity to remove almost completely one of the walls in the green piece, which placed the horizontal plane within the working distance of the objectives for inverted microscopy. After several trials had been done, an extra set of pieces was designed to evaluate different diameter sizes as well as the height of the pillars. This set consisted in a blue and green piece containing four different conditions (h = 0.4 or 1 mm and Ø = 0.5 or 1 mm) with three replicas for each condition.
RWPE-1 and WPE1-int were growth in KSFM (Life Technologies, Carlsbad) supplemented with 5 ng/mL of epidermal growth factor (EGF) and 50 mg/mL of bovine pituitary extract (Gibco) and 1% Penicillin-Streptomycin (Gibco). Cells were cultured up to 70% confluence and seeded in T25 Flasks at a density of 2 × 104 viable cells
In order to investigate the invasiveness processes, LNCaP cells were labelled with the Molecular Probes_CellTracker™_Green CMFDA (5-chloromethylfluorescein diacetate) (Life Technologies C7025) (3 μM, for 30 minutes) and then introduced into a monolayer of PNT-2 epithelial cells once they reached 90% of confluency. For immunostaining assays, cells were then incubated for 20 min at room temperature in PFA 4% in PBS then rinsed in PBS for 10 minutes and permeabilized 5 minutes in 0.5% Triton X100 in PBS. Cultures were finally incubated with Phalloidin-Cy3 (1:400) in PBS for 1 hour then washed 3x5 minutes and then nuclei were stained with Hoechst (2 μg/ml) for 10 minutes. Stained cells were imaged by fluorescence microscopy using a confocal spinning-disk inverted microscope (Nikon TI-E Eclipse) with a 20x air objective equipped with a Yokogawa CSU-1 Unit for z-stack acquisitions and an Evolve EM-CCD camera. Primary Renal Proximal Tubule Epithelial Cells (RPTEC) were kindly provided by I. Villegrain (CEA Grenoble) and were used as control for ELISPOT assay as non-PSA secreting epithelial cells.
Biochemical studies were carried out on secreted supernatants from cells grown in 2D to validate the use of our cell lines and the possibility to detect secreted proteins in the supernatants without having to apply purification or concentration steps. The 2D format offers very well controlled culture conditions to evaluate the minimum number of cells required to perform a secretomics-based Maldi analysis. Moreover, this number of cells and the corresponding volume of secretions need to be compatible with further microfluidic integration and sampling. PSA was chosen as “housekeeper” protein for the secretome.
Western Blot was performed with the same supernatant of cell culture used for MALDI analysis. Different quantities of supernatant were separated by electrophoresis on a NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Life Technologies) in MES buffer and then transferred onto a nitrocellulose membrane (Sigma-Aldrich). The membranes were blocked in 5% non-fat milk in Tris-buffered saline, 0.1% Tween 20 (TBS-T, Sigma) for 1 hour at room temperature and incubated with primary antibodies diluted at 1:1000 in 5% non-fat milk in TBS-T overnight at 4°C. Rabbit polyclonal antibodies directed against PSA (PSA/KLK3 (D6B1) XP® Rabbit mAb ref 5365, cell signalling technology) and GAPDH (Santa Cruz Biotechnology) were used. Membranes were washed three times for 10 min in TBS-T and incubated with secondary horseradish peroxidase-conjugated antibodies (anti-rabbit, eBiosciences) at a dilution of 1:10000 in TBS-T for 50 min. Membranes were washed three times for 10 min in TBS-T and revealed with ChemiDoc™ MP systems (BioRad). Chemiluminescence was analysed using a Chemi-Doc Touch Imaging System (BioRad).
ELISA tests were performed by using a Human Free PSA ELISA Kit from Anogen (EL10050) for the in vitro quantitative determination of free Prostate-Specific Antigen (fPSA) concentrations in the supernatant. Washing steps were performed with PBS, Tween 0.05% and milk 5% during 15 min. Incubation with IgG anti-PSA-ncam was performed during 30 min and incubation with a rabbit HRP (1:1000) during 30 min. Washing step was done with PBS Tween 0.05% during 15 min, twice.
The “Proteome Profiler Human XL Cytokine Array Kit” was used with 200 μl of the same supernatants used for biochemistry and MALDI analysis and following the supplier protocol and revealed with ChemiDoc™ MP systems (BioRad) with an exposure time of 150 seconds. For the calculation of the intensity of each of the spots, we used the Fiji software with the “Microarray profile” plugin. Each protein was spotted twice on the microarray and the histogram corresponds to the mean of the integrated density obtained for these two spots to which the value of the negative control has been subtracted.
