| 1. |
- Banijamali, Mahsan, et al.
(författare)
-
Characterizing single extracellular vesicles by droplet barcode sequencing for protein analysis
- 2022
-
Ingår i: Journal of Extracellular Vesicles. - : Wiley. - 2001-3078. ; 11:11
-
Tidskriftsartikel (refereegranskat)abstract
- Small extracellular vesicles (sEVs) have in recent years evolved as a source of biomarkers for disease diagnosis and therapeutic follow up. sEV samples derived from multicellular organisms exhibit a high heterogeneous repertoire of vesicles which current methods based on ensemble measurements cannot capture. In this work we present droplet barcode sequencing for protein analysis (DBS-Pro) to profile surface proteins on individual sEVs, facilitating identification of sEV-subtypes within and between samples. The method allows for analysis of multiple proteins through use of DNA barcoded affinity reagents and sequencing as readout. High throughput single vesicle profiling is enabled through compartmentalization of individual sEVs in emulsion droplets followed by droplet barcoding through PCR. In this proof-of-concept study we demonstrate that DBS-Pro allows for analysis of single sEVs, with a mixing rate below 2%. A total of over 120,000 individual sEVs obtained from a NSCLC cell line and from malignant pleural effusion (MPE) fluid of NSCLC patients have been analyzed based on their surface proteins. We also show that the method enables single vesicle surface protein profiling and by extension characterization of sEV-subtypes, which is essential to identify the cellular origin of vesicles in heterogenous samples.
|
|
| 2. |
- Cavallaro, Sara, et al.
(författare)
-
Label-Free Surface Protein Profiling of Extracellular Vesicles by an Electrokinetic Sensor
- 2019
-
Ingår i: ACS Sensors. - : AMER CHEMICAL SOC. - 2379-3694. ; 4:5, s. 1399-1408
-
Tidskriftsartikel (refereegranskat)abstract
- Small extracellular vesicles (sEVs) generated from the endolysosomal system, often referred to as exosomes, have attracted interest as a suitable biomarker for cancer diagnostics, as they carry valuable biological information and reflect their cells of origin. Herein, we propose a simple and inexpensive electrical method for label-free detection and profiling of sEVs in the size range of exosomes. The detection method is based on the electrokinetic principle, where the change in the streaming current is monitored as the surface markers of the sEVs interact with the affinity reagents immobilized on the inner surface of a silica microcapillary. As a proof-of-concept, we detected sEVs derived from the non-small-cell lung cancer (NSCLC) cell line H1975 for a set of representative surface markers, such as epidermal growth factor receptor (EGFR), CD9, and CD63. The detection sensitivity was estimated to be similar to 175000 sEVs, which represents a sensor surface coverage of only 0.04%. We further validated the ability of the sensor to measure the expression level of a membrane protein by using sEVs displaying artificially altered expressions of EGFR and CD63, which were derived from NSCLC and human embryonic kidney (HEK) 293T cells, respectively. The analysis revealed that the changes in EGFR and CD63 expressions in sEVs can be detected with a sensitivity in the order of 10% and 3%, respectively, of their parental cell expressions. The method can be easily parallelized and combined with existing microfluidic-based EV isolation technologies, allowing for rapid detection and monitoring of sEVs for cancer diagnosis.
|
|
| 3. |
- Haag, Daniel, et al.
(författare)
-
Nos2 inactivation promotes the development of medulloblastoma in Ptch1(+/-) mice by deregulation of Gap43-dependent granule cell precursor migration.
- 2012
-
Ingår i: PLOS Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 8:3
-
Tidskriftsartikel (refereegranskat)abstract
- Medulloblastoma is the most common malignant brain tumor in children. A subset of medulloblastoma originates from granule cell precursors (GCPs) of the developing cerebellum and demonstrates aberrant hedgehog signaling, typically due to inactivating mutations in the receptor PTCH1, a pathomechanism recapitulated in Ptch1(+/-) mice. As nitric oxide may regulate GCP proliferation and differentiation, we crossed Ptch1(+/-) mice with mice lacking inducible nitric oxide synthase (Nos2) to investigate a possible influence on tumorigenesis. We observed a two-fold higher medulloblastoma rate in Ptch1(+/-) Nos2(-/-) mice compared to Ptch1(+/-) Nos2(+/+) mice. To identify the molecular mechanisms underlying this finding, we performed gene expression profiling of medulloblastomas from both genotypes, as well as normal cerebellar tissue samples of different developmental stages and genotypes. Downregulation of hedgehog target genes was observed in postnatal cerebellum from Ptch1(+/+) Nos2(-/-) mice but not from Ptch1(+/-) Nos2(-/-) mice. The most consistent effect of Nos2 deficiency was downregulation of growth-associated protein 43 (Gap43). Functional studies in neuronal progenitor cells demonstrated nitric oxide dependence of Gap43 expression and impaired migration upon Gap43 knock-down. Both effects were confirmed in situ by immunofluorescence analyses on tissue sections of the developing cerebellum. Finally, the number of proliferating GCPs at the cerebellar periphery was decreased in Ptch1(+/+) Nos2(-/-) mice but increased in Ptch1(+/-) Nos2(-/) (-) mice relative to Ptch1(+/-) Nos2(+/+) mice. Taken together, these results indicate that Nos2 deficiency promotes medulloblastoma development in Ptch1(+/-) mice through retention of proliferating GCPs in the external granular layer due to reduced Gap43 expression. This study illustrates a new role of nitric oxide signaling in cerebellar development and demonstrates that the localization of pre-neoplastic cells during morphogenesis is crucial for their malignant progression.
