Analysis of Cancer Cell Line Secretomes: A Complementary Source of Disease-Specific Protein Biomarkers.

Various types of cancer cells enrich or condition the medium that they are cultured in by secreting or shedding proteins and small molecules. These secreted or shed factors are involved in key biological processes, including cellular communication, proliferation, and migration, and are represented by protein families, including cytokines, growth factors, and enzymes. The rapid development of high-resolution mass spectrometry and shotgun strategies for proteome analysis facilitates the identification of these factors in biological models and elucidation of their potential roles in pathophysiology. Hence, the following protocol provides details on how to prepare proteins present in conditioned media for mass spectrometry analysis.

Methodologies for Scalable Production of High-Quality Purified Small Extracellular Vesicles from Conditioned Medium.

The development of an extracellular vesicles (EV)-based therapeutic product requires the implementation of reproducible and scalable, purification protocols for clinical-grade EV. Commonly used isolation methods including ultracentrifugation, density gradient centrifugation, size exclusion chromatography, and polymer-based precipitation, faced limitations such as yield efficiency, EV purity, and sample volume. We developed a GMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving, tangential flow filtration (TFF). We applied this purification method for the isolation of EV from conditioned medium (CM) of cardiac stromal cells, namely cardiac progenitor cells (CPC) which has been shown to possess potential therapeutical application in heart failure. Conditioned medium collection and EV isolation using TFF demonstrated consistent particle recovery (~1013 particle/mL) enrichment of small/medium-EV subfraction (range size 120-140 nm). EV preparations achieved a 97% reduction of major protein-complex contaminant and showed unaltered biological activity. The protocol describes methods to assess EV identity and purity as well as procedures to perform downstream applications including functional potency assay and quality control tests. The large-scale manufacturing of GMP-grade EV represents a versatile protocol that can be easily applied to different cell sources for wide range of therapeutic areas.

In-Cell Labeling Coupled to Direct Analysis of Extracellular Vesicles in the Conditioned Medium to Study Extracellular Vesicles Secretion with Minimum Sample Processing and Particle Loss.

Extracellular vesicles (EVs) are involved in a multitude of physiological functions and play important roles in health and disease. The largest proportion of studies on EVs is based on the analysis and characterization of EVs secreted in the cell culture medium. These studies remain challenging due to the small size of the EV particles, a lack of universal EV markers, and sample loss or technical artifacts that are often associated with EV labeling for single particle tracking and/or separation techniques. To address these problems, we characterized and validated a method for in-cell EV labeling with fluorescent lipids coupled with direct analysis of lipid-labeled EVs in the conditioned medium by imaging flow cytometry (IFC). This approach significantly reduces sample processing and loss compared to established methods for EV separation and labeling in vitro, resulting in improved detection of quantitative changes in EV secretion and subpopulations compared to protocols that rely on EV separation by size-exclusion chromatography and ultracentrifugation. Our optimized protocol for in-cell EV labeling and analysis of the conditioned medium reduces EV sample processing and loss, and is well-suited for cell biology studies that focus on modulation of EV secretion by cells in culture.

The Growth Factors and Cytokines of Dental Pulp Mesenchymal Stem Cell Secretome May Potentially Aid in Oral Cancer Proliferation.

