Extracellular Vesicles in Chemoresistance.

Chemotherapy represents the current mainstay therapeutic approach for most types of cancer. Despite the development of targeted chemotherapeutic strategies, the efficacy of anti-cancer drugs is severely limited by the development of drug resistance. Multidrug resistance (MDR) consists of the simultaneous resistance to various unrelated cytotoxic drugs and is one of the main causes of anticancer treatment failure. One of the principal mechanisms by which cancer cells become MDR involves the overexpression of ATP Binding Cassette (ABC) transporters, such as P-glycoprotein (P-gp), mediating the active efflux of cytotoxic molecules from the cytoplasm. Extracellular vesicles (EVs) are submicron lipid-enclosed vesicles that are released by all cells and which play a fundamental role in intercellular communication in physiological and pathological contexts. EVs have fundamental function at each step of cancer development and progression. They mediate the transmission of MDR through the transfer of vesicle cargo including functional ABC transporters as well as nucleic acids, proteins and lipids. Furthermore, EVs mediate MDR by sequestering anticancer drugs and stimulate cancer cell migration and invasion. EVs also mediate the communication with the tumour microenvironment and the immune system, resulting in increased angiogenesis, metastasis and immune evasion. All these actions contribute directly and indirectly to the development of chemoresistance and treatment failure. In this chapter, we describe the many roles EVs play in the acquisition and spread of chemoresistance in cancer. We also discuss possible uses of EVs as pharmacological targets to overcome EV-mediated drug resistance and the potential that the analysis of tumour-derived EVs offers as chemoresistance biomarkers.

Membrane to cytosol redistribution of αII-spectrin drives extracellular vesicle biogenesis in malignant breast cells.

Spectrin is a ubiquitous cytoskeletal protein that provides structural stability and supports membrane integrity. In erythrocytes, spectrin proteolysis leads to the biogenesis of plasma membrane extracellular vesicles (EVs). However, its role in non-erythroid or cancer-derived plasma membrane EVs biogenesis is unknown. This study aims to examine the role of αII-spectrin in malignant and non-malignant plasma membrane vesiculation. We developed a custom, automated cell segmentation plugin for the image processor, Fiji, that provides an unbiased assessment of high resolution confocal microscopy images of the subcellular distribution of αII-spectrin. We show that, in low vesiculating non-malignant MBE-F breast cells, prominent cortical spectrin localises to the cell periphery at rest. In comparison, cortical spectrin is diminished in high vesiculating malignant MCF-7 breast cells at rest. A cortical distribution of spectrin correlates with increased biomechanical stiffness as measured by Atomic Force Microscopy. Furthermore, cortical spectrin can be induced in malignant MCF-7 cells by treatment with known vesiculation modulators including the calcium chelator, BAPTA-AM or the calpain inhibitor II (ALLM). These results demonstrate that the subcellular localisation of spectrin is distinctly different in malignant and non-malignant cells at rest and shows that the redistribution of cortical αII-spectrin to the cytoplasm supports plasma membrane-derived EV biogenesis in malignant cells.

Targeting respiratory diseases using miRNA inhibitor based nanotherapeutics: Current status and future perspectives.

MicroRNAs (miRNAs) play a fundamental role in the developmental and physiological processes that occur in both animals and plants. AntagomiRs are synthetic antagonists of miRNA, which prevent the target mRNA from suppression. Therapeutic approaches that modulate miRNAs have immense potential in the treatment of chronic respiratory disorders. However, the successful delivery of miRNAs/antagomiRs to the lungs remains a major challenge in clinical applications. A range of materials, namely, polymer nanoparticles, lipid nanocapsules and inorganic nanoparticles, has shown promising results for intracellular delivery of miRNA in chronic respiratory disorders. This review discusses the current understanding of miRNA biology, the biological roles of antagomiRs in chronic respiratory disease and the recent advances in the therapeutic utilization of antagomiRs as disease biomarkers. Furthermore our review provides a common platform to debate on the nature of antagomiRs and also addresses the viewpoint on the new generation of delivery systems that target antagomiRs in respiratory diseases.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Proteins Regulating Microvesicle Biogenesis and Multidrug Resistance in Cancer.

