The separation and identification of circulating small extracellular vesicles from endurance-trained, strength-trained and recreationally active men.

Small extracellular vesicles (EV) are membrane-encapsulated particles that carry bioactive cargoes, are released by all cell types and are present in all human biofluids. Changes in EV profiles and abundance occur in response to acute exercise, but this study investigated whether individuals with divergent histories of exercise training (recreationally active controls - CON; endurance-trained - END; strength-trained - STR) presented with varied abundances of small EVs in resting samples and whether the abundance of small EVs differed within each group across two measurement days. Participants (n = 38, all male; CON n = 12, END n = 13, STR n = 13) arrived at the lab on two separate occasions in a rested, overnight fasted state, with standardisation of time of day of sampling, recent dietary intake, time since last meal and time since last exercise training session (∼40 h). Whole blood samples were collected and separated into plasma from which small EVs were separated using size exclusion chromatography and identified in accordance with the Minimal Information For Studies of Extracellular Vesicles (MISEV) guidelines. No differences in the abundance of small EVs were observed within or between groups across multiple methods of small EV identification (nanoparticle tracking analysis, flow cytometry, immunoblot of specific EV markers). Targeted metabolomics of the small EV preparations identified 96 metabolites that were associated with the structure and function of small EVs, with no statistically significant differences in concentrations observed across groups. The results of the current study suggest that the abundance and metabolomic profile of small EVs derived from men with divergent histories of exercise training are similar to those in resting blood samples. KEY POINTS: Extracellular vesicles (EV) are membrane-encapsulated particles that are present in circulation and carry bioactive materials as 'cargo'. The abundance and profile of small EVs are responsive to acute exercise, but little is known about the relationship between small EVs and exercise training. This study examined the abundance, and a targeted metabolomic profile, of small EVs separated from the blood of endurance athletes, strength athletes and recreationally active controls at rest (∼40 h after the most recent exercise session) on two separate but identical lab visits. No differences were observed in the abundance or metabolomic profile of small EV preparations between the groups or between the lab visits within each group. Further research should determine whether the bioactive cargoes (e.g. RNA, protein and additional metabolites) carried within EVs are altered in individuals with divergent histories of exercise training or in response to exercise training interventions.

An extensive study of potential inhibitors of extracellular vesicles release in triple-negative breast cancer.

BACKGROUND: Cancer cells release heterogeneous populations of extracellular vesicles (EVs) that transmit aggressive phenotypic traits to recipient cells. We aimed to establish if the heterogenous EVs population or a sub-population is responsible, if we could block undesirable cell-to-cell communication by EVs, and, if some EVs continued to be released, would their undesirable influences on recipient cells continue. METHODS: Three triple-negative breast cancer (TNBC) cell lines were used. Non-toxic concentrations of calpeptin, Y27632, manumycin A, GW4869 and combinations thereof were tested to block EVs. Ultracentrifugation-based methods collected EVs, which were then characterised by nanoparticle tracking analysis, immunoblotting, and transmission electron microscopy. A quick screening flow cytometry method evaluated EVs in solution. The influences of EVs on recipient cells' migration was investigated. RESULTS: All EV sub-populations were apparently involved in transmitting undesirable phenotypic characteristics. All compounds/combinations significantly (64-98%) reduced EVs' release. Our quick screening broadly reflected our more comprehensive EVs analysis. The 2-36% of EVs that continued to be released caused less transmission to recipient cells, but not on a comparable scale to the reduction of EVs release achieved. CONCLUSION: Up to 98% inhibition of EVs' release was achieved. To prevent the transmission of undesirable phenotypic traits by EVs, their total inhibition may be necessary.

Functional assays reflective of cancer hallmarks in BT-549 cells are not impacted by media supplemented with exercise-trained plasma.