BACKGROUND: Growth factors and cytokines responsible for the regenerative potential of the dental pulp mesenchymal stem cell secretome (DPMSC-S) are implicated in oral carcinogenesis. The impact and effects of these secretory factors on cancer cells must be understood in order to ensure their safe application in cancer patients. OBJECTIVE: We aimed to quantify the growth factors and cytokines in DPMSC-S and assess their effect on oral cancer cell proliferation. MATERIALS AND METHODS: DPMSCs were isolated from patients with healthy teeth (n = 5) that were indicated for extraction for orthodontic reasons. The cells were characterized using flow cytometry and conditioned medium (DPMSC-CM) was prepared. DPMSC-CM was subjected to a bead-based array to quantify the growth factors and cytokines that may affect oral carcinogenesis. The effect of DPMSC-CM (20%, 50%, 100%) on the proliferation of oral cancer cells (AW123516) was evaluated using a Ki-67-based assay at 48 h. AW13516 cultured in the standard growth medium acted as the control. RESULTS: VEGF, HCF, Ang-2, TGF-α, EPO, SCF, FGF, and PDGF-BB were the growth factors with the highest levels in the DPMSC-CM. The highest measured pro-inflammatory cytokine was TNF-α, followed by CXCL8. The most prevalent anti-inflammatory cytokine in the DPMSC-CM was IL-10, followed by TGF-ß1 and IL-4. Concentrations of 50% and 100% DPMSC-CM inhibited Ki-67 expression in AW13516, although the effect was non-significant. Moreover, 20% DPMSC-CM significantly increased Ki-67 expression compared to the control. CONCLUSIONS: The increased Ki-67 expression of oral cancer cells in response to 20% DPMSC-CM indicates the potential for cancer progression. Further research is needed to identify their effects on other carcinogenic properties, including apoptosis, stemness, migration, invasion, adhesion, and therapeutic resistance.

Consistent results of non-invasive PGT-A of human embryos using two different techniques for chromosomal analysis.

RESEARCH QUESTION: Are discordances in non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) results attributable to the technique used for chromosomal analysis? DESIGN: A prospective blinded study was performed (September 2018 to December 2019). In total 302 chromosomal analyses were performed: 92 trophectoderm PGT-A biopsies and their corresponding spent embryo culture medium (SCM) evaluated by two methods (n = 184), negative controls (n = 8), and trophectoderm and inner cell mass biopsies from trophectoderm-aneuploid embryos (n = 18). Trophectoderm analyses were carried out using Veriseq (Illumina), and SCM was analysed using Veriseq and NICS (Yikon). RESULTS: Genetic results were obtained for 96.8% of trophectoderm samples versus 92.4% for both SCM techniques. The mosaicism rate was higher for SCM regardless of the technique used: 30.4% for SCM-NICS and 28.3% for SCM-Veriseq versus 14.1% for trophectoderm biopsies (P = 0.013, P = 0.031, respectively). No significant differences in diagnostic concordance were seen between the two SCM techniques (74.6% for SCM-NICS versus 72.3% for SCM-Veriseq; P = 0.861). For embryos biopsied on day 6, these rates reached 92.0% and 86.5%, respectively. On reanalysing trophectoderm-aneuploid embryos, the discrepancies were shown to be due to maternal DNA contamination (55.6%; 5/9), embryo mosaicism (22.2%; 2/9) and low resolution in SCM-NICS (11.1%; 1/9) and in both SCM techniques (11.1%; 1/9). CONCLUSIONS: This is the first study evaluating the consistency of different chromosomal analysis techniques for niPGT-A. In conclusion, the diagnostic concordance between PGT-A and niPGT-A seems independent of the technique used. Optimization of culture conditions and medium retrieval provides a potential target to improve the reliability of niPGT-A.

Engineering Functional Rat Ovarian Spheroids Using Granulosa and Theca Cells.

Although menopausal hormone therapy (MHT) is the most effective approach to managing the loss of ovarian activity, serious side effects have been reported. Cell-based therapy is a promising alternative for MHT. This study constructed engineered ovarian cell spheroids and investigated their endocrine function. Theca and granulosa cells were isolated from ovaries of 10-week-old rats. Two types of engineered ovarian cell spheroids were fabricated through forced aggregation in microwells, multilayered spheroids with centralized granulosa aggregates surrounded by an outer layer of theca cells and mixed ovarian spheroids lacking spatial rearrangement. The ovarian cell spheroids were encapsulated into a collagen gel. Non-aggregated ovarian cells served as controls. The endocrine function of the engineered ovarian spheroids was assessed over 30 days. The structure of the spheroids was well maintained during culture. The secretion of 17ß-estradiol from both types of engineered ovarian cell spheroids was higher than in the control group and increased continuously in a time-dependent manner. Secretion of 17ß-estradiol in the multi-layered ovarian cell spheroids was higher than in the non-layered constructs. Increased secretion of progesterone was detected in the multi-layered ovarian cell spheroids at day 5 of culture and was sustained during the culture period. The initial secretion level of progesterone in the non-layered ovarian cell spheroids was similar to those from the controls and increased significantly from days 21 to 30. An in vitro rat model of engineered ovarian cell spheroids was developed that was capable of secreting sex steroid hormones, indicating that the hormone secreting function of ovaries can be recapitulated ex vivo and potentially adapted for MHT.

