Imaging of Light-Enhanced Extracellular Vesicle-Mediated Delivery of Oxaliplatin to Colorectal Cancer Cells via Laser Ablation, Inductively Coupled Plasma Mass Spectrometry.

Extracellular vesicles (EVs) are lipid bilayer structures released by all cells that mediate cell-to-cell communication via the transfer of bioactive cargo. Because of the natural origin of EVs, their efficient uptake by recipient cells, capacity to stabilize and transport biomolecules and their potential for cell/tissue targeting and preferential uptake by cancer cells, they have enormous potential for bioengineering into improved and targeted drug delivery systems. In this work, we investigated the use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) as a tool to measure the loading of platinum-based chemotherapeutic agents. The EV loading of oxaliplatin via co-incubation was demonstrated, and LA-ICP-MS imaging showed greater efficiency of delivery to colorectal cancer cells compared to free oxaliplatin, leading to enhanced cytotoxic effect. Further, the impact of EV co-loading with a porphyrin (C5SHU, known as 'C5') photosensitizer on oxaliplatin delivery was assessed. Fluorescence analysis using nano-flow cytometry showed dose-dependent EV loading as well as a trend towards the loading of larger particles. Exposure of OXA-C5-EV-treated colorectal cancer cells to light indicated that delivery was enhanced by both light exposure and porphyrins, with a synergistic effect on cell viability observed between oxaliplatin, EVs and light exposure after the delivery of the co-loaded EVs. In summary, this work demonstrates the utility of LA-ICP-MS and mass spectrometry imaging in assessing the loading efficiency and cellular delivery of platinum-based therapeutics, which would also be suitable for agents containing other elements, confirms that EVs are more efficient at delivery compared to free drugs, and describes the use of light exposure in optimizing delivery and therapeutic effects of EV-mediated drug delivery both in combination and independently of porphyrin-based photosensitizers.

Water-Soluble Truncated Fatty Acid-Porphyrin Conjugates Provide Photo-Sensitizer Activity for Photodynamic Therapy in Malignant Mesothelioma.

Clinical trials evaluating intrapleural photodynamic therapy (PDT) are ongoing for mesothelioma. Several issues still hinder the development of PDT, such as those related to the inherent properties of photosensitizers. Herein, we report the synthesis, photophysical, and photobiological properties of three porphyrin-based photosensitizers conjugated to truncated fatty acids (C5SHU to C7SHU). Our photosensitizers exhibited excellent water solubility and high PDT efficiency in mesothelioma. As expected, absorption spectroscopy confirmed an increased aggregation as a consequence of extending the fatty acid chain length. In vitro PDT activity was studied using human mesothelioma cell lines (biphasic MSTO-211H cells and epithelioid NCI-H28 cells) alongside a non-malignant mesothelial cell line (MET-5A). The PDT effect of these photosensitizers was initially assessed using the colorimetric WST-8 cell viability assay and the mode of cell death was determined via flow cytometry of Annexin V-FITC/PI-stained cells. Photosensitizers appeared to selectively localize within the non-nuclear compartments of cells before exhibiting high phototoxicity. Both apoptosis and necrosis were induced at 24 and 48 h. As our pentanoic acid-derivatized porphyrin (C5SHU) induced the largest anti-tumor effect in this study, we put this forward as an anti-tumor drug candidate in PDT and photo-imaging diagnosis in mesothelioma.

Elemental Mapping of Human Malignant Mesothelioma Tissue Samples Using High-Speed LA-ICP-TOFMS Imaging.