Microvesicles (MV) are emerging as important mediators of intercellular communication. While MVs are important signaling vectors for many physiological processes, they are also implicated in cancer pathology and progression. Cellular activation is perhaps the most widely reported initiator of MV biogenesis, however, the precise mechanism remains undefined. Uncovering the proteins involved in regulating MV biogenesis is of interest given their role in the dissemination of deleterious cancer traits. MVs shed from drug-resistant cancer cells transfer multidrug resistance (MDR) proteins to drug-sensitive cells and confer the MDR phenotype in a matter of hours. MDR is attributed to the overexpression of ABC transporters, primarily P-glycoprotein and MRP1. Their expression and functionality is dependent on a number of proteins. In particular, FERM domain proteins have been implicated in supporting the functionality of efflux transporters in drug-resistant cells and in recipient cells during intercellular transfer by vesicles. Herein, the most recent research on the proteins involved in MV biogenesis and in the dissemination of MV-mediated MDR are discussed. Attention is drawn to unanswered questions in the literature that may prove to be of benefit in ongoing efforts to improve clinical response to chemotherapy and circumventing MDR.

Role of the Tristetraprolin (Zinc Finger Protein 36 Homolog) Gene in Cancer.

Cancer is a complicated transformational progression that fiercely changes the appearance of cell physiology as well as cells' relations with adjacent tissues. Developing an oncogenic characteristic requires a wide range of modifications in a gene expression at a cellular level. This can be achieved by activation or suppression of the gene regulation pathway in a cell. Tristetraprolin (TTP or ZFP36) associated with the initiation and development of tumors are regulated at the level of mRNA decay, frequently through the activity of AU-rich mRNA-destabilizing elements (AREs) located in their 3'-untranslated regions. TTP is an attractive target for therapeutic use and diagnostic tools due to its characteristic appearance in cancer tissue alone. Thus, the illumination of TTP in diverse types of cancer might deliver additional effective remedies in the coming era for cancer patients. The objective of this review is to familiarize the reader with the TTP proteins, focus on efficient properties that endow them with their effective oncogenic potential, describe their physiological role in cancer cells, and review the unique properties of TT, and of TTP-driven cancer.

Circulating tumor DNA - Current state of play and future perspectives.

Cancer management paradigms are shifting towards a personalized approach thanks to the advent of the -omics technologies. Liquid biopsies, consisting in the sampling of blood and other bodily fluids, are emerging as a valid alternative to circulating tumor biomarkers and tumor tissue biopsies for cancer diagnosis, routine monitoring and prognostication. The content of a liquid biopsy is referred to as the "tumor circulome". Among its components, circulating tumor DNA (ctDNA), including both cell-free and exosome-associated DNA, is the most widely characterized element. ctDNA analysis has a tremendous capability in the diagnostic arena. Its potential has been demonstrated at each level of disease staging and management and supported by a recent FDA approval for companion diagnostic, and the investments being made by pharmaceutical companies in this sector are numerous. The approaches available for ctDNA analysis allow both quantitative and qualitative studies and range from PCR and dPCR-mediated single/multiple gene mutational assessment to whole genome next generation sequencing and methylation mapping. Although the principal object of a liquid biopsy is blood, other body fluids such as urine and saliva show potential as complementary DNA sources for tumor analysis. In this review we provide a synopsis on the state of play of current ctDNA application. We discuss the clinical significance of ctDNA analysis and review the state of the art of technologies being currently developed to this aim. We also discuss the current issues limiting ctDNA application and highlight the promising approaches being developed to overcome these.

Microparticles in cancer: A review of recent developments and the potential for clinical application.

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 donor cells to recipient cells. Although microparticles (MPs) contribute to the pathogenesis of cancer, their unique characteristics can also be exploited in the context of cancer management. 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 these readily accessible systemic biomarkers has the potential to circumvent the need for invasive biopsy procedures. In addition, the autologous nature of MPs may allow them to be used as novel drug delivery carriers. Consequently, the modulation of MP vesiculation to treat disease, 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.

Microparticles shed from multidrug resistant breast cancer cells provide a parallel survival pathway through immune evasion.