NEW FINDINGS: What is the central question of this study? Little is known regarding the effects of media supplemented with resting plasma from exercise-trained individuals, despite the established bioactive effects of acutely exercised samples. Does media supplemented with resting plasma from endurance-trained, strength-trained or recreationally active controls impact hallmarks of cancer in BT-549 cells? What is the main finding and its importance? Supplementing media with plasma from these trained athletes did not impact proliferation, migration, invasion or anoikis resistance compared to plasma from recreationally-active controls. These findings suggest that 'anti-cancer' effects of exercise are not present in resting blood samples of exercise-trained individuals. ABSTRACT: Media supplemented with sera from acutely exercised men has been shown to have 'anti-cancer' effects on prostate and breast cancer cell lines. This study investigated whether media supplemented with plasma samples taken at rest (≥30 h since the most recent exercise session) from men who were endurance-trained (END), strength-trained (STR) or recreationally active controls (CON) impacted the results of four assays that mimic hallmarks of cancer (proliferation, migration, extracellular matrix invasion and anoikis resistance) in the BT-549 breast cancer cell line. Compared to control conditions of either serum-free media or fetal bovine serum as appropriate, BT-549 cells cultured with plasma-supplemented media regardless of group resulted in greater cell proliferation (∼20-50%) and cell migration (∼15-20%), and lower extracellular matrix invasion (∼10-20%) and anoikis resistance (∼15-20%). Supplementing media with plasma from END or STR did not impact any outcomes of these assays compared to plasma from CON. Media supplemented with human plasma can impact functional assays reflective of cancer hallmarks in BT-549 cells, but effects of exercise on proliferation, migration, extracellular matrix invasion and anoikis resistance were not evident in resting blood samples of individuals with a prior history of exercise training.

Within-Subject Variability and the Influence of Exercise Training History on the Resting Plasma Metabolome in Men.

This study investigated within-subject variability in the circulating metabolome under controlled conditions, and whether divergent exercise training backgrounds were associated with alterations in the circulating metabolome assessed in resting samples. Thirty-seven men comprising of endurance athletes (END; body mass, 71.0 ± 6.8 kg; fat-free mass index, 16.9 ± 1.1 kg/m2), strength athletes (STR; 94.5 ± 8.8 kg; 23.0 ± 1.8 kg/m2), and recreationally active controls (CON; 77.6 ± 7.7 kg; 18.1 ± 1.0 kg/m2) provided blood samples after an overnight fast on two separate occasions controlled for time of day of sampling, recent dietary intake, time since last meal, and time since last exercise training session. A targeted profile of metabolites, performed using liquid chromatography and mass spectrometry on plasma samples, identified 166 individual metabolites and metabolite features, which were analyzed with intraclass correlation coefficients, a multilevel principal component analysis, and univariate t tests adjusted for multiple comparisons. The median intraclass correlation coefficient was .49, with 46 metabolites displaying good reliability and 31 metabolites displaying excellent reliability. No difference in the abundance of any individual metabolite was identified within groups when compared between visits, but a combined total of 44 metabolites were significantly different (false discovery rate <0.05) between groups (END vs. CON, 42 metabolites; STR vs. CON, 10 metabolites; and END vs. STR, five metabolites). Under similar measurement conditions, the reliability of resting plasma metabolite concentrations varies largely at the level of individual metabolites with ∼48% of metabolites displaying good-to-excellent reliability. However, a history of exercise training was associated with alterations in the abundance of ∼28% of metabolites in the targeted profile employed in this study.

Proteomics profiling identifies extracellular vesicles' cargo associated with tumour cell induced platelet aggregation.

BACKGROUND: Cancer patients have an increased risk of developing venous thromboembolism, with up to 30% dying within a month of their development. Some cancer cells are known to induce platelet aggregation, and this interaction is understood to contribute to thrombosis and haematogenous metastasis. Many researchers have reported on extracellular vesicles (EVs) released from platelets. However, less is known about how cancer cells' EVs may affect platelet function. Here EVs released by triple-negative breast cancer (TNBC) cell line variants were extensively investigated in this regard. METHODS: EVs were separated from conditioned media of TNBC Hs578T and Hs578Ts(i)8 cells using filtration and ultracentrifugation and were characterised by nanoparticle tracking analysis, immunoblots, and transmission electron microscopy. Blood samples from consenting donors were procured, and their platelets collected by differential centrifugation. Light transmission aggregometry and optical microscopy evaluated the potential interaction of TNBC cells and their EVs with platelets. Global proteomic analysis was performed on the EVs, by in-solution digestion and mass spectrometry. Data analysis included the use of Perseus, FunRich, and Vesiclepedia. Immunoblotting was used as a secondary method to investigate some key EV cargo proteins identified by the global proteomics approach. RESULTS: Both TNBC cell variants induced platelet aggregation. Increasing cell numbers significantly reduced the time taken for platelet aggregation to occur. EVs released by the cells also resulted in platelet aggregation. The time to induce platelet aggregation was EV dose-dependent. Proteomics profiling and immunoblotting of the EVs' cargo identified candidate proteins (including uPAR and PDGFRß) that may be involved during this process. CONCLUSIONS: TNBC cells induce platelet aggregation. Furthermore, the cell-free EVs induced this undesirable effect. A number of EV cargo proteins were identified that may be relevant as therapeutic targets.