This is the first report of the use of laser ablation-inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS) to analyze human malignant pleural mesothelioma (MPM) samples at the cellular level. MPM is an aggressive, incurable cancer associated with asbestos exposure, with a long latency and poor overall survival. Following careful optimization of the laser fluence, the simultaneous ablation of soft biological tissue and hard mineral fibers was possible, allowing the spatial detection of elements such as Si, Mg, Ca, and Fe, which are also present in the glass substrate. A low-dispersion LA setup was employed, which provided the high spatial resolution necessary to identify the asbestos fibers and fiber fragments in the tissue and to characterize the metallome at the cellular level (a pixel size of 2 µm), with a high speed (at 250 Hz). The multielement LA-ICP-TOFMS imaging approach enabled (i) the detection of asbestos fibers/mineral impurities within the MPM tissue samples of patients, (ii) the visualization of the tissue structure with the endogenous elemental pattern at high spatial resolution, and (iii) obtaining insights into the metallome of MPM patients with different pathologies in a single analysis run. Asbestos and other mineral fibers were detected in the lung and pleura tissue of MPM patients, respectively, based on their multielement pattern (Si, Mg, Ca, Fe, and Sr). Interestingly, strontium was detected in asbestos fibers, suggesting a link between this potential toxic element and MPM pathogenesis. Furthermore, monitoring the metallome around the talc deposit regions (characterized by elevated levels of Al, Mg, and Si) revealed significant tissue damage and inflammation caused by talc pleurodesis. LA-ICP-TOFMS results correlated to Perls' Prussian blue and histological staining of the corresponding serial sections. Ultimately, the ultra-high-speed and high-spatial-resolution capabilities of this novel LA-ICP-TOFMS setup may become an important clinical tool for simultaneous asbestos detection, metallome monitoring, and biomarker identification.

Scorpion Venom Antimicrobial Peptides Induce Caspase-1 Dependant Pyroptotic Cell Death.

Within the last decade, several peptides have been identified according to their ability to inhibit the growth of microbial pathogens. These antimicrobial peptides (AMPs) are a part of the innate immune system of all living organisms. Many studies on their effects on prokaryotic microorganisms have been reported; some of these peptides have cytotoxic properties although the molecular mechanisms underlying their activity on eukaryotic cells remain poorly understood. Smp24 and Smp43 are novel cationic AMPs which were identified from the venom of the Egyptian scorpion Scorpio maurus palmatus. Smp24 and Smp43 showed potent activity against both Gram-positive and Gram-negative bacteria as well as fungi. Here we describe cytotoxicity of these peptides towards two acute leukaemia cell lines (myeloid (KG1-a) and lymphoid (CCRF-CEM) leukaemia cell lines) and three non-tumour cell lines CD34+ (hematopoietic stem progenitor from cord blood), HRECs (human renal epithelial cells) and HaCaT (human skin keratinocytes). Smp24 and Smp43 (4-256 µg/ml) decreased the viability of all cell lines, although HaCaT cells were markedly less sensitive. With the exception HaCaT cells, the caspase-1 gene was uniquely up-regulated in all cell lines studied. However, all cell lines showed an increase in downstream interleukin-1ß (IL-1ß) expression. Transmission electron microscope studies revealed the formation of cell membrane blebs and the appearance of autolysosomes and lipid droplets in all cell lines; KG1-a leukemia cells also showed the unique appearance of glycogen deposits. Our results reveal a novel mechanism of action for scorpion venom AMPs, activating a cascade of events leading to cell death through a programmed pyroptotic mechanism.

Comorbid Conditions, Mental Health and Cognitive Functions in Adults with Fibromyalgia.

This study examined age group differences across adulthood in comorbid conditions, mental health, and cognitive function in people with fibromyalgia. Participants completed an online survey about how fibromyalgia affects their everyday life. Chi square analyses were conducted to examine associations between age groups and (a) comorbid conditions and (b) severity of anxiety and depression. ANOVA analyses examined age group differences on aspects of self-report cognitive function. The greatest prevalence of comorbid conditions was found in middle adulthood. Early adulthood was associated with more cases of severe anxiety with the lowest number of cases being in the oldest age group. Middle adulthood was associated with worse self-report pain compared to the youngest age group. Older adults showed better self-report cognitive function compared to younger adults. Distinct age profiles based on comorbid conditions, mental health, and symptom severity across adulthood in fibromyalgia have been demonstrated.

Laser ablation inductively coupled plasma mass spectrometry as a novel clinical imaging tool to detect asbestos fibres in malignant mesothelioma.