LC-MS grade Water, Acetonitrile (99.9%), and trifluoroacetic acid (99%) were obtained from Sigma-Aldrich. α-Cyano-4-hydroxycinnamic acid and calibration standard mix (BTS) were purchased from Bruker Daltonics. For each spot, 0.6 μl of cell supernatant was mixed with 0.6 μl of α-Cyano-4-hydroxycinnamic acid (10mg/ml in acetonitrile/water/trifluoroacetic acid 50:49,8:0,2 v/v/v) and deposited onto a 384 MTP AnchorChip Target (Bruker Daltonics).
MALDI mass spectrometry profiling was performed on an Ultraflex III MALDI-TOF/TOF (Bruker Daltonics) equipped with a Smart-beam laser operating at 1000 Hz pulse rate. Ions were detected in positive linear mode at +20 kV accelerating potential, in the mass range of m/z 1000-20000 with ion suppression up to 950 Da. On each sample spots, 5000 spectra were summed with automatic spectral quality evaluation.
Spectra were preprocessed (tophat baseline removal and smoothing) and normalized by total ion current (TIC) in Bruker Clinprotool software v3.0. 2D peak distribution and Principal Component Analysis (PCA) was performed in Clinprotool. Unsupervised hierarchical clustering was performed on exported spectra from Clinprotool, using log transformation of the intensities and clustering via uncentered correlation similarity metric (Gene cluster v3.0). For each condition, 3 biological replicates were analysed 3 times.
Cells at day 8 were well-organized at the bottom of the PE scaffolds and form a monolayer with intercellular tight junctions revealed by immunofluorescence (
In order to validate the exocrine function of prostatic cells grown at the bottom of the wells, we verified that cells did secrete PSA. For this, the medium present above 3 moulds was harvested in 3 conditions. WPE1-int unstimulated with DHT, WPE1-int stimulated with DHT, but also 3 PDMS mould with RPTEC cells (renal cells, as negative control). A positive control was made with 3 ng of pure PSA (
To validate our hypothesis that cancerous cell lines can be discriminated on the basis of their secretome, we performed biochemical analysis from their supernatant. As expected and confirmed with ELISA test, PSA secretions were detected from LNCaP, with or without stimulation (15) by contrast to the other cell lines that are described as not secreting PSA (16) (
To avoid any interference of FCS within MALDI analysis, FCS was replaced with BSA 2g/L, as previously described in the literature (17, 18), during the incubation phase used to recover the secretory fluid. In the final point after five days, a Live/Dead staining showed no cell mortality and no alteration in cell morphology (data not shown). After biochemistry validations, supernatants from LNCaP, PNT2 and PC3 cell cultured as described above were analyzed by MALDI MS in the range of 1000 to 20000 Da for peptides/small proteins profiling. The amount of supernatant spotted was normalized to approximately 300 cells per sample.
To assess the ability to differentiate a healthy PNT2 culture from a PNT2 and LNCaP co-culture, different numbers of LNCaP cells (1,000, 10,000 and 50,000) have been seeded in a PNT2 culture and MALDI profiling analysis has been performed on the supernatant. We first observed that spiked cancerous cells were properly incorporated in the non-cancerous cell monolayer (
On the basis of the results obtained with PE-coated hemispheres, we developed incremental systems to construct self-supporting finger-like scaffolds providing an access to both side of the acinus, basal and luminal. Alginate flowing was used to create a sacrificial 3D shaped structure in top of which polyelectrolyte membrane would be created. The idea was then to obtain a homogeneous region containing several alginate pillars. Moreover, the use of a programmable software controlled syringe pump would allow a fine optimisation and automatisation of the process. Nevertheless, alginate flowing through the 3D printed UroSCAF pieces resulted in irregular and non-uniform extrusions of alginate from the holes; sometimes extrusions appeared from only one hole. Moreover, when the procedure was repeated with the same piece, alginate came out from a different hole. These results may have been due to microfluidic physical flow-resistance issues, which might be due to slight differences in the shape between different holes in the same piece that was 3D-printed.
As this first device (see
New UroBOXES pieces were then carefully designed (see
In this study we demonstrate how to discriminate non-cancerous from cancerous cell lines by MALDI profiling of secretions following 48h of cell growth, with no concentration or purification steps and without any a priori on the knowledge of targeted proteins. The analysis of the crude supernatants of 3 different cell lines (PNT2, PC3 and LNCaP) demonstrated the capacity of the MALDI MS profiling approach to identify specific MS signatures. This result encouraged us to further perform the analysis of supernatant samples from PNT2 normal cells co-cultured with small amount of LNCaP tumor cells to detect a specific MS signature of a tumor invasion mimicking condition. As expected, the discriminative ability of MALDI profiling was less significant in our experimental settings. However, the data obtained in the condition with PNT2 co-cultured with 50,000 LNCaP cells remain promising. Considering the low concentration of cells secretome in the supernatant and the presence of highly abundant BSA in the culture media, several optimisation steps could be implemented to improve MALDI MS detection of pertinent markers. We envision that future developments including pre-concentration and enrichment of cells secretome in specific microfluidic devices will greatly enhance the MALDI detection of specific secreted markers.