International Society for Extracellular Vesicles and International Society for Cell and Gene Therapy statement on extracellular vesicles from mesenchymal stromal cells and other cells: considerations for potential therapeutic agents to suppress coronavirus disease-19.

STATEMENT: The International Society for Cellular and Gene Therapies (ISCT) and the International Society for Extracellular Vesicles (ISEV) recognize the potential of extracellular vesicles (EVs, including exosomes) from mesenchymal stromal cells (MSCs) and possibly other cell sources as treatments for COVID-19. Research and trials in this area are encouraged. However, ISEV and ISCT do not currently endorse the use of EVs or exosomes for any purpose in COVID-19, including but not limited to reducing cytokine storm, exerting regenerative effects or delivering drugs, pending the generation of appropriate manufacturing and quality control provisions, pre-clinical safety and efficacy data, rational clinical trial design and proper regulatory oversight.

Development of acquired resistance to lapatinib may sensitise HER2-positive breast cancer cells to apoptosis induction by obatoclax and TRAIL.

BACKGROUND: Lapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells. METHODS: We examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer. RESULTS: In this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells. CONCLUSIONS: Our findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies.

Neuromedin U alters bioenergetics and expands the cancer stem cell phenotype in HER2-positive breast cancer.

Neuromedin U (NmU) is a neuropeptide belonging to the neuromedin family. Recently, we reported a significant association between NmU and breast cancer, particularly correlating with increased aggressiveness, resistance to HER2-targeted therapies and overall significantly poorer outcome for patients, although the mechanism through which it exerts this effect remained unexplained. Investigating this, here we found that ectopic over-expression of NmU in HER2-positive breast cancer cells induced aberrant metabolism, with increased glycolysis, likely due to enhanced pyruvate dehydrogenase kinase activity. Similar results were observed in HER2-targeted drug-resistant cell variants, which we had previously shown to display increased levels of NmU. Overexpression of NmU also resulted in upregulation of epithelial-mesenchymal transition markers and increased IL-6 secretion which, together with aberrant metabolism, have all been associated with the cancer stem cell (CSC) phenotype. Flow cytometry experiments confirmed that NmU-overexpressing and HER2-targeted drug-resistant cells showed an increased proportion of cells with CSC phenotype (CD44+ /CD24- ). Taken together, our results report a new mechanism of action for NmU in HER2-overexpressing breast cancer that enhances resistance to HER2-targeted drugs through conferring CSC characteristics and expansion of the CSC phenotype.

Neratinib resistance and cross-resistance to other HER2-targeted drugs due to increased activity of metabolism enzyme cytochrome P4503A4.

BACKGROUND: Neratinib is in Phase 3 clinical trials but, unfortunately, the development of resistance is inevitable. Here, we investigated the effects of acquired neratinib resistance on cellular phenotype and the potential mechanism of this resistance. METHODS: Neratinib-resistant variants of HER2-positive breast cancer cells were developed and their cross-resistance investigated using cytotoxicity assays. Similarly, sensitivity of trastuzumab-resistant and lapatinib-resistant cells to neratinib was assessed. Cellular phenotype changes were evaluated using migration, invasion and anoikis assays. Immunoblotting for HER family members and drug efflux pumps, as well as enzyme activity assays were performed. RESULTS: Neratinib resistance conferred cross-resistance to trastuzumab, lapatinib and afatinib. Furthermore, the efficacy of neratinib was reduced in trastuzumab- and lapatinib-resistant cells. Neratinib-resistant cells were more aggressive than their drug-sensitive counterparts, with increased CYP3A4 activity identified as a novel mechanism of neratinib resistance. CONCLUSIONS: The potential of increased CYP3A4 activity as a biomarker and/or target to add value to neratinib warrants investigation.

Neuromedin U: a multifunctional neuropeptide with pleiotropic roles.

