Microparticles (MPs) play a vital role in cell communication by facilitating the horizontal transfer of cargo between cells. Recently, we described a novel “non-genetic” mechanism for the acquisition of multidrug resistance (MDR) in cancer cells by intercellular transfer of functional P-gp, via MPs. MDR is caused by the overexpression of the efflux transporters P-glycoprotein (P-gp) and Multidrug Resistance-Associated Protein 1 (MRP1). These transporters efflux anticancer drugs from resistant cancer cells and maintain sublethal intracellular drug concentrations. By conducting MP transfer experiments, we show that MPs derived from DX breast cancer cells selectively transfer P-gp to malignant MCF-7 breast cells only, in contrast to VLB100 leukaemic cell-derived MPs that transfer P-gp and MRP1 to both malignant and non-malignant cells. The observed transfer selectivity is not the result of membrane restrictions for intercellular exchange, limitations in MP binding to recipient cells or the differential expression of the cytoskeletal protein, Ezrin. CD44 (isoform 10) was found to be selectively present on the breast cancer-derived MPs and not on leukaemic MPs and may contribute to the observed selective transfer of P-gp to malignant breast cells observed. Using the MCF-7 murine tumour xenograft model we demonstrated the stable transfer of P-gp by MPs in vivo, which was found to localize to the tumour core as early as 24 hours post MP exposure and to remain stable for at least 2 weeks. These findings demonstrate a remarkable capacity by MPs to disseminate a stable resistant trait in the absence of any selective pressure.

Once thought of as inert remnants of cellular processes, the significance of membrane vesicles is now expanding as their capacity to package and transfer bioactive molecules during intercellular communication is established. This ability to serve as vectors in the trafficking of cellular cargo is of mounting interest in the context of cancer, particularly in the dissemination of deleterious cancer traits from parent cells to recipient cells. Although microparticles (MPs) contribute to the pathogenesis of cancer, their unique characteristics can also be also exploited in the context of cancer treatment. The detection of MPs in body fluids has the potential to provide an effective means for the diagnosis, prognosis and surveillance of cancer patients. The use of such easily accessible systemic biomarkers that reflect the characteristics of the parent cells circumvents the need for invasive biopsy procedures. In addition, the autologous nature of MPs may allow them to be used as novel therapeutic drug carriers. Thus the modulation of MP vesiculation, the detection of MPs in disease monitoring, and the application of MPs as therapeutic delivery vehicles present prospective clinical interventions in the treatment of cancer. Revision Notes1. The authors overviewed the contribution of microparticles (MPs) to the pathogenesis of cancer and the potential for clinical application of MPs. In contrast to the title of the manuscript "Microparticles in Cancer: A Review of Recent Developments and the Potential for Clinical Application.", a large part of the content of this manuscript occupied the refer and discussed on the publications of the authors' group. The authors should discuss more generally on state-of-the-art for clinical application of MPs or some other extracellular vesicles (EVs). The authors have now included an extra section on Page 10 discussing the clinical applications of extracellular vesicles in cancer.2. The authors should refer to sufficient publications in general, especially in cancer-derived extracellular vesicles.Other references relevant to this study have now been added.3. The term "microparticles" mainly used in this manuscript is not well documented. Is there any specific differences between MPs and EVs? If so please describe those appropriately in the text. An explanation has now been added on Page 3 to describe the different membrane vesicles.4. The authors described "exosomes" in the text in the section 4. However, there is no mentioned the differ from MPs. This is a very confusing. An explanation of the different membrane vesicles has now been included on Page 3.5. The content of the figure is a very limited information. Modify the figure more generously including more other findings on tumor metastasis and drug resistance. Additional figures have now been included. Revision NotesAbbreviations: Matrix metalloproteinases, MMPs; mesenchymal stem cells, MSC; MPs, microparticles; miRNA, microRNA; microvesicles, MVs; MDR, multidrug resistance; non-small cell lung carcinoma, NSCLC; P-gp, P...

