INMdok (Leibniz Institute for New Materials)
Not a member yet
931 research outputs found
Sort by
Freestanding Films of Reduced Graphene Oxide Fully Decorated with Prussian Blue Nanoparticles for Hydrogen Peroxide Sensing
Get PDFDeveloping thin, freestanding electrodes that work simultaneously as a current collector and electroactive material is pivotal to integrating portable and wearable chemical sensors. Herein, we have synthesized graphene/Prussian blue (PB) electrodes for hydrogen peroxide detection (H2O2) using a two-step method. First, an reduced graphene oxide/PAni/Fe2O3 freestanding film is prepared using a doctor blade technique, followed by the electrochemical deposition of PB nanoparticles over the films. The iron oxide nanoparticles work as the iron source for the heterogeneous electrochemical deposition of the nanoparticles in a ferricyanide solution. The size of the PB cubes electrodeposited over the graphene-based electrodes was controlled by the number of voltammetric cycles. For H2O2 sensing, the PB10 electrode achieved the lowest detection and quantification limits, 2.00 and 7.00 μM, respectively. The findings herein evidence the balance between the structure of the graphene/PB-based electrodes with the electrochemical performance for H2O2 detection and pave the path for developing new freestanding electrodes for chemical sensors
Nanostructured lipid carrier formulation for delivering poorly water-soluble ITF3756 HDAC inhibitor
Get PDFHistone deacetylases (HDACs) are enzymes that play crucial roles in cellular processes by hydrolyzing acetyl-L-lysine side chains in core histones, thereby regulating gene expression and maintaining homeostasis. Histone deacetylase inhibitors (HDACi) have emerged as promising agents, particularly in cancer treatment, due to their ability to induce cytotoxic and pro-apoptotic effects. Selective HDAC6 inhibitors, such as ITF3756, have shown low off-target toxicity and promising pharmacological activities, but their poor water solubility limits their application in nanoparticulate drug delivery systems. Here, we optimized a nanostructured lipid carrier (NLC) formulation for delivering ITF3756 using the design of experiments (DOE) and response surface methodology (RSM). An interaction between the factor surfactant and formulation volume was observed, thus demonstrating that the surfactant concentration impacts the NLC size. It can be speculated that the higher the amount of the drug in the formulation, the lower the polydispersion index (PDI), thus resulting in more stable nanostructures. The optimized ITF3756-NLC demonstrated a size of 51.1 ± 0.3 nm, 8.85 ± 4.71 mV charge, and high entrapment efficiency (EE%), maintaining stability for 60 days. Moreover, ITF3756-NLC enhanced α-tubulin acetylation in melanoma, lung, and brain cancer cell lines, indicating retained or improved bioactivity. The ITF3756-NLC formulation offers a viable approach for enhancing the bioavailability and therapeutic efficacy of HDAC6 inhibitors, demonstrating potential for clinical applications in cancer immunotherapy
A screening setup to streamline in vitro Engineered Living Material cultures with the host
Get PDFEngineered living materials (ELMs), which usually comprise bacteria, fungi, or animal cells entrapped in polymeric matrices, offer limitless possibilities in fields like drug delivery or biosensing. To determine the conditions that sustain ELM performance while ensuring ELM-host compatibility is essential before testing them in vivo. This is critical to reduce animal experimentation and can be achieved through in vitro investigations. Towards this goal, we designed a 96-well plate-based screening method to streamline ELM growth across culture conditions and determine their compatibility potential in vitro. We showed proliferation of three bacterial species encapsulated in hydrogels over time and screened six different cell culture media. We fabricated ELMs in bilayer and monolayer formats and tracked bacterial leakage. After screening, an appropriate medium was selected that sustained growth of an ELM, and it was used to study cytocompatibility in vitro. ELM cytotoxicity on murine fibroblasts and human monocytes was studied by adding ELM supernatants and measuring cell membrane integrity and live/dead staining, respectively, proving ELM cytocompatibility. Our work illustrates a simple setup to streamline the screening of compatible environmental conditions of ELMs with the host