Interactome Analysis of iPSC Secretome and Its Effect on Macrophages In Vitro.

Induced pluripotent stem cell secretome (iPSC-CM) mitigate organ injury and help in repair. Macrophages play a critical role in tissue repair and regeneration and can be directed to promote tissue repair by iPSC-CM, although the exact mechanisms are not known. In the current investigative study, we evaluated the possible mechanism by which iPSC-CM regulates the phenotype and secretory pattern of macrophages in vitro. Macrophages were obtained from human peripheral blood mononuclear cells and differentiated to various subpopulations and treated with either iPSC-CM or control media in vitro. Macrophage phenotype was assessed by flow cytometry, gene expression changes by qRT PCR and secretory pattern by multiplex protein analysis. The protein and gene interaction network revealed the involvement of Amyloid precursor protein (APP) and ELAV-like protein 1 (ELAVL-1) both present in the iPSC-CM to play an important role in regulating the macrophage phenotype and their secretory pattern. This exploratory study reveals, in part, the possible mechanism and identifies two potential targets by which iPSC-CM regulate macrophages and help in repair and regeneration.

Extraction and characterization of fungal chitin nanofibers from Mucor indicus cultured in optimized medium conditions.

Chitin nanofibers (ChNFs) were extracted from Mucor indicus by a purification method followed by a mechanical treatment, cultivated under obtained optimum culture medium conditions to improve fungal chitin production rate. A semi synthetic media containing 50 g/l glucose was used for cultivation of the fungus in shake flasks. The cell wall analysis showed that N-acetyl glucosamine (GlcNAc) content, which is an indication of chitin content, was maximum in presence of 2.5 g/l H3PO4, 2.5 g/l of NaOH, 1 g/l of yeast extract, 1 mg/l of plant hormones, and 1 ml/l of trace metals. The chemical characterizations demonstrated that the isolated fibers had a degree of deacetylation lower than of 10%, and were phosphate free. The FTIR results revealed successful removal of different materials during the purification step. The FE-SEM of fibrillated chitin illustrated an average diameter of 28 nm. Finally, X-ray diffraction results showed that the crystallinity index of nanofibers was 82%.

Short chain fatty acids produced by Cutibacterium acnes inhibit biofilm formation by Staphylococcus epidermidis.

Biofilm formation by bacterial pathogens is associated with numerous human diseases and can confer resistance to both antibiotics and host defenses. Many strains of Staphylococcus epidermidis are capable of forming biofilms and are important human pathogens. Since S. epidermidis coexists with abundant Cutibacteria acnes on healthy human skin and does not typically form a biofilm in this environment, we hypothesized that C. acnes may influence biofilm formation of S. epidermidis. Culture supernatants from C. acnes and other species of Cutibacteria inhibited S. epidermidis but did not inhibit biofilms by Pseudomonas aeruginosa or Bacillus subtilis, and inhibited biofilms by S. aureus to a lesser extent. Biofilm inhibitory activity exhibited chemical properties of short chain fatty acids known to be produced from C. acnes. The addition of the pure short chain fatty acids propionic, isobutyric or isovaleric acid to S. epidermidis inhibited biofilm formation and, similarly to C. acnes supernatant, reduced polysaccharide synthesis by S. epidermidis. Both short chain fatty acids and C. acnes culture supernatant also increased sensitivity of S. epidermidis to antibiotic killing under biofilm-forming conditions. These observations suggest the presence of C. acnes in a diverse microbial community with S. epidermidis can be beneficial to the host and demonstrates that short chain fatty acids may be useful to limit formation of a biofilm by S. epidermidis.

Extracellular non-coding RNA signatures of the metacestode stage of Echinococcus multilocularis.