|
|
| 4. |
- Haag, Petra, et al.
(författare)
-
Caspase-2 is a mediator of apoptotic signaling in response to gemtuzumab ozogamicin in acute myeloid leukemia
- 2022
-
Ingår i: Cell Death Discovery. - : Springer Nature. - 2058-7716. ; 8:1
-
Tidskriftsartikel (refereegranskat)abstract
- The antibody conjugate gemtuzumab ozogamicin (GO; Mylotarg((R))) provides targeted therapy of acute myeloid leukemia (AML), with recent approvals for patients with CD33-positive disease at diagnosis or relapse, as monotherapy or combined with chemotherapeutics. While its clinical efficacy is well documented, the molecular routes by which GO induces AML cell death warrant further analyses. We have earlier reported that this process is initiated via mitochondria-mediated caspase activation. Here we provide additional data, focusing on the involvement of caspase-2 in this mechanism. We show that this enzyme plays an important role in triggering apoptotic death of human AML cells after exposure to GO or its active moiety calicheamicin. Accordingly, the caspase-2 inhibitor z-VDVAD-fmk reduced GO-induced caspase-3 activation. This finding was validated with shRNA and siRNA targeting caspase-2, resulting in reduced caspase-3 activation and cleavage of poly [ADP-ribose] polymerase 1 (PARP-1). We previously demonstrated that GO-induced apoptosis included a conformational change of Bax into a pro-apoptotic state. Present data reveal that GO-treatment also induced Bid cleavage, which was partially reduced by caspase-2 specific inhibition while the effect on GO-induced Bax conformational change remained unaltered. In mononuclear cells isolated from AML patients that responded to GO treatment in vitro, processing of caspase-2 was evident, whereas in cells from an AML patient refractory to treatment no such processing was seen. When assessing diagnostic samples from 22 AML patients, who all entered complete remission (CR) following anthracycline-based induction therapy, and comparing patients with long versus those with short CR duration no significant differences in baseline caspase-2 or caspase-3 full-length protein expression levels were found. In summary, we demonstrate that GO triggers caspase-2 cleavage in human AML cells and that the subsequent apoptosis of these cells in part relies on caspase-2. These findings may have future clinical implications.
|
|
| 5. |
- Inwinkl, Petra, et al.
(författare)
-
Currency transaction tax and its conformity with the free movement of capital and monetary policy
- 2012
-
Ingår i: Steuer und Wirtschaft. - 0341-2954. ; 10:1
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Taxation of financial transactions and currency exchange has been discussed and debated by economists and politicians ever since John Keynes first called for a taxation of stock market transactions. The financial crisis 2008-2009 and the vast expansion of currency exchange markets have intensified discussion on introducing regulatory practice to the currency exchange in the EU. By imposing a currency transaction tax, even at a very low rate, large sums of money could be collected to supplement the EU budget, meet UN Millennium Goals, or create a common rescue fund for bailing out economies in financial distress. However, imposing a tax on currency exchange is not possible without taking the supranational legal body of the EU into consideration. This study takes an initial look at the legal aspects of implementing a currency transaction tax, namely: Is implementation of a currency transaction tax compatible with the free movement of capital and the ESCB’s exclusive right to set monetary policy in the Euro zone?
|
|
| 6. |
- Sahu, Siddharth S., et al.