RATIONALE: Malignant pleural mesothelioma is an extremely aggressive and incurable malignancy associated with prior exposure to asbestos fibres. Difficulties remain in relation to early diagnosis, notably due to impeded identification of asbestos in lung tissue. This study describes a novel laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) imaging approach to identify asbestos within mesothelioma models with clinical significance. METHODS: Human mesothelioma cells were exposed to different types of asbestos fibres and prepared on plastic slides for LA-ICP-MS analysis. No further sample preparation was required prior to analysis, which was performed using an NWR Image 266 nm laser ablation system coupled to an Element XR sector-field ICP mass spectrometer, with a lateral resolution of 2 µm. Data was processed using LA-ICP-MS ImageTool v1.7 with the final graphic production made using DPlot software. RESULTS: Four different mineral fibres were successfully identified within the mesothelioma samples based on some of the most abundant elements that make up these fibres (Si, Mg and Fe). Using LA-ICP-MS as an imaging tool provided information on the spatial distribution of the fibres at cellular level, which is essential in asbestos detection within tissue samples. Based on the metal counts generated by the different types of asbestos, different fibres can be identified based on shape, size, and elemental composition. Detection of Ca was attempted but requires further optimisation. CONCLUSIONS: Detection of asbestos fibres in lung tissues is very useful, if not necessary, to complete the pathological dt9iagnosis of asbestos-related malignancies in the medicolegal field. For the first time, this study demonstrates the successful application of LA-ICP-MS imaging to identify asbestos fibres and other mineral fibres within mesothelioma samples. Ultimately, high-resolution, fast-speed LA-ICP-MS analysis has the potential to be integrated into clinical workflow to aid earlier detection and stratification of mesothelioma patient samples.

Headspace analysis of mesothelioma cell lines differentiates biphasic and epithelioid sub-types.

Malignant mesothelioma (MM) is an incurable cancer. MM is often misdiagnosed, with a poor 5-year survival and limited treatment options. The discovery of endogenous volatile organic compounds (VOCs) is required in order to accelerate the development of a breath test as an alternative to conventional MM diagnosis. For the first time, this study used solid-phase microextraction and gas chromatography-mass spectrometry to identify VOCs released directly from the biphasic MM cell line MSTO-211H and the epithelioid MM cell line NCI-H28 as well as the non-malignant mesothelial cell line MET-5A. Multivariate statistical analysis showed separation between MSTO-211H, NCI-H28 and MET-5A results. 2-ethyl-1-hexanol was significantly increased in both MSTO-211H and NCI-H28 cells compared to MET-5A controls. In addition, ethyl propionate and cyclohexanol were significantly increased in MSTO-211H cells and dodecane was significantly increased in NCI-H28 cells. This is the first study reporting headspace analysis of these MM cell lines and the first to consider the effects of mesothelioma sub-type on VOC profile. Current results further highlight the potential for a diagnostic mesothelioma breath test as well as providing proof of concept for the differentiation between biphasic and epithelioid mesothelioma based on VOC profiles.

Polyphenols enhance the activity of alkylating agents in leukaemia cell lines.

Polyphenols have been shown to sensitize solid tumours to alkylating agents such as cisplatin, and induce apoptosis and/or cell-cycle arrest. Here, we assess the effects of five polyphenols alone and in combination with three alkylating agents: cisplatin, cyclophosphamide and chlorambucil in lymphoid and myeloid leukaemia cells lines, and non-tumour control cells. In lymphoid leukaemia cell lines there was a synergistic reduction in ATP and glutathione levels, an induction of cell cycle arrest, DNA damage and apoptosis when quercetin, apigenin, emodin and rhein were combined with cisplatin and cyclophosphamide; and when apigenin and rhein were combined with chlorambucil. In myeloid leukaemia cells quercetin, apigenin and emodin showed a similar synergistic effect with all alkylating agents; however antagonistic effects were observed with some or all alkylating agents when combined with emodin, rhein and cis-stilbene. All synergistic effects were associated with reduced glutathione levels, DNA damage and apoptosis; whilst during antagonism the reverse effects were observed. The combination of alkylating agents, particularly cisplatin with polyphenols could be promising for the treatment of lymphoid leukaemias, with apigenin showing the greatest effects. Likewise in myeloid cells apigenin also synergised the action of all alkylating agents, suggesting that apigenin may also be beneficial in myeloid leukaemias.

Patients' perspective of the effectiveness and acceptability of pharmacological and non-pharmacological treatments of fibromyalgia.