Extracellular RNAs (ex-RNAs) are secreted by cells through different means that may involve association with proteins, lipoproteins or extracellular vesicles (EV). In the context of parasitism, ex-RNAs represent new and exciting communication intermediaries with promising potential as novel biomarkers. In the last years, it was shown that helminth parasites secrete ex-RNAs, however, most work mainly focused on RNA secretion mediated by EV. Ex-RNA study is of special interest in those helminth infections that still lack biomarkers for early and/or follow-up diagnosis, such as echinococcosis, a neglected zoonotic disease caused by cestodes of the genus Echinococcus. In this work, we have characterised the ex-RNA profile secreted by in vitro grown metacestodes of Echinococcus multilocularis, the casuative agent of alveolar echinococcosis. We have used high throughput RNA-sequencing together with RT-qPCR to characterise the ex-RNA profile secreted towards the extra- and intra-parasite milieus in EV-enriched and EV-depleted fractions. We show that a polarized secretion of small RNAs takes place, with microRNAs mainly secreted to the extra-parasite milieu and rRNA- and tRNA-derived sequences mostly secreted to the intra-parasite milieu. In addition, we show by nanoparticle tracking analyses that viable metacestodes secrete EV mainly into the metacestode inner vesicular fluid (MVF); however, the number of nanoparticles in culture medium and MVF increases > 10-fold when metacestodes show signs of tegument impairment. Interestingly, we confirm the presence of host miRNAs in the intra-parasite milieu, implying their internalization and transport through the tegument towards the MVF. Finally, our assessment of the detection of Echinococcus miRNAs in patient samples by RT-qPCR yielded negative results suggesting the tested miRNAs may not be good biomarkers for this disease. A comprehensive study of the secretion mechanisms throughout the life cycle of these parasites will help to understand parasite interaction with the host and also, improve current diagnostic tools.

iPS-Derived Early Oligodendrocyte Progenitor Cells from SPMS Patients Reveal Deficient In Vitro Cell Migration Stimulation.

The most challenging aspect of secondary progressive multiple sclerosis (SPMS) is the lack of efficient regenerative response for remyelination, which is carried out by the endogenous population of adult oligoprogenitor cells (OPCs) after proper activation. OPCs must proliferate and migrate to the lesion and then differentiate into mature oligodendrocytes. To investigate the OPC cellular component in SPMS, we developed induced pluripotent stem cells (iPSCs) from SPMS-affected donors and age-matched controls (CT). We confirmed their efficient and similar OPC differentiation capacity, although we reported SPMS-OPCs were transcriptionally distinguishable from their CT counterparts. Analysis of OPC-generated conditioned media (CM) also evinced differences in protein secretion. We further confirmed SPMS-OPC CM presented a deficient capacity to stimulate OPC in vitro migration that can be compensated by exogenous addition of specific components. Our results provide an SPMS-OPC cellular model and encouraging venues to study potential cell communication deficiencies in the progressive form of multiple sclerosis (MS) for future treatment strategies.

Comparison of methods for the isolation of cell-free DNA from cell culture supernatant.

In vitro characterization of cell-free DNA using two-dimensional cell culture models is emerging as an important step toward an improved understanding of the physical and biological characteristics of cell-free DNA in human biology. However, precise measurement of the cell-free DNA in cell culture medium is highly dependent on the efficacy of the method used for DNA purification, and is often a juncture of experimental confusion. Therefore, in this study, we compared six commercially available cell-free DNA isolation kits for the recovery of cell-free DNA from the cell culture supernatant of a human bone cancer cell line (143B), including two magnetic bead-based manual kits, one automated magnetic bead-based extraction method, and three manual spin-column kits. Based on cell-free DNA quantitation and sizing, using the Qubit dsDNA HS assay and Bioanalyzer HS DNA assay, respectively, the different methods showed significant variability concerning recovery, reproducibility, and size discrimination. These findings highlight the importance of selecting a cell-free DNA extraction method that is appropriate for the aims of a study. For example, mutational analysis of cell-free DNA may be enhanced by a method that favors a high yield or is biased toward the isolation of short cell-free DNA fragments. In contrast, quantitative analysis of cell-free DNA in a comparative setting (e.g. measuring the fluctuation of cell-free DNA levels over time) may require the selection of a cell-free DNA isolation method that forgoes a high recovery for high reproducibility and minimal size bias.

