Extracellular vesicles swarm the cancer microenvironment: from tumor–stroma communication to drug intervention

Intercellular communication sets the pace for transformed cells to survive and to thrive. Extracellular vesicles (EVs), such as exosomes, microvesicles and large oncosomes, are involved in this process shuttling reciprocal signals and other molecules between transformed and stromal cells, including fibroblasts, endothelial and immune cells. As a result, these cells are adapted or recruited to a constantly evolving cancer microenvironment. Moreover, EVs take part in the response to anticancer therapeutics not least by promoting drug resistance throughout the targeted tumor. Finally, circulating EVs can also transport important molecules to remote destinations in order to prime metastatic niches in an otherwise healthy tissue. Although the understanding of EV biology remains a major challenge in the field, their characteristics create new opportunities for advances in cancer diagnostics and therapeutics

[Letter to editor] Shedding of bevacizumab in tumour cells derived extracellular vesicles as a new therapeutic escape mechanism in glioblastoma

Glioblastoma (GBM) is the most aggressive type of primary brain tumours. Anti-angiogenic therapies (AAT), such as bevacizumab, have been developed to target the tumour blood supply. However, GBM presents mechanisms of escape from AAT activity, including a speculated direct effect of AAT on GBM cells. Furthermore, bevacizumab can alter the intercellular communication of GBM cells with their direct microenvironment. Extracellular vesicles (EVs) have been recently described as main acts in the GBM microenvironment, allowing tumour and stromal cells to exchange genetic and proteomic material. Herein, we examined and described the alterations in the EVs produced by GBM cells following bevacizumab treatment. Interestingly, bevacizumab that is able to neutralise GBM cells-derived VEGF-A, was found to be directly captured by GBM cells and eventually sorted at the surface of the respective EVs. We also identified early endosomes as potential pathways involved in the bevacizumab internalisation by GBM cells. Via MS analysis, we observed that treatment with bevacizumab induces changes in the EVs proteomic content, which are associated with tumour progression and therapeutic resistance. Accordingly, inhibition of EVs production by GBM cells improved the anti-tumour effect of bevacizumab. Together, this data suggests of a potential new mechanism of GBM escape from bevacizumab activity

Sodium silicate particle size measurements using time-resolved fluorescence anisotropy

Sodium silicates are versatile inorganic chemicals produced by combining silica sand and soda ash (sodium carbonate) under high temperature. When in aqueous solution, they are often used in coating and bonding applications. Additionally, they exhibit a range of attractive characteristics, such as being odorless and non-toxic, high strength and rigidity, resistance to high temperatures and low-cost [1].The important characteristics of silicates are the correlation between the ratio of silica to soda concentrations and the size of the species. Traditionally, the particle sizes of nanoparticles are determined using methods such as Dynamic Light Scattering (DLS) [2], Small-Angle X-Ray Scattering (SAXS) [3], Small Angle Neutron Scattering (SANS) [4] and Transmission Electron Microscopy (TEM) [5]. All these methods are far from ideal and have significant drawbacks: DLS becomes difficult for particles below 10 nm, SAXS and SANS are expensive and complex, and TEM requires complex sample preparations which can lead to alterations of particle sizes [6-8].Here, we present a new way of determining the particle sizes of sodium silicate liquors at high pH using time-resolved fluorescence anisotropy. Different from previous approach of using a single dye label, two fluorescent labels were used in this work [9,10]. Rotational times of the non-binding rhodamine B and electrostatically binding rhodamine 6G were used to determine the medium microviscosity and the silicate particle radius, respectively. This approach of using two dyes ensures that the microviscosity stays accurate in time, unlike in the case when a single dye was used. Applying this method to samples of various pH (prepared by diluting the stock solution of silicate to the concentrations of NaOH ranging from 0.2M to 2M) and different temperatures (10°C to 55°C), therecovered average particle size was found to have an upper limit of 7.0±1.2

Sodium silicate particle size measurements using time-resolved fluorescence anisotropy

We present a development of the method for determining the particle sizes in sodium silicate liquors at high pH by using the measurement of time-resolved fluorescence anisotropy. Rather than the previous approach of using a single dye label, we demonstrate the use and advantages of using two fluorescent labels. Rotational times of the non-binding rhodamine B and electrostatically binding rhodamine 6G are used to independently determine the medium microviscosity and the silicate particle radius, respectively. The anisotropy measurements were performed on the range of samples prepared by diluting the stock solution of silicate to the concentrations ranging between 0.2M and 2M of NaOH and on the stock solution at different temperatures. The recovered average particle size has an upper limit of 7.0±1.2Å