(författare)
-
Exploiting Electrostatic Interaction for Highly Sensitive Detection of Tumor-Derived Extracellular Vesicles by an Electrokinetic Sensor
- 2021
-
Ingår i: ACS Applied Materials and Interfaces. - : American Chemical Society (ACS). - 1944-8244 .- 1944-8252. ; 13:36, s. 42513-42521
-
Tidskriftsartikel (refereegranskat)abstract
- We present an approach to improve the detection sensitivity of a streaming current-based biosensor for membrane protein profiling of small extracellular vesicles (sEVs). The experimental approach, supported by theoretical investigation, exploits electrostatic charge contrast between the sensor surface and target analytes to enhance the detection sensitivity. We first demonstrate the feasibility of the approach using different chemical functionalization schemes to modulate the zeta potential of the sensor surface in a range -16.0 to -32.8 mV. Thereafter, we examine the sensitivity of the sensor surface across this range of zeta potential to determine the optimal functionalization scheme. The limit of detection (LOD) varied by 2 orders of magnitude across this range, reaching a value of 4.9 x 10(6) particles/mL for the best performing surface for CD9. We then used the optimized surface to profile CD9, EGFR, and PD-L1 surface proteins of sEVs derived from non-small cell lung cancer (NSCLC) cell-line H1975, before and after treatment with EGFR tyrosine kinase inhibitors, as well as sEVs derived from pleural effusion fluid of NSCLC adenocarcinoma patients. Our results show the feasibility to monitor CD9, EGFR, and PD-L1 expression on the sEV surface, illustrating a good prospect of the method for clinical application.
|
|
| 7. |
- Sahu, Siddharth S., et al.
(författare)
-
Multi-marker profiling of extracellular vesicles using streaming current and sequential electrostatic labeling
- 2023
-
Ingår i: Biosensors & bioelectronics. - : Elsevier BV. - 0956-5663 .- 1873-4235. ; 227
-
Tidskriftsartikel (refereegranskat)abstract
- High heterogeneity in the membrane protein expression of small extracellular vesicles (sEVs) means that bulk methods relying on antibody-based capture for expression analysis have a drawback that each type of antibody may capture a different sub-population. An improved approach is to capture a representative sEV population, without any bias, and then perform a multiplexed protein expression analysis on this population. However, such a possibility has been largely limited to fluorescence-based methods. Here, we present a novel electrostatic labelling strategy and a microchip-based all-electric method for membrane protein analysis of sEVs. The method allows us to profile multiple surface proteins on the captured sEVs using alternating charge labels. It also permits the comparison of expression levels in different sEV-subtypes. The proof of concept was tested by capturing sEVs both non-specifically (unbiased) as well as via anti-CD9 capture probes (biased), and then profiling the expression levels of various surface proteins using the charge labelled antibodies. The method is the first of its kind, demonstrating an all-electrical and microchip based method that allows for unbiased analysis of sEV membrane protein expression, comparison of expression levels in different sEV subsets, and fractional estimation of different sEV sub-populations. These results were also validated in parallel using a single-sEV fluorescence technique.
|
|
| 8. |
- Stiller, Christiane, et al.
(författare)
-
Detection of Tumor-Associated Membrane Receptors on Extracellular Vesicles from Non-Small Cell Lung Cancer Patients via Immuno-PCR
- 2021
-
Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 13:4
-
Tidskriftsartikel (refereegranskat)abstract
- Simple Summary Lung cancer is often detected at late stages when metastases are present and the genomic make-ups of the tumors are heterogeneous. Analyses of genomic alterations in non-small-cell lung cancer (NSCLC) have revealed mutated tumor-associated membrane receptors and fusion proteins, which can be targeted via tyrosine kinase inhibitors (TKIs). TKIs initially often have a good effect, but a fraction of the tumor lesions may develop resistance through additional mutations in the targeted kinases or by increased expression/function of other membrane receptors. Detection of TKI-bypassing mechanisms is difficult in tissue biopsies as these analyze only a subpart of tumors or lesions. Liquid biopsies based on tumor-secreted small extracellular vesicles (sEVs) into body fluids can assess tumor heterogeneity. We present an immuno-PCR method for the detection of the epidermal growth factor receptor (EGFR), the human epidermal growth factor receptor 2 (HER2), and the insulin-like growth factor 1 receptor (IGF-1R) on sEVs. Initial investigations of sEVs from EGFR-mutant NSCLC tumor cells or pleural effusion (PE) fluid from patients with NSCLC or benign diseases showed different protein profiles for individual sEV samples. Further development of the immuno-PCR could complement DNA/mRNA-based assays detecting kinase mutations to allow longitudinal treatment monitoring of diverse TKI-bypassing mechanisms. Precision cancer medicine for non-small-cell lung cancer (NSCLC) has increased patient survival. Nevertheless, targeted agents towards tumor-associated membrane receptors only result in partial remission for a limited time, calling for approaches which allow longitudinal treatment monitoring. Rebiopsy of tumors in the lung is challenging, and metastatic lesions may have heterogeneous signaling. One way ahead is to use liquid biopsies such as circulating tumor DNA or small extracellular vesicles (sEVs) secreted by the tumor into blood or other body fluids. Herein, an immuno-PCR-based detection of the tumor-associated membrane receptors EGFR, HER2, and IGF-1R on CD9-positive sEVs from NSCLC cells and pleural effusion fluid (PE) of NSCLC patients is developed utilizing DNA conjugates of antibody mimetics and affibodies, as detection agents. Results on sEVs purified from culture media of NSCLC cells treated with anti-EGFR siRNA, showed that the reduction of EGFR expression can be detected via immuno-PCR. Protein profiling of sEVs from NSCLC patient PE samples revealed the capacity to monitor EGFR, HER2, and IGF-1R with the immuno-PCR method. We detected a significantly higher EGFR level in sEVs derived from a PE sample of a patient with an EGFR-driven NSCLC adenocarcinoma than in sEVs from PE samples of non-EGFR driven adenocarcinoma patients or in samples from patients with benign lung disease. In summary, we have developed a diagnostic method for sEVs in liquid biopsies of cancer patients which may be used for longitudinal treatment monitoring to detect emerging bypassing resistance mechanisms in a noninvasive way.