BACKGROUND: Neuromedin U (NmU) belongs to the neuromedin family, comprising a series of neuropeptides involved in the gut-brain axis and including neuromedins B and C (bombesin-like), K (neurokinin B), L (neurokinin A or neurotensin), N, S, and U. CONTENT: Although initially isolated from porcine spinal cord on the basis of their ability to induce uterine smooth muscle contraction, these peptides have now been found to be expressed in several different tissues and have been ascribed numerous functions, from appetite regulation and energy balance control to muscle contraction and tumor progression. NmU has been detected in several species to date, particularly in mammals (pig, rat, rabbit, dog, guinea pig, human), but also in amphibian, avian, and fish species. The NmU sequence is highly conserved across different species, indicating that this peptide is ancient and plays an important biological role. Here, we summarize the main structural and functional characteristics of NmU and describe its many roles, highlighting the jack-of-all-trades nature of this neuropeptide. SUMMARY: NmU involvement in key processes has outlined the possibility that this neuropeptide could be a novel target for the treatment of obesity and cancer, among other disorders. Although the potential for NmU as a therapeutic target is obvious, the multiple functions of this molecule should be taken into account when designing an approach to targeting NmU and/or its receptors.

The Role of Exosomes in Breast Cancer.

BACKGROUND: Although it has been long realized that eukaryotic cells release complex vesicular structures into their environment, only in recent years has it been established that these entities are not merely junk or debris, but that they are tailor-made specialized minimaps of their cell of origin and of both physiological and pathological relevance. These exosomes and microvesicles (ectosomes), collectively termed extracellular vesicles (EVs), are often defined and subgrouped first and foremost according to size and proposed origin (exosomes approximately 30-120 nm, endosomal origin; microvesicles 120-1000 nm, from the cell membrane). There is growing interest in elucidating the relevance and roles of EVs in cancer. CONTENT: Much of the pioneering work on EVs in cancer has focused on breast cancer, possibly because breast cancer is a leading cause of cancer-related deaths worldwide. This review provides an in-depth summary of such studies, supporting key roles for exosomes and other EVs in breast cancer cell invasion and metastasis, stem cell stimulation, apoptosis, immune system modulation, and anti-cancer drug resistance. Exosomes as diagnostic, prognostic, and/or predictive biomarkers and their potential use in the development of therapeutics are discussed. SUMMARY: Although not fully elucidated, the involvement of exosomes in breast cancer development, progression, and resistance is becoming increasingly apparent from preclinical and clinical studies, with mounting interest in the potential exploitation of these vesicles for breast cancer biomarkers, as drug delivery systems, and in the development of future novel breast cancer therapies.

miR-630 targets IGF1R to regulate response to HER-targeting drugs and overall cancer cell progression in HER2 over-expressing breast cancer.

BACKGROUND: While the treatment of HER2 over-expressing breast cancer with recent HER-targeted drugs has been highly effective for some patients, primary (also known as innate) or acquired resistance limits the success of these drugs. microRNAs have potential as diagnostic, prognostic and predictive biomarkers, as well as replacement therapies. Here we investigated the role of microRNA-630 (miR-630) in breast cancer progression and as a predictive biomarker for response to HER-targeting drugs, ultimately yielding potential as a therapeutic approach to add value to these drugs. METHODS: We investigated the levels of intra- and extracellular miR-630 in cells and conditioned media from breast cancer cell lines with either innate- or acquired- resistance to HER-targeting lapatinib and neratinib, compared to their corresponding drug sensitive cell lines, using qPCR. To support the role of miR-630 in breast cancer, we examined the clinical relevance of this miRNA in breast cancer tumours versus matched peritumours. Transfection of miR-630 mimics and inhibitors was used to manipulate the expression of miR-630 to assess effects on response to HER-targeting drugs (lapatinib, neratinib and afatinib). Other phenotypic changes associated with cellular aggressiveness were evaluated by motility, invasion and anoikis assays. TargetScan prediction software, qPCR, immunoblotting and ELISAs, were used to assess miR-630's regulation of mRNA, proteins and their phosphorylated forms. RESULTS: We established that introducing miR-630 into cells with innate- or acquired- resistance to HER-drugs significantly restored the efficacy of lapatinib, neratinib and afatinib; through a mechanism which we have determined to, at least partly, involve miR-630's regulation of IGF1R. Conversely, we demonstrated that blocking miR-630 induced resistance/insensitivity to these drugs. Cellular motility, invasion, and anoikis were also observed as significantly altered by miR-630 manipulation, whereby introducing miR-630 into cells reduced cellular aggression while inhibition of miR-630 induced a more aggressive cellular phenotype. CONCLUSIONS: Taken together, our findings suggest miR-630 as a key regulator of cancer cell progression in HER2 over-expressing breast cancer, through targeting of IGF1R. This study supports miR-630 as a diagnostic and a predictive biomarker for response to HER-targeted drugs and indicates that the therapeutic addition of miR-630 may enhance and improve patients' response to HER-targeting drugs.