Background and aims Fibromyalgia is a complex condition characterised by widespread pain, sleep disturbance, fatigue and cognitive impairment, with a global mean prevalence estimated at 2.7%. There are inconsistencies in guidelines on the treatment of fibromyalgia leading to dissatisfaction from patients and healthcare professionals. This study investigated patient-reported outcomes of pharmacological and non-pharmacological treatment usage and effectiveness with an assessment of acceptability. Methods Nine hundred and forty-one participants completed a self-administered anonymous questionnaire giving quantitative data of demographics, treatment usage and treatment outcomes. Participant-reported effectiveness and side effects were compared in the following treatment classes: analgesics, antidepressants, gabapentinoids, gastrointestinal treatments, activity interventions, dietary-based treatments, and psychological, physical and alternative therapies. Participants also reported whether they knew about or had tried different treatments. Results The results from the online survey indicated that the range of mean effectiveness ratings were similar for pharmacological and non-pharmacological treatments, whereas non-pharmacological treatments had lower side effects ratings and higher acceptability relative to pharmacological treatments. Participants were not aware of some treatment options. Conclusions The results show lower side effects ratings and higher acceptability for non-pharmacological treatments compared to pharmacological treatments despite similar effectiveness ratings. Implications This article presents results from a large online survey on fibromyalgia patient perspectives of pharmacological and non-pharmacological treatments. Results will inform healthcare professionals and patients about optimal treatments based on ratings of effectiveness, side effects and acceptability that are tailored to patient symptom profiles. Some participants were unaware of treatment options highlighting the importance of patient education allowing collaboration between patients and healthcare professionals to find optimal treatments.

What causes fibromyalgia? An online survey of patient perspectives.

Fibromyalgia is a severe chronic pain condition that affects every aspect of life. Causes of the condition remain unclear, and quantitative research cannot account for patients' personal illness narratives and perceptions. This online survey gathered qualitative accounts of the perceived causes of their condition from 596 people with fibromyalgia, which were analyzed thematically. Themes were "Bodily assault, ill-health, and change"; "Emotional trauma and distress"; "Stress and vulnerability"; and "Explaining and authenticating fibromyalgia." Discussion focuses on the complexity of causation, the importance of understanding and having symptoms validated, and the potential for benefiting from patient expertise in building better practitioner-client relationships.

Dietary polyphenols influence antimetabolite agents: methotrexate, 6-mercaptopurine and 5-fluorouracil in leukemia cell lines.

Polyphenols have been previously shown to sensitize leukemia cell lines to topoisomerase inhibitors. Here, we assess the effects of five polyphenols when used alone and in combination with antimetabolites: methotrexate, 6-mercaptopurine and 5-fluorouracil; in lymphoid and myeloid leukemia cells lines, and non-tumor control cells. The effects of combined treatments were investigated on ATP and glutathione levels, cell-cycle progression, DNA damage and apoptosis. Polyphenols antagonized methotrexate and 6-mercaptopurine induced cell-cycle arrest and apoptosis in most leukemia cell lines. This was associated with reduced DNA damage and increased glutathione levels, greater than that seen following individual treatments alone. In contrast, 5-fluorouracil when combined with quercetin, apigenin and rhein caused synergistic decrease in ATP levels, induction of cell-cycle arrest and apoptosis in some leukemia cell lines. However, antagonistic effects were observed when 5-fluorouracil was combined with rhein and cis-stilbene in myeloid cell lines. The effects were dependant on polyphenol type and chemotherapy agent investigated, and cell type treated. Interestingly treatment of non-tumor control cells with polyphenols protected cells from antimetabolite treatments. This suggests that polyphenols modulate the action of antimetabolite agents; more importantly they antagonized methotrexate and 6-mercaptopurine actions, thus suggesting the requirement of polyphenol-exclusion during their use.

"A Future Amalgamation Between the Scientist and the Clinician?"

Personalized medicine is gaining momentum and analytical methods such as MS are ideally situated to provide coherent imaging of human disease. The cancer research field is already starting to benefit from the MS imaging applications; non-cancerous diseases will hopefully enjoy the same success. Often, the way forward is to embrace several techniques, which can complement and verify each other. This approach can be seen as less confrontational as everyone can play a part in the development of a new working practice. Stakeholders, professional bodies, and instrument manufacturers will be open to maximizing the patient benefit; investment is more likely given the past performance and reputation of the collaborative highly skilled team.With this in mind, how close are we to a future amalgamation between the scientist and the clinician? Can we accelerate the integration of innovative bench technologies into the clinical setting and bring state-of-the-art imaging diagnostics to the patient bedside or General Practitioners treatment room?