Selenium Disulfide from Sustainable Resources: An Example of “Redneck” Chemistry with a Pinch of Salt
Get PDFSelenium disulfide (often referred to as SeS2) encompasses a family of mixed selenium-sulfide eight-membered rings, traditionally used as an anti-dandruff agent in shampoos. SeS2 can be produced by reacting hydrogen sulfide (H2S) with selenite (SeO32−) under acidic conditions. This chemistry is also possible with natural spring waters that are rich in H2S, thus providing an avenue for the more sustainable, green production of high-quality SeS2 particles from an abundant natural source. The orange material obtained this way consists of small globules with a diameter in the range of 1.1 to 1.2 µm composed of various SexS8−x chalcogen rings. It shows the usual composition and characteristics of a Se-S interchalcogen compound in EDX and Raman spectroscopy. Since the mineral water from Bad Nenndorf is also rich in salts, the leftover brine has been evaporated to yield a selenium-enriched salt mixture similar to table salt. As the water from Bad Nenndorf—in comparison to other bodies of water around the world—is still rather modest in terms of its H2S content, especially when compared with volcanic waters, this approach may be refined further to become economically and ecologically viable, especially as a regional business model for small and medium-sized enterprises
Response of immune killer cells to mechanical cues and living therapeutic materials
Get PDFNK cells are one of the major immune killer cell types exhibiting anti-tumor activity. During immune surveillance NK cells infiltrate into tissues and come in contact with cells and organs with varied stiffness. It has been shown that tumor cells with a lower elasticity modulus than their counterparts to have a higher metastatic potential. Whether the change in tumor cell stiffness affects the functionality of NK cells is not well understood. In this work, to test the effect of substrate stiffness on NK responses, PAAm-co-AA hydrogels of varied stiffness (2 kPa,12 kPa, 50 kPa) functionalized with biotinylated NKp46 activating antibody, prepared by Dr. Jingnan Zhang (research group of Prof. del Campo, INM-Leibniz Institute for New Materials) were used. Stiffness of substrate indeed played a huge role in modulating NK responses with stiffer substrates (12 kPa, 50 kPa) eliciting a stronger response in most donors whereas the soft substrates (2 kPa) failed to do so. To further test the impact of target cell stiffness on NK cytolytic activity, stiffness of target cells was altered using blebbistatin (made cells stiffer) and DMSO (made cells softer). Cytotoxicity of NK cells was boosted against stiffened tumor cells and impaired against softened tumor cells. In addition, the time required for NK cell to detach from the stiffened target cell after apoptosis or necrosis of the latter was significantly shorter, also contributing to a more effective cytotoxicity. To further decipher the role of mechanosensing in killing processes, functions of mechanosensitive ion channels was blocked using unspecific antagonizers (gadolinium and nifedipine), and it was found that blockage of mechanosensing substantially impaired NK mediated cytotoxicity as determined by 2D and 3D killing assays. Regarding the responsible mechanosensor, we have identified from the microarray data of our lab (by Dr. Eva Schwarz) that PIEZO1 are the predominately expressed mechanosensitive ion channels in NK cells. Blockade of PIEZO1 in NK cells by GsMTx4 impaired NK mediated cytotoxicity and activation of PIEZO1, using its specific agonist Yoda-1, potentiated NK mediated killing of the target cells. Blockade of PIEZO1 was shown to decrease the infiltration of NK cells into 3D collagen matrices, and activation of PIEZO1 boosted the infiltration of NK cells into 3D collagen matrix. As the role of PIEZO1 to be a major mechanosensor in NK cells was established, its role in sensing the stiffness of substrates was explored. Perturbation of PIEZO1 channels abrogated NK responses to substrate stiffness. Together, these data emphasize the role of mechanosensing in regulating NK cytotoxicity and the central role of tumor cell stiffness in evading immune surveillance. To fight cancer and other infective diseases, living therapeutic materials (LTMs) offer