Non-invasive molecular assessment of human embryo development and implantation potential.

In vitro fertilization (IVF) is the most common assisted reproductive technology used to treat infertility. Embryo selection for transfer in IVF cycles relies on the morphological evaluation by embryologists, either by conventional microscopic assessment or more recently by time-lapse imaging systems. Despite the introduction of time-lapse imaging improvements in IVF success rates have failed to materialize, therefore alternative approaches are needed. Recent studies have shown that embryos resulting in successful pregnancy differ in their secretome and metabolism compared to embryos that fail to implant, suggesting that molecular analysis of embryo culture medium could assist in non-invasive single embryo selection. However, this approach has yet to be adopted clinically due to the lack of appropriate highly sensitive screening technologies needed to assess volume-limited samples. Here we report the detection of hCGß, IL-8 and TNFα from conditioned culture media of single human embryos using electrochemical impedance spectroscopy. The impedimetric immunosensors revealed that morphologically non-viable embryos produce higher levels of IL-8 and TNFα, associated with abnormal cell division and cell death, respectively. More importantly, hCGß detection was able to discriminate apparently morphologically identical viable embryos. This work brings an objective dimension to embryo selection, which could overcome the major limitations of morphology-based embryo selection for implantation. Future work should include the validation of these biomarkers in a large patient cohort.

Capillary-assisted microfluidic biosensing platform captures single cell secretion dynamics in nanoliter compartments.

Cancer cells continuously secrete inflammatory biomolecules which play significant roles in disease progression and tumor metastasis toward secondary sites. Despite recent efforts to capture cancer cells' intercellular secretion heterogeneity using microfluidics, the challenges in operation of these systems as well as the complexity of designing a biosensing assay for long-term and real-time measurement of single cell secretions have become grand research barriers. Here, we present a new capillary-based microfluidic biosensing approach to easily and reliably capture ~500 single cells inside isolated dead-end nanoliter compartments using simple pipette injection, and quantify their individual secretion dynamics at the single cell resolution over a long period of culture (~16 h). We first present a detailed investigation of the fluid mechanics underlying the formation of nanoliter compartments in the microfluidic system. Based on the measurement of single cell capture efficiency, we employ a one-step FRET-based biosensor which monitors the single cancer cells' protease activity. The sensor reports the fluorescent signal as a product of amino acid chain cleavage and reduction in its quenching capability. Using the single cell protease secretion data, we identified modes of cell secretion dynamics in our cell sample. While most of the cells had low secretion levels, two other smaller and more aggressive secretion dynamics were cells with secretion modes that include sharp spikes or slow but progressive trend. The method presented here overcomes the difficulties associated with performing single cell secretion assays, enabling a feasible and reliable technique for high throughput measurement of metabolic activities in cancer cells.

Antibody-Free Multiplex Measurement of 23 Human Cytokines in Primary Cell Culture Secretome Using Targeted Mass Spectrometry.

Cytokines are commonly measured by immunoassays; however, these have limited multiplexing capacity, are costly, and can exhibit cross-reactivity. Multiple reaction monitoring (MRM) mass spectrometry is a robust method to quantify analytes with high specificity and multiplexing ability, hence we aimed to investigate its suitability as an alternative cost-effective method for cytokine measurement. Human keratinocyte conditioned media spiked with recombinant cytokines was used as an experimental system to evaluate sensitivity, linearity, and reproducibility of an MRM assay targeting 79 peptides representing 23 human cytokines. Our MRM method was able to identify 21 cytokines by two or more unique peptides and two cytokines by a single unique peptide. In a serum-free matrix, the median LOD and LOQ for cytokine peptides was 130 and 433 pg/mL, respectively. The presence of serum increased median LOD and LOQ by about 2.3-fold. The assay shows excellent replicate consistency with 8% intra- and 12% interday coefficient of variations. We found high pH reversed-phase fractionation a useful tool to increase assay sensitivity with the drawback of increasing its variability by approximately 10%. Overall, our results suggest utility of a multiplex cytokine MRM for routine measurement of secreted cytokines in cellular experiments under low serum conditions. Additional enrichment steps will be required in high complexity matrices such as serum.