Nanoparticle metrology of silicates using time-resolved multiplexed dye fluorescence anisotropy, small angle x-ray scattering and molecular dynamics simulations

We investigate the nanometrology of sub-nanometre particle sizes in industrially manufactured sodium silicate liquors at high pH using time-resolved fluorescence anisotropy. Rather than the previous approach of using a single dye label, we investigate and quantify the advantages and limitations of multiplexing two fluorescent dye labels. Rotational times of the non-binding rhodamine B and adsorbing rhodamine 6G dyes are used to independently determine the medium microviscosity and the silicate particle radius, respectively. The anisotropy measurements were performed on the range of samples prepared by diluting the stock solution of silicate to concentrations ranging between 0.2 M and 2 M of NaOH and on the stock solution at different temperatures. Additionally, it was shown that the particle size can also be measured using a single excitation wavelength when both dyes are present in the sample. The recovered average particle size has an upper limit of 7.0 ± 1.2 Å. The obtained results were further verified using small-angle X-ray scattering, with the recovered particle size equal to 6.50 ± 0.08 Å. To disclose the impact of the dye label on the measured complex size, we further investigated the adsorption state of rhodamine 6G on silica nanoparticles using molecular dynamics simulations, which showed that the size contribution is strongly impacted by the size of the nanoparticle of interest. In the case of the higher radius of curvature (less curved) of larger particles, the size contribution of the dye label is below 10%, while in the case of smaller and more curved particles, the contribution increases significantly, which also suggests that the particles of interest might not be perfectly spherical

PDGF-R inhibition induces glioblastoma cell differentiation via DUSP1/p38MAPK signalling

Glioblastoma (GBM) is the most common and fatal primary brain tumour in adults. Considering that resistance to current therapies leads to limited response in patients, new therapeutic options are urgently needed. In recent years, differentiation therapy has been proposed as an alternative for GBM treatment, with the aim of bringing cancer cells into a post-mitotic/differentiated state, ultimately limiting tumour growth. As an integral component of cancer development and regulation of differentiation processes, kinases are potential targets of differentiation therapies. The present study describes how the screening of a panel of kinase inhibitors (KIs) identified PDGF-Rα/β inhibitor CP-673451 as a potential differentiation agent in GBM. We show that targeting PDGF-Rα/β with CP-673451 in vitro triggers outgrowth of neurite-like processes in GBM cell lines and GBM stem cells (GSCs), suggesting differentiation into neural-like cells, while reducing proliferation and invasion in 3D hyaluronic acid hydrogels. In addition, we report that treatment with CP-673451 improves the anti-tumour effects of temozolomide in vivo using a subcutaneous xenograft mouse model. RNA sequencing and follow-up proteomic analysis revealed that upregulation of phosphatase DUSP1 and consecutive downregulation of phosphorylated-p38MAPK can underlie the pro-differentiation effect of CP-673451 on GBM cells. Overall, the present study identifies a potential novel therapeutic option that could benefit GBM patients in the future, through differentiation of residual GSCs post-surgery, with the aim to limit recurrence and improve quality of life

'Being there' for women with metastatic breast cancer: a pan-European patient survey

BACKGROUND: Understanding their experiences of diagnosis is integral to improving the quality of care for women living with advanced/metastatic breast cancer. METHODS: A survey, initiated in March 2011, was conducted in two stages. First, the views of 47 breast cancer-related patient groups in eight European countries were sought on standards of breast cancer care and unmet needs of patients. Findings were used to develop a patient-centric survey to capture personal experiences of advanced breast cancer to determine insights into the ‘trade-off' between extending overall survival and side effects associated with its treatment. The second online survey was open to women with locally advanced or metastatic breast cancer, or their carers, and responders were recruited through local patient groups. Data were collected via anonymous local language questionnaires. RESULTS: The online stage II survey received a total of 230 responses from 17 European countries: 94% of respondents had locally advanced or metastatic breast cancer and 6% were adult carers. Although the overall experience of care was generally good/excellent (77%), gaps were still perceived in terms of treatment choice and information provision. Treatment choice for patients was felt to be lacking by 32% of responders. In addition, 68% of those who responded would have liked more information about future medical treatments and research, with 57% wishing to receive this information from their oncologist. Two-thirds (66%) of women with advanced breast cancer, or their carers, believed life-extending treatment to be important so that they can spend more time with family and friends, and 67% said that the treatment was worthwhile, despite potential associated side effects. CONCLUSION: These findings show a continuing need to provide women with advanced breast cancer with better information and emphasise the importance that these patients often place on prolonging survival