BACKGROUND: Breast cancer is the most frequently diagnosed cancer in women. Resident macrophages at distant sites provide a highly responsive and immunologically dynamic innate immune response against foreign infiltrates. Despite extensive characterization of the role of macrophages and other immune cells in malignant tissues, there is very little known about the mechanisms which facilitate metastatic breast cancer spread to distant sites of immunological integrity. The mechanisms by which a key healthy defense mechanism fails to protect distant sites from infiltration by metastatic cells in cancer patients remain undefined. Breast tumors, typical of many tumor types, shed membrane vesicles called microparticles (MPs), ranging in size from 0.1-1 µm in diameter. MPs serve as vectors in the intercellular transfer of functional proteins and nucleic acids and in drug sequestration. In addition, MPs are also emerging to be important players in the evasion of cancer cell immune surveillance. METHODS: A comparative analysis of effects of MPs isolated from human breast cancer cells and non-malignant human brain endothelial cells were examined on THP-1 derived macrophages in vitro. MP-mediated effects on cell phenotype and functionality was assessed by cytokine analysis, cell chemotaxis and phagocytosis, immunolabelling, flow cytometry and confocal imaging. Student's t-test or a one-way analysis of variance (ANOVA) was used for comparison and statistical analysis. RESULTS: In this paper we report on the discovery of a new cellular basis for immune evasion, which is mediated by breast cancer derived MPs. MPs shed from multidrug resistant (MDR) cells were shown to selectively polarize macrophage cells to a functionally incapacitated state and facilitate their engulfment by foreign cells. CONCLUSIONS: We propose this mechanism may serve to physically disrupt the inherent immune response prior to cancer cell colonization whilst releasing mediators required for the recruitment of distant immune cells. These findings introduce a new paradigm in cancer cell biology with significant implications in understanding breast cancer colonization at distant sites. Most importantly, this is also the first demonstration that MPs serve as conduits in a parallel pathway supporting the cellular survival of MDR cancer cells through immune evasion.

Synthesis and in vitro biological evaluation of thiosulfinate derivatives for the treatment of human multidrug-resistant breast cancer.

Organosulfur compounds derived from Allium vegetables have long been recognized for various therapeutic effects, including anticancer activity. Allicin, one of the main biologically active components of garlic, shows promise as an anticancer agent; however, instability makes it unsuitable for clinical application. The aim of this study was to investigate the effect of stabilized allicin derivatives on human breast cancer cells in vitro. In this study, a total of 22 stabilized thiosulfinate derivatives were synthesized and screened for their in vitro antiproliferative activities against drug-sensitive (MCF-7) and multidrug-resistant (MCF-7/Dx) human adenocarcinoma breast cancer cells. Assays for cell death, apoptosis, cell cycle progression and mitochondrial bioenergetic function were performed. Seven compounds (4b, 7b, 8b, 13b, 14b, 15b and 18b) showed greater antiproliferative activity against MCF-7/Dx cells than allicin. These compounds were also selective towards multidrug-resistant (MDR) cells, a consequence attributed to collateral sensitivity. Among them, 13b exhibited the greatest anticancer activity in both MCF-7/Dx and MCF-7 cells, with IC50 values of 18.54±0.24 and 46.50±1.98 µmol/L, respectively. 13b altered cellular morphology and arrested the cell cycle at the G2/M phase. Additionally, 13b dose-dependently induced apoptosis, and inhibited cellular mitochondrial respiration in cells at rest and under stress. MDR presents a significant obstacle to the successful treatment of cancer clinically. These results demonstrate that thiosulfinate derivatives have potential as novel anticancer agents and may offer new therapeutic strategies for the treatment of chemoresistant cancers.

The Role of CD44 and ERM Proteins in Expression and Functionality of P-glycoprotein in Breast Cancer Cells.

Multidrug resistance (MDR) is often attributed to the over-expression of P-glycoprotein (P-gp), which prevents the accumulation of anticancer drugs within cells by virtue of its active drug efflux capacity. We have previously described the intercellular transfer of P-gp via extracellular vesicles (EVs) and proposed the involvement of a unique protein complex in regulating this process. In this paper, we investigate the role of these mediators in the regulation of P-gp functionality and hence the acquisition of MDR following cell to cell transfer. By sequentially silencing the FERM domain-binding proteins, Ezrin, Radixin and Moesin (ERM), as well as CD44, which we also report a selective packaging in breast cancer derived EVs, we have established a role for these proteins, in particular Radixin and CD44, in influencing the P-gp-mediated MDR in whole cells. We also report for the first time the role of ERM proteins in the vesicular transfer of functional P-gp. Specifically, we demonstrate that intercellular membrane insertion is dependent on Ezrin and Moesin, whilst P-gp functionality is governed by the integrity of all ERM proteins in the recipient cell. This study identifies these candidate proteins as potential new therapeutic targets in circumventing MDR clinically.

MRP1 and its role in anticancer drug resistance.

The phenomenon of multidrug resistance (MDR) in cancer is associated with the overexpression of the ATP-binding cassette (ABC) transporter proteins, including multidrug resistance-associated protein 1 (MRP1) and P-glycoprotein. MRP1 plays an active role in protecting cells by its ability to efflux a vast array of drugs to sub-lethal levels. There has been much effort in elucidating the mechanisms of action, structure and substrates and substrate binding sites of MRP1 in the last decade. In this review, we detail our current understanding of MRP1, its clinical relevance and highlight the current environment in the search for MRP1 inhibitors. We also look at the capacity for the rapid intercellular transfer of MRP1 phenotype from spontaneously shed membrane vesicles known as microparticles and discuss the clinical and therapeutic significance of this in the context of cancer MDR.