Conditioned medium derived from FGF-2-modified GMSCs enhances migration and angiogenesis of human umbilical vein endothelial cells.

BACKGROUND: Angiogenesis plays an important role in tissue repair and regeneration, and conditioned medium (CM) derived from mesenchymal stem cells (MSC-CM) possesses pro-angiogenesis. Nevertheless, the profile and concentration of growth factors in MSC-CM remain to be optimized. Fibroblast growth factor-2 (FGF-2) has been proven to be an effective angiogenic factor. Thus, the aim of this study was to verify whether FGF-2 gene overexpression optimized CM from human gingival mesenchymal stem cells (hGMSCs) and whether such optimized CM possessed more favorable pro-angiogenesis effect. METHODS: First, FGF-2 gene-modified hGMSCs were constructed using lentiviral transfection technology (LV-FGF-2+-hGMSCs) and the concentration of angiogenesis-related factors in LV-FGF-2+-hGMSC-CM was determined by ELISA. Then, human umbilical vein endothelial cells (HUVECs) were co-cultured for 3 days with LV-FGF-2+-hGMSC-CM, and the expression level of placenta growth factor (PLGF), stem cell factor (SCF), vascular endothelial growth factor receptor 2 (VEGFR2) in HUVECs were determined by qRT-PCR, western blot, and cellular immunofluorescence techniques. The migration assay using transwell and in vitro tube formation experiments on matrigel matrix was conducted to determine the chemotaxis and angiogenesis enhanced by LV-FGF-2+-hGMSC-CM. Finally, NOD-SCID mice were injected with matrigel mixed LV-FGF-2+-hGMSC-CM, and the plug sections were analyzed by immunohistochemistry staining with anti-human CD31 antibody. RESULTS: LV-FGF-2+-hGMSC-CM contained significantly more FGF-2, vascular endothelial growth factor A (VEGF-A), and transforming growth factor ß (TGF-ß) than hGMSC-CM. HUVECs pretreated with LV-FGF-2+-hGMSC-CM expressed significantly more PLGF, SCF, and VEGFR2 at gene and protein level than hGMSC-CM pretreated HUVECs. Compared with hGMSC-CM, LV-FGF-2+-hGMSC-CM presented significantly stronger chemotaxis to HUVECs and significantly strengthened HUVECs mediated in vitro tube formation ability. In vivo, LV-FGF-2+-hGMSC-CM also possessed stronger promoting angiogenesis ability than hGMSC-CM. CONCLUSIONS: Overexpression of FGF-2 gene promotes hGMSCs paracrine of angiogenesis-related growth factors, thereby obtaining an optimized conditioned medium for angiogenesis promotion.

Quantitation of Cyclosporin A in Cell Culture Media by Differential Mobility Mass Spectrometry (DMS-MS/MS).

Cell permeability is an important factor in determining the bioavailability of therapeutics that is usually measured by cell culture testing. The concentration of pharmaceutical in a medium such as Hank's Balanced Salt Solution with HEPES organic buffer (HBSS-HEPES) is measured at a series of time points, making simplicity and high throughput of the analytical method important characteristics. We report an electrospray differential mobility spectrometry mass spectrometry method (nanoESI-DMS-MS) for the rapid determination of cyclosporin A (CsA, cyclosporine) concentration in such a buffer. DMS technology provides gas phase atmospheric pressure ion filtration for small-molecule bioanalytical methods that suppresses interfering ions and reduces chemical noise, without the use of chromatography. This allows simplified sample preparation, fast calibration curve development, and shortened analysis times. It has also been noted that the DMS prefilter can reduce contamination of the mass spectrometer by salts, thereby extending mass spectrometer system uptime.In the application described here, DMS-MS/MS is applied to cyclosporine A (CsA) in cell medium. Sample preparation is limited to dilution with an ammonium acetate-methanol-water mobile phase and the addition of CsA-d4 internal standard. The isotope ratio data are obtained in DMS-MS MRM mode observing NH3 loss from the ammonium adduct of the two species. A calibration curve with high linearity (R2 = 0.998) is rapidly obtained with nearly zero intercept, while it was found that a liquid chromatography LC-MS method required a preliminary SPE step to obtain a linear calibration curve. The time for data acquisition in the DMS-MS MRM method with flow injection (FIA) or infusion introduction at ESI flow of 400 nL/min is typically 30 s leading to a cycle time of less than 1 min.