miR-34a is an intracellular and exosomal predictive biomarker for response to docetaxel with clinical relevance to prostate cancer progression.

BACKGROUND: Docetaxel-resistance limits successful treatment of castration resistant prostate cancer. We previously demonstrated that extracellular vesicles (exosomes) may play a role in regulating docetaxel resistance. Here, we investigated intracellular and extracellular (exosomal) miRNAs related to docetaxel resistance. METHODS: Following global miRNA profiling of cell line models of docetaxel-resistance and their corresponding exosomes, we investigated the clinical relevance of four selected miRNAs (miR-598, miR-34a, miR-146a, miR-148a) in four publically available clinical cohorts representing both primary and advanced disease in tissue and urine specimens. One of these miRNAs, miR-34a was selected for functional evaluation by miRNA inhibition and over-expression in vitro. We further assessed the panel of miRNAs for their combined clinical relevance as a biomarker signature by examining their common predicted targets. RESULTS: A strong correlation was found between the detection of miRNAs in exosomes and their corresponding cells of origin. Of the miRNAs chosen for further validation and clinical assessment, decreased miR-34a levels showed substantial clinical relevance and so was chosen for further analysis. Manipulating miR-34a in prostate cancer cells confirms that this miRNA regulates BCL-2 and may, in part, regulate response to docetaxel. When combined, these miRNAs are predicted to regulate a range of common mRNA targets, two of which (e.g., SNCA, SCL7A5) demonstrate a strong relationship with prostate cancer progression and poor prognosis. CONCLUSIONS: This study supports the extracellular environment as an important source of minimally invasive predictive biomarkers representing their cellular origin. Using miR-34a as example, we showed that biomarkers identified in this manner may also hold functional relevance.

Effect of In Vitro Enzyme Digestion and Bile Treatment on Milk Extracellular Vesicles Stability.

SCOPE: Milk extracellular vesicles (EVs) are nanosized particles with potential immune bioactivities. This study examines their fate during in vitro infant gastrointestinal digestion (GI). METHODS AND RESULTS: Bovine milk is digested using the in vitro INFOGEST method, adjusted for the infant. To unravel the contribution of digestive enzymes from bile, milk is treated with digestive enzymes, bile, or a combination of both. EVs are collected posttreatment using differential ultracentrifugation. EVs characterization includes electrophoresis, immunoblotting, nanoparticle tracking analysis, and atomic force microscopy. EVs protein markers programmed cell death 6-interacting protein (ALIX), tumor susceptibility gene 101 (TSG101), cluster of differentiation 9 (CD9), and xanthine dehydrogenase (XDH) are detected after gastric digestion (G60), but their signal intensity is significantly reduced by intestinal conditions (p < 0.05). Enzyme digestion, compared to bile treatment (I60 + bile), results in a significant reduction of signal intensities for TSG101 and CD9 (p < 0.05). Nanoparticle tracking analysis shows a significant reduction (p < 0.05) of EV numbers at the end of the intestinal phase. EVs are detected by atomic force microscopy at the end of the intestinal phase, showing that intact EVs can survive upper gut digestion. CONCLUSION: Intact EVs can be found at the end of the intestinal phase. However, digestive enzymes and bile reduce the quantity and characteristics of EVs, with digestive enzymes playing a larger role.

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

Correlating transcriptional networks to breast cancer survival: a large-scale coexpression analysis.