|
|
| 9. |
- Stridfeldt, Fredrik, et al.
(författare)
-
Analyses of single extracellular vesicles from non-small lung cancer cells to reveal effects of epidermal growth factor receptor inhibitor treatments
- 2023
-
Ingår i: Talanta. - : Elsevier BV. - 0039-9140 .- 1873-3573. ; 259
-
Tidskriftsartikel (refereegranskat)abstract
- Precision cancer medicine has changed the treatment landscape of non-small cell lung cancer (NSCLC) as illustrated by the introduction of tyrosine kinase inhibitors (TKIs) towards mutated epidermal growth factor receptor (EGFR). However, as responses to EGFR-TKIs are heterogenous among NSCLC patients, there is a need for ways to early monitor changes in treatment response in a non-invasive way e.g., in patient's blood samples. Recently, extracellular vesicles (EVs) have been identified as a source of tumor biomarkers which could improve on non-invasive liquid biopsy-based diagnosis of cancer. However, the heterogeneity in EVs is high. Putative biomarker candidates may be hidden in the differential expression of membrane proteins in a subset of EVs hard to identify using bulk techniques. Using a fluorescence-based approach, we demonstrate that a single-EV tech-nique can detect alterations in EV surface protein profiles. We analyzed EVs isolated from an EGFR-mutant NSCLC cell line, which is refractory to EGFR-TKIs erlotinib and responsive to osimertinib, before and after treatment with these drugs and after cisplatin chemotherapy. We studied expression level of five proteins; two tetraspanins (CD9, CD81), and three markers of interest in lung cancer (EGFR, programmed death-ligand 1 (PD-L1), human epidermal growth factor receptor 2 (HER2)). The data reveal alterations induced by the osimertinib treatment compared to the other two treatments. These include the growth of the PD-L1/HER2-positive EV population, with the largest increase in vesicles exclusively expressing one of the two proteins. The expression level per EV decreased for these markers. On the other hand, both the TKIs had a similar effect on the EGFR-positive EV population.
|
|
| 10. |
- Zovko, Ana, et al.
(författare)
-
Marine Sponge Cribrochalina vasculum Compounds Activate Intrinsic Apoptotic Signaling and Inhibit Growth Factor Signaling Cascades in Non-Small Cell Lung Carcinoma
- 2014
-
Ingår i: Molecular Cancer Therapeutics. - 1535-7163 .- 1538-8514. ; 13:12, s. 2941-2954
-
Tidskriftsartikel (refereegranskat)abstract
- Marine-derived compounds have been explored and considered as possible antitumor agents. In this study, we analyzed extracts of the sponge Cribrochalina vasculum for their ability to inhibit tumor cell proliferation. Screening identified two acetylenic compounds of similar structure that showed strong tumor-specific toxicity in non-small cell lung carcinoma (NSCLC) cells and small-cell lung carcinoma cells, and less prominent toxicity in ovarian carcinoma, while having no effect on normal cells. These acetylenic compounds were found to cause a time-dependent increase in activation of apoptotic signaling involving cleavage of caspase-9, caspase-3, and PARP, as well as apoptotic cell morphology in NSCLC cells, but not in normal fibroblasts. Further analysis demonstrated that these compounds caused conformational change in Bak and Bax, and resulted in loss of mitochondrial potential and cytochrome c release in NSCLC cells. Moreover, a decreased phosphorylation of the growth factor signaling kinases Akt, mTOR, and ERK was evident and an increased phosphorylation of JNK was observed. Thus, these acetylenic compounds hold potential as novel therapeutic agents that should be further explored for NSCLC and other tumor malignancies.
|
|