possibilities to release therapeutics in a sustainable and tunable manner. LTMs contain genetically engineered biological component encapsulated in a polymeric material such as hydrogels to contain their exposure in the host. LTMs are being extensively researched for their use in treatment of cancer with many studies reinforcing the beneficial effects of using smart materials. To contain the exposure of living component such as bacteria and to protect it from adverse environmental conditions of the host and to avoid a direct contact with immune cells, they are often encapsulated. However, one major concern for LTMs is that they may trigger an immune response and create a pro-inflammatory milieu in the host which could lead to critical situations if unregulated. So, the second part of my thesis is to characterize the immune response of PBMCs to PluDA hydrogel encapsulated E.coli and ClearColi bacteria. This work was carried out in collaboration with the group of Dr. Shrikrishnan Sankaran, Bioprogramable materials, INM-Leibniz Institute for New Materials, Saarbrücken. The ClearColi strain was produced from E.coli after genetically deleting LPS. ClearColi was encapsulated in Pluronic F127-based hydrogels (PluDA). It has to be noted that the bacteria were not in direct contact with the host so any immune reaction elicited would be due to the release of soluble factors and metabolites. The release of pro-inflammatory cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, TNFα and IFNγ) and cytotoxic proteins (granzyme A, granzyme B, perforin, granulysin, sFas, and sFasL) by PBMCs in response to bacteria and bacteria encapsulated gels was checked along with its influence on immune killer cells’ subtypes. Interestingly, PBMCs from the blood donors could be grouped in to two groups, donors with low spontaneous IL-2 and high spontaneous IL-2 release, based on the IL-2 release when PBMCs were cultured alone. Our results show that co-incubation of PluDA blank gels with PBMCs did not alter their profiles of cytokines and cytotoxic proteins, and had no influence on differentiation of NK cells, CD4+ and CD8+ T cells in donors with low spontaneous release of IL-2. ClearColi elicited release of IL-6 and IFNγ from PBMCs. Interestingly, the predominantly released cytokine was IL-6 for low spontaneous IL-2 release donors, but IFNγ for high spontaneous IL-2 release donors. Both the transwell condition and the encapsulated gel condition showed the same tendency. When the bacteria were in direct contact with the PBMCs they triggered the apoptosis of PBMCs on day 3 but encapsulation of the bacteria in PluDA gels completely abolished this effect.NK-Zellen sind eine der wichtigsten Arten von Killerzellen des Immunsystems im Kampf gegen Krebs. . Während der Immunüberwachung infiltrieren NK-Zellen ins Gewebe und kommen mit Zellen und Organen unterschiedlicher Steifigkeit in Kontakt. Es hat sich gezeigt, dass Tumorzellen mit einem niedrigeren Elastizitätsmodul ein höheres Metastasierungspotenzial haben als solche mit einem höheren Elastizitätsmodul. Ob sich die veränderte Steifigkeit der Tumorzellen auf die Funktionalität der NK-Zellen auswirkt, ist noch nicht ausreichend geklärt. In dieser Arbeit wurden PAAm-co-AA-Hydrogele unterschiedlicher Steifigkeit (2 kPa, 12 kPa, 50 kPa), die mit einem biotinylierten, NKp46 aktivierenden Antikörper funktionalisiert waren, von Dr. Jingnan Zhang (Forschungsgruppe von Prof. del Campo, INM-Leibniz-Institut für Neue Materialien) verwendet, um die Auswirkungen der Substratsteifigkeit auf NK-Zellfunktionen zu untersuchen. Die Steifigkeit des Substrats spielte in der Tat eine große Rolle bei der Modulation der NKFunktionalität, wobei steifere Substrate (12 kPa, 50 kPa) bei den meisten Spendern eine stärkere Reaktion auslösten, während dies bei den weichen Substraten (2 kPa) nicht der Fall war. Um die Auswirkungen der Steifigkeit der Zielzellen auf die zytotoxische Aktivität der NK-Zellen weiter zu testen, wurde die Steifigkeit der Zielzellen mit Hilfe von Blebbistatin (machte die Zellen steifer) und DMSO (machte die Zellen weicher) verändert. Die Zytotoxizität der NK-Zellen gegen steifere Tumorzellen war stärker als gegen weichere Tumorzellen. Darüber hinaus war die Zeit, die die NK-Zellen benötigten, um sich nach der Apoptose oder Nekrose einer steiferen Krebszelle von dieser zu lösen, deutlich kürzer als bei einer weicheren