The structure-function relationship of oncogenic LMTK3

Elucidating signaling driven by lemur tyrosine kinase 3 (LMTK3) could help drug development. Here, we solve the crystal structure of LMTK3 kinase domain to 2.1Å resolution, determine its consensus motif and phosphoproteome, unveiling in vitro and in vivo LMTK3 substrates. Via high-throughput homogeneous time-resolved fluorescence screen coupled with biochemical, cellular, and biophysical assays, we identify a potent LMTK3 small-molecule inhibitor (C28). Functional and mechanistic studies reveal LMTK3 is a heat shock protein 90 (HSP90) client protein, requiring HSP90 for folding and stability, while C28 promotes proteasome-mediated degradation of LMTK3. Pharmacologic inhibition of LMTK3 decreases proliferation of cancer cell lines in the NCI-60 panel, with a concomitant increase in apoptosis in breast cancer cells, recapitulating effects of LMTK3 gene silencing. Furthermore, LMTK3 inhibition reduces growth of xenograft and transgenic breast cancer mouse models without displaying systemic toxicity at effective doses. Our data reinforce LMTK3 as a druggable target for cancer therap

Risk management to prioritise the eradication of new and emerging invasive non-native species

Robust tools are needed to prioritise the management of invasive non-native species (INNS). Risk assessment is commonly used to prioritise INNS, but fails to take into account the feasibility of management. Risk management provides a structured evaluation of management options, but has received little attention to date. We present a risk management scheme to assess the feasibility of eradicating INNS that can be used, in conjunction with existing risk assessment schemes, to support prioritisation. The Non-Native Risk Management scheme (NNRM) can be applied to any predefined area and any taxa. It uses semi-quantitative response and confidence scores to assess seven key criteria: Effectiveness, Practicality, Cost, Impact, Acceptability, Window of opportunity and Likelihood of re-invasion. Scores are elicited using expert judgement, supported by available evidence, and consensus-building methods. We applied the NNRM to forty-one INNS that threaten Great Britain (GB). Thirty-three experts provided scores, with overall feasibility of eradication assessed as ‘very high’ (8 species), ‘high’ (6), ‘medium’ (8), ‘low’ (10) and ‘very low’ (9). The feasibility of eradicating terrestrial species was higher than aquatic species. Lotic freshwater and marine species scored particularly low. Combining risk management and existing risk assessment scores identified six established species as priorities for eradication. A further six species that are not yet established were identified as priorities for eradication on arrival as part of contingency planning. The NNRM is one of the first INNS risk management schemes that can be used with existing risk assessments to prioritise INNS eradication in any area

LMTK3 confers chemo-resistance in breast cancer

Lemur tyrosine kinase 3 (LMTK3) is an oncogenic kinase that is involved in different types of cancer (breast, lung, gastric, colorectal) and biological processes including proliferation, invasion, migration, chromatin remodeling as well as innate and acquired endocrine resistance. However, the role of LMTK3 in response to cytotoxic chemotherapy has not been investigated thus far. Using both 2D and 3D tissue culture models, we found that overexpression of LMTK3 decreased the sensitivity of breast cancer cell lines to cytotoxic (doxorubicin) treatment. In a mouse model we showed that ectopic overexpression of LMTK3 decreases the efficacy of doxorubicin in reducing tumor growth. Interestingly, breast cancer cells overexpressing LMTK3 delayed the generation of double strand breaks (DSBs) after exposure to doxorubicin, as measured by the formation of γH2AX foci. This effect was at least partly mediated by decreased activity of ataxia-telangiectasia mutated kinase (ATM) as indicated by its reduced phosphorylation levels. In addition, our RNA-seq analyses showed that doxorubicin differentially regulated the expression of over 700 genes depending on LMTK3 protein expression levels. Furthermore, these genes were found to promote DNA repair, cell viability and tumorigenesis processes / pathways in LMTK3-overexpressing MCF7 cells. In human cancers, immunohistochemistry staining of LMTK3 in pre- and postchemotherapy breast tumor pairs from four separate clinical cohorts revealed a significant increase of LMTK3 following both doxorubicin and docetaxel based chemotherapy. In aggregate, our findings show for the first time a contribution of LMTK3 in cytotoxic drug resistance in breast cancer