An analysis of the therapeutic benefits of genotyping in pediatric hematopoietic stem cell transplantation.

Hematopoietic stem cell transplantation is a high-risk procedure that is offered, with curative intent, to patients with malignant and nonmalignant disease. The clinical benefits of personalization of therapy by genotyping have been demonstrated by the reduction in transplant related mortality from donor-recipient HLA matching. However, defining the relationship between genotype and transplant conditioning agents is yet to be translated into clinical practice. A number of the therapeutic agents used in stem cell transplant preparative regimens have pharmacokinetic parameters that predict benefit of incorporating pharmacogenomic data into dosing strategies. Busulfan, cyclophosphamide, thio-TEPA and etoposide have well-described drug metabolism pathways, however candidate gene studies have identified there is a gap in the identification of pharmacogenomic data that can be used to improve transplant outcomes. Incorporating pharmacogenomics into pharmacokinetic modeling may demonstrate the therapeutic benefits of genotyping in transplant preparative regimen agents.

Inhibition of the multidrug resistance P-glycoprotein: time for a change of strategy?

P-glycoprotein (P-gp) is a key player in the multidrug-resistant phenotype in cancer. The protein confers resistance by mediating the ATP-dependent efflux of an astonishing array of anticancer drugs. Its broad specificity has been the subject of numerous attempts to inhibit the protein and restore the efficacy of anticancer drugs. The general strategy has been to develop compounds that either compete with anticancer drugs for transport or act as direct inhibitors of P-gp. Despite considerable in vitro success, there are no compounds currently available to "block" P-gp-mediated resistance in the clinic. The failure may be attributed to toxicity, adverse drug interaction, and numerous pharmacokinetic issues. This review provides a description of several alternative approaches to overcome the activity of P-gp in drug-resistant cells. These include 1) drugs that specifically target resistant cells, 2) novel nanotechnologies to provide high-dose, targeted delivery of anticancer drugs, 3) compounds that interfere with nongenomic transfer of resistance, and 4) approaches to reduce the expression of P-gp within tumors. Such approaches have been developed through the pursuit of greater understanding of resistance mediators such as P-gp, and they show considerable potential for further application.

Cellular communication via microparticles: role in transfer of multidrug resistance in cancer.

Multidrug resistance (MDR) continues to be a major impediment to the successful treatment of cancer. The two efflux transporters, P-glycoprotein (P-gp) and MRP1 are major contributors to cancer MDR clinically. The upregulation of P-gp leading to MDR was initially understood to occur via pre- and post-transcriptional mechanisms only. However, we demonstrated that microparticles mediate the intercellular exchange and trafficking of bioactive material, including functional P-gp and selected modulatory miRNAs. This exchange of P-gp leads to the dissemination of MDR within a cancer cell population. These findings have significant implications in understanding the cellular basis governing the intercellular acquisition of deleterious traits in cancers, serving to substantially advance our understanding of the molecular basis of the emergence of MDR in cancer clinically.

Ciprofloxacin is actively transported across bronchial lung epithelial cells using a Calu-3 air interface cell model.

Ciprofloxacin is a well-established broad-spectrum fluoroquinolone antibiotic that penetrates well into the lung tissues; still, the mechanisms of its transepithelial transport are unknown. The contributions of specific transporters, including multidrug efflux transporters, organic cation transporters, and organic anion-transporting polypeptide transporters, to the uptake of ciprofloxacin were investigated in vitro using an air interface bronchial epithelial model. Our results demonstrate that ciprofloxacin is subject to predominantly active influx and a slight efflux component.

Microparticle-associated nucleic acids mediate trait dominance in cancer.

Drug resistance is a major cause of cancer treatment failure, with multidrug resistance (MDR) being the most serious, whereby cancer cells display cross-resistance to structurally and functionally unrelated drugs. MDR is caused by overexpression of the efflux transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1). These transporters act to maintain sublethal intracellular drug concentrations within the cancer cell, making the population treatment unresponsive. Recently, we discovered a novel nongenetic basis to MDR whereby microparticles (MPs) transfer P-gp intercellularly from MDR donor cells to drug-sensitive recipient cells. MPs isolated from MDR leukemia and breast cancer cells were cocultured with their drug-sensitive counterparts. P-gp transfer was assessed by direct immunolabeling, and acquired transcripts and regulatory microRNAs by quantitative real-time PCR. We show that MDR MPs incorporate nucleic acids; MPs change recipient cells' transcriptional environment to reflect donor MDR phenotype, and distinct pathways exist among cancers of different origin that may be dependent on donor cells' ABCB1 overexpression. We demonstrate that this pathway exists for both hematological and nonhematological malignancies. By conferring MDR and "retemplating" the transcriptional landscape of recipient cells, MPs provide a novel pathway, having implications in the dissemination and acquisition of deleterious traits in clinical oncology.