Krebszelle, was ebenfalls zu einer effektiveren Zytotoxizität beitrug. Um die Rolle der Mechanosensorik im Tötungsprozess weiter zu entschlüsseln, wurden die Funktionen mechanosensitiver Ionenkanäle mit Hilfe unspezifischer Antagonisten (Gadolinium und Nifedipin) blockiert. Es zeigte sich, dass die Blockierung der Mechanosensorik die durch NK-Zellen vermittelte Zytotoxizität erheblich beeinträchtigte, wie durch 2D- und 3D-Zytotoxizitätsassays ermittelt wurde. Was den verantwortlichen Mechanosensor betrifft, so haben wir anhand bereits vorhandener Microarray-Daten (von Dr. Eva Schwarz) unseres Labors festgestellt, dass PIEZO1 Kanäle die vorwiegend exprimierten mechanosensitiven Ionenkanäle in NK-Zellen sind. Die Blockade von PIEZO1 in NK-Zellen durch GsMTx4 beeinträchtigte die NK-vermittelte Zytotoxizität, und die Aktivierung von PIEZO1 durch seinen spezifischen Agonisten Yoda-1 verstärkte die NK-vermittelte Abtötung der Zielzellen. Die Blockade von PIEZO1 verringerte die Infiltration von NK-Zellen in 3D-Kollagenmatrizen, während die Aktivierung von PIEZO1 die Infiltration von NK-Zellen in die 3D-Kollagenmatrix verstärkte. Da die Rolle von PIEZO1 als wichtiger Mechanosensor in NK-Zellen somit nachgewiesen wurde, wurde seine Rolle bezüglich der Steifigkeit von Substraten untersucht. Eine Inhibierung der PIEZO1-Kanäle verminderte die Reaktionen der NK auf die Steifigkeit des Substrats. Zusammengenommen unterstreichen diese Daten die Rolle der Mechanosensorik bei der Regulierung der NK-Zytotoxizität und die zentrale Rolle der Steifigkeit von Tumorzellen bei der Umgehung der Immunüberwachung. Zur Bekämpfung von Krebs und anderen Infektionskrankheiten bieten lebende therapeutische Materialien (LTM) die Möglichkeit, Therapeutika auf nachhaltige und abstimmbare Weise freizusetzen. LTMs enthalten gentechnisch veränderte biologische Komponenten, die in einem Polymermaterial wie Hydrogelen eingekapselt sind, um ihre Exposition im Wirt zu begrenzen. LTMs werden derzeit intensiv für ihren Einsatz in der Krebsbehandlung erforscht, wobei viele Studien die positiven Auswirkungen der Verwendung intelligenter Materialien unterstreichen. Um die Exposition von lebenden Komponenten wie Bakterien einzudämmen, sie vor ungünstigen Milieubedingungen im Wirt zu schützen und einen direkten Kontakt mit Immunzellen zu vermeiden, werden sie häufig eingekapselt. Eine große Sorge bei LTMs ist jedoch, dass sie eine Immunreaktion auslösen und ein entzündungsförderndes Milieu im Wirt schaffen können, das zu kritischen Situationen führen könnte, wenn es nicht reguliert wird. Der zweite Teil meiner Dissertation besteht darin, die Immunantwort von PBMCs auf in PluDA-Hydrogel eingekapselte E.coli und ClearColi Bakterien zu charakterisieren. Diese Arbeit wurde in Zusammenarbeit mit der Gruppe von Dr. Shrikrishnan Sankaran (Bio-programmierbare Materialien, INM-Leibniz- Institut für Neue Materialien, Saarbrücken) durchgeführt. Der ClearColi-Stamm wurde aus E.coli nach genetischer Deletion von LPS hergestellt. ClearColi wurde in Hydrogelen auf der Basis von Pluronic F127 (PluDA) eingekapselt. Somit war gewährleistet, dass die Bakterien nicht in direktem Kontakt mit dem Wirt standen, so dass jede ausgelöste Immunreaktion auf die Freisetzung von löslichen Faktoren und Metaboliten zurückzuführen ist. Die Freisetzung von proinflammatorischen Zytokinen (IL-2, IL-4, IL-6, IL-10, IL-17A, TNFα und IFNγ) und zytotoxischen Proteinen (Granzym A, Granzym B, Perforin, Granulysin, sFas und sFasL) durch PBMCs als Reaktion auf Bakterien und bakterienverkapselte Gele wurde zusammen mit ihrem Einfluss auf die Subtypen der Immunkillerzellen untersucht. Unerwarteterweise konnten die PBMCs der Blutspender in zwei Gruppen eingeteilt werden: Spender mit geringer spontaner IL-2- Freisetzung und Spender mit hoher spontaner IL-2-Freisetzung, basierend auf der IL-2-Freisetzung von PBMCs ohne Stimulus. Unsere Ergebnisse zeigen, dass die Co-Inkubation von PluDA-Gelen mit PBMCs deren Profile von Zytokinen und zytotoxischen Proteinen nicht veränderte und keinen Einfluss auf die Differenzierung von NK-Zellen, CD4+ und CD8+ T-Zellen bei Spendern mit geringer spontaner IL-2-Freisetzung hatte. ClearColi löste die Freisetzung von IL-6 und IFNγ aus PBMCs aus. Interessanterweise war das vorwiegend freigesetzte Zytokin IL-6 bei Spendern mit geringer spontaner IL-2-Freisetzung, aber IFNγ bei Spendern mit hoher spontaner IL-2- Freisetzung. Sowohl bei der Transwell-Bedingung als auch bei der verkapselten Gel-Bedingung zeigte sich die gleiche Tendenz. Wenn die Bakterien in direktem Kontakt mit den PBMCs waren, lösten sie am dritten Tag die Apoptose der PBMCs aus, die Verkapselung der Bakterien in PluDAGelen hob diesen Effekt jedoch vollständig auf