A qualitative exploration of the experiences of living with and being treated for fibromyalgia.

This study explores the life and treatment experience of people in the United Kingdom with fibromyalgia in order to inform the development of treatments which are both effective and acceptable to users. Qualitative interviews were conducted with 14 participants with interpretative phenomenological analysis used as the theoretical framework and analytical method. The themes identified were as follows: Inauthenticity of fibromyalgia, An Unconventional healthcare experience, Re-creating support networks, Challenging the working identity, Threatening the family dynamic and Fighting, accepting or accommodating? The biopsychosocial impacts of fibromyalgia disrupted the identity, lifestyle, roles and relationships of our participants with such challenges further exacerbated by the contested nature of the illness.

Recombinant " IMS TAG" proteins--a new method for validating bottom-up matrix-assisted laser desorption/ionisation ion mobility separation mass spectrometry imaging.

RATIONALE: Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) provides a methodology to map the distribution of peptides generated by in situ tryptic digestion of biological tissue. It is challenging to correlate these peptides to the proteins from which they arise because of the many potentially overlapping and hence interfering peptide signals generated. METHODS: A recombinant protein has been synthesised that when cleaved with trypsin yields a range of peptide standards for use as identification and quantification markers for multiple proteins in one MALDI-IMS-MSI experiment. Mass spectrometry images of the distribution of proteins in fresh frozen and formalin-fixed paraffin-embedded tissue samples following in situ tryptic digestion were generated by isolating signals on the basis of their m/z value and ion mobility drift time, which were correlated to matching peptides in the recombinant standard. RESULTS: Tryptic digestion of the IMS-TAG protein and MALDI-MS analysis yielded m/z values and ion mobility drift time for the signature peptides included in it. MALDI-IMS-MSI images for the distribution of the proteins HSP90 and vimentin, in FFPE EMT6 mouse tumours, and HSP90 and plectin in a fresh frozen mouse fibrosarcoma, were generated by extracting ion images at the corresponding m/z value and drift time from the tissue samples. CONCLUSIONS: The IMS-TAG approach provides a new means to confirm the identity of peptides generated by in situ digestion of biological tissue.

Differential effects of polyphenols on proliferation and apoptosis in human myeloid and lymphoid leukemia cell lines.

BACKGROUND: Mortality rates for leukemia are high despite considerable improvements in treatment. Since polyphenols exert pro-apoptotic effects in solid tumors, our study investigated the effects of polyphenols in haematological malignancies. The effect of eight polyphenols (quercetin, chrysin, apigenin, emodin, aloe-emodin, rhein, cis-stilbene and trans-stilbene) were studied on cell proliferation, cell cycle and apoptosis in four lymphoid and four myeloid leukemic cells lines, together with normal haematopoietic control cells. METHODS: Cellular proliferation was measured by CellTiter-Glo(®) luminescent assay; and cell cycle arrest was assessed using flow cytometry of propidium iodide stained cells. Apoptosis was investigated by caspase-3 activity assay using flow cytometry and apoptotic morphology was confirmed by Hoescht 33342 staining. RESULTS: Emodin, quercetin, and cis-stilbene were the most effective polyphenols at decreasing cell viability (IC50 values of 5-22 µM, 8-33 µM, and 25-85 µM respectively) and inducing apoptosis (AP50 values (the concentration which 50% of cells undergo apoptosis) of 2-27 µM, 19-50 µM, and 8-50 µM respectively). Generally, lymphoid cell lines were more sensitive to polyphenol treatment compared to myeloid cell lines, however the most resistant myeloid (KG-1a and K562) cell lines were still found to respond to emodin and quercetin treatment at low micromolar levels. Non-tumor cells were less sensitive to all polyphenols compared to the leukemia cells. CONCLUSIONS: These findings suggest that polyphenols have anti-tumor activity against leukemia cells with differential effects. Importantly, the differential sensitivity of emodin, quercetin, and cis-stilbene between leukemia and normal cells suggests that polyphenols are potential therapeutic agents for leukemia.