Microparticles mediate MRP1 intercellular transfer and the re-templating of intrinsic resistance pathways.

Multidrug resistance (MDR) is a major impediment to the overall success of chemotherapy in clinical oncology. MDR has been primarily attributed by the ATP-dependent transmembrane proteins, P-glycoprotein (P-gp, ABCB1) and Multidrug Resistance-Associated Protein 1 (MRP1, ABCC1). These proteins maintain sublethal concentrations of intracellular chemotherapeutics by virtue of their drug efflux capacity. In this study, we report the acquisition and dissemination of functional MRP1 via microparticle (MP) mediated intercellular transfer. After we showed the transfer and functionality of P-gp in drug sensitive recipient cells, we report the transfer and time-dependent functionality of MRP1 in drug sensitive leukaemia cells following exposure to MPs shed by MRP1-overexpressing MDR cells. We also demonstrate a remarkable capacity for MPs shed from cells with a P-gp dominant resistance profile to re-template a pre-existing MRP1 dominant profile in recipient cells. These findings have significance in understanding the molecular basis for tumour dominant phenotypes and introduce potential new strategies and targets for the acquisition of MDR and other deleterious traits.

Glioma microvesicles carry selectively packaged coding and non-coding RNAs which alter gene expression in recipient cells.

Interactions between glioma cells and their local environment are critical determinants of brain tumor growth, infiltration and neovascularisation. Communication with host cells and stroma via microvesicles represents one pathway by which tumors can modify their surroundings to achieve a tumor-permissive environment. Here we have taken an unbiased approach to identifying RNAs in glioma-derived microvesicles, and explored their potential to regulate gene expression in recipient cells. We find that glioma microvesicles are predominantly of exosomal origin and contain complex populations of coding and noncoding RNAs in proportions that are distinct from those in the cells from which they are derived. Microvesicles show a relative depletion in microRNA compared with their cells of origin, and are enriched in unusual or novel noncoding RNAs, most of which have no known function. Short-term exposure of brain microvascular endothelial cells to glioma microvesicles results in many gene expression changes in the endothelial cells, most of which cannot be explained by direct delivery of transcripts. Our data suggest that the scope of potential actions of tumor-derived microvesicles is much broader and more complex than previously supposed, and highlight a number of new classes of small RNA that remain to be characterized.

Salbutamol sulfate absorption across Calu-3 bronchial epithelia cell monolayer is inhibited in the presence of common anionic NSAIDs.

PURPOSE: The aim of this study was to characterize the permeability kinetics of salbutamol sulfate, a commonly used ß2-agonist in the treatment of asthma exacerbation, across Calu-3 respiratory epithelial cell monolayers in the presence of non-steroidal anti-inflammatory drugs (NSAIDs), as they have been implicated to be able to modulate organic cation transporters (OCTs). METHODS: Calu-3 cell monolayers were grown in a liquid covered culture (LCC) configuration on 0.33 cm(2) Transwell polyester cell culture supports. Monolayers, cultured between 11 and 14 days were evaluated for epithelial resistance, tight junction integrity, and expression of OCT using Western blot analysis. The transport of salbutamol across the monolayer was studied as a function of concentration. Directional transport was investigated by assessing apical-basal (a-b) and basal-apical (b-a) directions. The influence of a non-specific OCT inhibitor (tetraethylammonium, TEA) and three NSAIDs (aspirin, ibuprofen, and indomethacin) on the uptake of salbutamol was studied. RESULTS: The flux of salbutamol sulfate increased with increasing concentration before reaching a plateau, suggesting the involvement of a transport-mediated uptake mechanism. Western blot analysis detected the presence of OCT1-3 and N1 and N2 sub-types, suggesting the presence of functioning transporters. The apparent permeability (P(app)) of 0.1 mM salbutamol across the epithelial monolayer displayed directional transport in the a-b direction which was inhibited by ˜70% in the presence of TEA, suggesting OCT-mediated uptake. Likewise, the uptake of 0.1 mM salbutamol was decreased in the presence of all the three NSAIDs, supporting a mechanism whereby NSAIDs inhibit absorption of salbutamol across the bronchial epithelium via effects on the OCT transporters. CONCLUSION: This study demonstrates that NSAIDs influence the uptake kinetics of salbutamol in an in vitro Calu-3 cell system.