It is known that tumour cells change their genetic expression pattern during the stages of increasing malignancy (19). That leads to differential secretion of peptides or proteins from the tumour cells but only in very low amounts. Existing mass spectrometry analysis is variable due to the need to measure these small amounts of secreted peptides and proteins in whole body fluids. Nonetheless, the principle of the change in levels of secreted peptides and proteins has been demonstrated by measuring the supernatant of cultured carcinoma cells using mass spectrometry (19). Those published reports from cell culture supernatant augurs well for the approach taken by UroLOC, since the perspective of UroLOC device is to collect the in-line secretions of tumour cells, in a controlled microenvironment, for analysis of the secreted peptides and proteins using mass spectrometry. In our approach, the status of cancerous cells is determined by comparing the analysis of secretions from a preformed layer of normal urothelial cells (“before”) to the change in secretions induced after “seeding” the urothelial cells into the pre-formed layer (“after”). Thus, unlike previous reported systems where the markers in secreted fluids are detected by fixed-cell immunostaining, thus implying the death of the cells (5), our UroLOC lab-on-chip device makes it possible to analyse in real-time the composition of the secretions from the living cells. (For example, the analysis of secreted fluid can be made over several days for carrying out kinetic studies, while maintaining the viability and the functionality of cells over time). As a first example to mimic an exocrine gland to demonstrate the function our new approach to a 3D lab-on-chip device, we have taken the medical problem of measuring changes in the secretions from the acinar cells of the prostate gland and then to consider the use of detecting these small changes in secretions as an early indicator/diagnostic for prostate cancer.
We also demonstrated in this study the feasibility of 3D shaping a polyelectrolyte membrane resulting in a 3D self-supporting nanostructured membrane. At this stage, the polyelectrolyte membrane was floating and its manipulation was difficult and the realisation of closed finger-like structure in all the holes of the pieces still need optimisation for complete reproducibility. However, our design along with first validation are promising in the way to closely mimic the functional conditions of the secretory ductal/acinar structures. Continuing this developmental path provides a significant advantage, since it is well-known that the function of secretory cells changes dramatically from the classical 2D culture plates to even Matrigel® cultures. Indeed, 3D cultures are essential for investigating more natural responses from exocrine tissue such as breast epithelial cells.
Tumour cells change their genetic expression pattern as they progress to states of increasing malignancy (19). Investigations at the DNA and RNA level alone cannot provide all the information resulting after the translation and processing of the corresponding proteins, which is one reason for a poor correlation between mRNA and the respective protein abundance. In diagnostics, differentially expressed peptides or proteins are important markers for the early detection of cancer. Unfortunately, tumour cells secrete peptides and proteins in only very low amounts, making mass spectrometric determination very difficult. As reported by Peter et al (19), methods have been developed for the effective enrichment and cleanup of substances secreted by cultivated cancer cells. To obviate peptides from fetal calf serum used in cell culture, a serum surrogate was developed, which maintained growth of the cancer cells. After the binding of substances from cell-culture supernatants to custom-made magnetic reversed-phase particles, the substances were eluted and separated by capillary high-performance liquid chromatography. Fractions were spotted directly on a MALDI target, and MALDI-TOF mass spectrometric data acquisition was performed in automatic mode. This technology was used to detect substances secreted by two mammary carcinoma cell lines differing in their malignancy (MCF-7, MDA-MB231). Unequivocal differences in the peptide secretion patterns were observed. In conclusion, this system allows the sensitive investigation of peptides secreted by cancer cells in culture and provides a valuable tool for the investigation of cancer cells in different states of malignancy (20).
Our concept to collect secretions directly from the urothelial cells (e.g. prostate) is radically different from the attempt to purify these secretions from whole urine. The existing use of whole urine as the sample from which to detect molecules secreted by cancerous urothelial cells is an arduous task for several reasons. Tumour cells secrete peptides and proteins in only very low amounts (19). Urine is variable in volume, pH, type of background protein “noise”, and is particularly variable depending on the time of day of collection (21). Consequently, it is very difficult to purify urine in a reliable way in order to detect the small amount of molecules secreted by urothelial cells. To have access to those low amounts of peptides and proteins in whole urine, existing procedures require complex enrichment and cleanup protocols prior to mass spectrometric analysis of whole urine.
In conclusion, our work hence anticipates the use of microfluidics connected to our nanostructured scaffolds to obtain concentrated secretions without the need of complex filtration (such as would be needed by using whole urine). It opens also new opportunities in terms of liquid-biopsy based diagnosis (