The accurate quantification of changes in the abundance of proteins is one of the main applications of proteomics. The maintenance of accuracy can be affected by bias and error that can occur at many points in the experimental process, and normalization strategies are crucial to attempt to overcome this bias and return the sample to its regular biological condition, or normal state. Much work has been published on performing normalization on data post-acquisition with many algorithms and statistical processes available. However, there are many other sources of bias that can occur during experimental design and sample handling that are currently unaddressed. This article aims to cast light on the potential sources of bias and where normalization could be applied to return the sample to its normal state. Throughout we suggest solutions where possible but, in some cases, solutions are not available. Thus, we see this article as a starting point for discussion of the definition of and the issues surrounding the concept of normalization as it applies to the proteomic analysis of biological samples. Specifically, we discuss a wide range of different normalization techniques that can occur at each stage of the sample preparation and analysis process.

Cancer-derived extracellular vesicles are known to play a role in the progression of the disease. In this rapidly-growing field, there are many reports of phenotypic changes in cells following exposure to cancer-derived extracellular vesicles. This study examines the protein contents of vesicles derived from three well-known breast cancer cell lines, MCF-7, MDA-MB-231 and T47D, using peptide-centric LC-MS/MS and cytokine multiplex immunoassay analysis to understand the molecular basis of these changes. Through these techniques a large number of proteins within these vesicles were identified. A large proportion of these proteins are known to be important in cancer formation and progression and associated with cancer signaling, angiogenesis, metastasis and invasion and immune regulation. This highlights the importance of extracellular vesicles (EVs) in cancer communications and shows some of the mechanisms the vesicles use to assist in cancer progression.

Mesenchymal stem cells are a continually expanding area in research and clinical applications. Their usefulness and capacity to differentiate into various cells, particularly neural types, has driven the research area for several years. Neural differentiation has considerable usefulness. There are several successful differentiation techniques of mesenchymal stem cells that employ the use of small molecules, growth factors and commercially available kits and supplements. Phenotyping, molecular biology, genomics and proteomics investigation revealed a wealth of data about these cells during neurogenic differentiation. However, there remain large gaps in the knowledge base, particularly related to cytokines and how their role, drive mechanisms and the downstream signalling processes change with their varied expression throughout the differentiation process. In this study, adult mesenchymal stem cells were induced with neurogenic differentiation media, the cellular changes monitored by live-cell microscopy and the changes in cytokine expression in the intracellular region, secretion into the media and in the extracellular vesicle cargo were examined and analysed bioinformatically. Through this analysis, the up-regulation of key cytokines was revealed, and several neuroprotective and neurotrophic roles were displayed. Statistically significant molecules IFN-G, IL1B, IL6, TNF-A, have roles in astrocyte development. Furthermore, the cytokine bioinformatics suggests the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway is upregulated, supporting differentiation toward an astroglial lineage.

Cytokines are multifunctional small proteins that have a vital influence on inflammatory states of tissues and play a role in signalling and cellular control mechanisms. Cytokine expression has primarily been viewed as a form of direct secretion of molecules through an active transportation; however, other forms of active transport such as extracellular vesicles are at play. This is particularly important in stem cells where signalling molecules are key to communication managing the levels of proliferation, migration, and differentiation into mature cells. This study investigated cytokines from intracellular content, direct cellular secretions, and extracellular vesicles from adult adipose-derived stem cells isolated from three distinct anatomical locations: abdomen, thigh, and chin. The cells were cultured investigated using live cell microscopy, cytokine assays, and bioinformatics analysis. The cytokines quantified and examined from each sample type showed a distinct difference between niche areas and sample types. The varying levels of TNF-alpha, IL-6 and IL-8 cytokines were shown to play a crucial role in signalling pathways such as MAPK, ERK1/2 and JAK-STAT in cells. On the other hand, the chemotactic cytokines IL-1rn, Eotaxin, IP-10 and MCP-1 showed the most prominent changes across extracellular vesicles with roles in noncanonical signalling. By examining the local and tangential roles of cytokines in stem cells, their roles in signalling and in regenerative mechanisms may be further understood.