Weighted gene coexpression network analysis (WGCNA) is a powerful 'guilt-by-association'-based method to extract coexpressed groups of genes from large heterogeneous messenger RNA expression data sets. We have utilized WGCNA to identify 11 coregulated gene clusters across 2342 breast cancer samples from 13 microarray-based gene expression studies. A number of these transcriptional modules were found to be correlated to clinicopathological variables (e.g. tumor grade), survival endpoints for breast cancer as a whole (disease-free survival, distant disease-free survival and overall survival) and also its molecular subtypes (luminal A, luminal B, HER2+ and basal-like). Examples of findings arising from this work include the identification of a cluster of proliferation-related genes that when upregulated correlated to increased tumor grade and were associated with poor survival in general. The prognostic potential of novel genes, for example, ubiquitin-conjugating enzyme E2S (UBE2S) within this group was confirmed in an independent data set. In addition, gene clusters were also associated with survival for breast cancer molecular subtypes including a cluster of genes that was found to correlate with prognosis exclusively for basal-like breast cancer. The upregulation of several single genes within this coexpression cluster, for example, the potassium channel, subfamily K, member 5 (KCNK5) was associated with poor outcome for the basal-like molecular subtype. We have developed an online database to allow user-friendly access to the coexpression patterns and the survival analysis outputs uncovered in this study (available at http://glados.ucd.ie/Coexpression/).

EGFR and HER2 inhibition in pancreatic cancer.

The aim of this study was to investigate the effect of lapatinib, a selective inhibitor of EGFR/HER2 tyrosine kinases, on pancreatic cancer cell lines both alone and in combination with chemotherapy. Two cell lines, BxPc-3 and HPAC, displayed the greatest sensitivity to lapatinib (IC(50)<2 µM). Lapatinib also demonstrated some activity in three K-Ras mutated pancreatic cancer cell lines which displayed resistance to erlotinib. Drug effect/combination index (CI) isobologram analysis was used to study the interactions of lapatinib with gemcitabine, cisplatin and 5'deoxy-5'fluorouridine. Concentration-dependent anti-proliferative effects of lapatinib in combination with chemotherapy were observed. To evaluate the potential effect of lapatinib in pancreatic cancer tumours, and to identify a subset of patient most likely to benefit from lapatinib, expression of EGFR and HER2 were investigated in 72 pancreatic cancer tumour specimens by immunohistochemistry. HER2 membrane expression was observed in only 1 % of cases, whereas 44 % of pancreatic tumours expressed EGFR. Based on our in vitro results, lapatinib may provide clinical benefit in EGFR positive pancreatic ductal adenocarcinoma.

Doxorubicin Loading into Milk and Mesenchymal Stem Cells' Extracellular Vesicles as Drug Delivery Vehicles.

Extracellular vesicles (EVs) have great potential as drug delivery vehicles. While mesenchymal/stromal stem cell (MSC) conditioned medium (CM) and milk are potentially safe and scalable sources of EVs for this purpose, the suitability of MSC EVs and milk EVs as drug delivery vehicles has never been compared and so was the objective of this study. Here EVs were separated from MSCs' CM and from milk and were characterised by nanoparticle tracking analysis, transmission electron microscopy, total protein quantification, and immunoblotting. An anti-cancer chemotherapeutic drug, doxorubicin (Dox), was then loaded into the EVs by one of three methods: by passive loading or by active loading by either electroporation or sonication. Dox-loaded EVs were analysed by fluorescence spectrophotometer, high-performance liquid chromatography (HPLC), and imaging flow cytometer (IFCM). Our study showed that EVs were successfully separated from the milk and MSC CM, with significantly (p < 0.001) higher yields of milk EVs/mL starting material compared to MSC EVs/mL of starting material. Using a fixed amount of EVs for each comparison, electroporation achieved significantly more Dox loading when compared to passive loading (p < 0.01). Indeed, of 250 µg of Dox made available for loading, electroporation resulted in 90.1 ± 12 µg of Dox loading into MSC EVs and 68.0 ± 10 µg of Dox loading into milk EVs, as analysed by HPLC. Interestingly, compared to the passive loading and electroporation approach, after sonication significantly fewer CD9+ EVs/mL (p < 0.001) and CD63+ EVs/mL (p < 0.001) existed, as determined by IFCM. This observation indicates that sonication, in particular, may have detrimental effects on EVs. In conclusion, EVs can be successfully separated from both MSC CM and milk, with milk being a particularly rich source. Of the three methods tested, electroporation appears to be superior for achieving maximum drug loading while not causing damage to EV surface proteins.