Analysis of Secreted MIF from Cultured Murine Bone Marrow-Derived Immune Cells.

Macrophage migration inhibitory factor (MIF) is expressed and released ubiquitously by numerous cell types and tissues. MIF is detected and constitutively expressed at the protein level both intra- and extracellularly. This chapter outlines methods for cultivating, purifying, detecting, and quantifying concentrations of MIF from murine primary derived macrophages and dendritic cell culture supernatants.

Dietary polyunsaturated fatty acids modulate adipose secretome and is associated with changes in mammary epithelial stem cell self-renewal.

Chronic low-grade adipose inflammation, characterized by aberrant adipokine production and pro-inflammatory macrophage activation/polarization is associated with increased risk of breast cancer. Adipocyte fatty acid composition is influenced by dietary availability and may regulate adipokine secretion and adipose inflammation. After feeding F344 rats for 20 weeks with a Western diet or a fish oil-supplemented diet, we cultured primary rat adipose tissue in a three-dimensional explant culture and collected the conditioned medium. The rat adipose tissue secretome was assayed using the Proteome Profiler Cytokine XL Array, and adipose tissue macrophage polarization (M1/M2 ratio) was assessed using the iNOS/ARG1 ratio. We then assessed the adipokine's effects upon stem cell self-renewal using primary human mammospheres from normal breast mammoplasty tissue. Adipose from rats fed the fish oil diet had an ω-3:ω-6 fatty acid ratio of 0.28 compared to 0.04 in Western diet rats. The adipokine profile from the fish oil-fed rats was shifted toward adipokines associated with reduced inflammation compared to the rats fed the Western diet. The M1/M2 macrophage ratio decreased by 50% in adipose of fish oil-fed rats compared to that from rats fed the Western diet. Conditioned media from rats fed the high ω-6 Western diet increased stem cell self-renewal by 62%±9% (X¯%±SD) above baseline compared to only an 11%±11% increase with the fish oil rat adipose. Modulating the adipokine secretome with dietary interventions therefore may alter stromal-epithelial signaling that plays a role in controlling mammary stem cell self-renewal.

In vitro characteristics of an airway barrier-disrupting factor secreted by Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is a major contributor to the pathophysiology of chronic rhinosinusitis (CRS). Previous research has shown that S. aureus-secreted products disrupt the airway barrier. METHODS: S. aureus ATCC 13565 and 25923 strains were grown at exponential, postexponential, and stationary phases. Microbial conditioned media (CM) was collected from the cultures and ultrafiltered (UF). Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was performed on the UF-CM. UF-CM was subjected to heat and protease treatment, size fractionation, and ultracentrifugation (UC) separation. Human nasal epithelial cells grown at air-liquid interface (HNEC-ALI) cultures were exposed to purified alpha hemolysin (Hla), staphylococcal enterotoxin A (SEA), lipoteichoic acid (LTA), and UF-CM. Barrier function outcomes were measured by transepithelial electrical resistance (TEER) and apparent permeability (Papp). UC fraction exposed cultures were subjected to immunofluorescence microscopy for tight junction (TJ) protein zonula occludens-1 (ZO-1). RESULTS: LC-ESI-MS/MS identified 107 proteins, with Hla being most abundant. Hla, SEA, and LTA did not alter the HNEC-ALI barrier as measured by TEER or Papp. Barrier disruption caused by UF-CM peaked in the postexponential phase, was sensitive to heat and protease treatment, >30-kDa in size, and enriched in the UC fraction. HNEC-ALI exposed to UF-CM and UC demonstrated loss of ZO-1 localization. CONCLUSION: These results suggest that the S. aureus factor responsible for TJ disruption in HNEC-ALI cultures is either a protein-macromolecule or a combination of secreted factors. The product is enriched in the UC fraction, suggesting it is associated with large structures such as membrane components or vesicles.