Engineering probiotic bacteria as living therapeutic agents
Get PDFLiving bacterial therapeutics represent an exciting frontier for achieving controlled drug release within the body. However, genetic modules require improvement to control the production and release of therapeutic biomolecules in medically relevant strains. Model probiotic strains like E. coli Nissle 1917 have extensive genetic toolkits but still lack rapidly responsive and stringent genetic switches to regulate drug release. On the other hand, probiotic bacteria from the Lactobacilli family have broader applicability in the body but remain as non-model strains with restrictive genetic programmability. This thesis addresses both these limitations. Firstly, I developed a strategy to achieve strict control over the release of an enzymatically synthesized antibiotic (darobactin) from E. coli Nissle 1917. By combining parts from pre-established genetic switches, I created a thermo-amplifier circuit that released darobactin at pathogen inhibitory levels within a few hours. Secondly, I expanded the genetic toolbox of the probiotic Lactiplantibacillus plantarum WCFS1 strain with two genetic parts - a strong constitutive promoter (PtlpA) and several type II toxin-antitoxin (TA)-based plasmid retention systems. The performance of these genetic modules in recombinant plasmids was verified using reporter proteins such as mCherry and Staphylococcal nuclease without the need for antibiotic-based selection pressure.Lebende bakterielle Therapeutika stellen eine spannsende Möglichkeit dar, um eine kontrollierte Freisetzung von Medikamenten im Körper zu erreichen. Die genetischen Module müssen jedoch verbessert werden, um die Produktion und Freisetzung therapeutischer Biomoleküle in medizinisch relevanten Stämmen zu kontrollieren. Probiotische Modellstämme wie E. coli Nissle 1917 verfügen zwar über ein umfangreiches genetisches Instrumentarium, doch fehlt es ihnen noch an schnell reagierenden und stringenten genetischen Schaltern zur Steuerung der Wirkstofffreisetzung. Andererseits sind probiotische Bakterien aus der Familie der Laktobazillen zwar breiter im Körper einsetzbar, bleiben aber als Nicht Modellstämme mit restriktiver genetischer Programmierbarkeit bestehen. Die vorliegende Arbeit befasst sich mit diesen beiden Einschränkungen. Erstens habe ich eine Strategie entwickelt, um die Freisetzung eines enzymatisch synthetisierten Antibiotikums (Darobactin) aus E. coli Nissle 1917 streng zu kontrollieren. Durch die Kombination von Teilen bereits etablierter genetischer Schalter schuf ich einen Thermo-Verstärker-Schaltkreis, der Darobactin innerhalb weniger Stunden in pathogenhemmenden Mengen freisetzt. Zweitens habe ich den genetischen Werkzeugkasten des probiotischen Lactiplantibacillus plantarum WCFS1- Stammes um zwei genetische Teile erweitert - einen starken konstitutiven Promotor (PtlpA) und mehrere Plasmider haltung systeme auf der Basis von Typ-II-Toxin-Antitoxin (TA). Die Leistungsfähigkeit dieser genetischen Module in rekombinanten Plasmiden wurde mit Reporterproteinen wie mCherry und Staphylokokken-Nuklease ohne antibiotischen Selektionsdruck überprüft
Metabolite-responsive Control of Transcription by Phase Separation-based Synthetic Organelles
No full textLiving natural materials have remarkable sensing abilities that translate external cues into functional changes of the material. The reconstruction of such sensing materials in bottom-up synthetic biology provides the opportunity to develop synthetic materials with life-like sensing and adaptation ability. Key to such functions are material modules that translate specific input signals into a biomolecular response. Here, we engineer a synthetic organelle based on liquid-liquid phase separation that translates a metabolic signal into the regulation of gene transcription. To this aim, we engineer the pyruvate-dependent repressor PdhR to undergo liquid-liquid phase separationin vitroby fusion to intrinsically disordered regions. We demonstrate that the resulting coacervates bind DNA harbouring PdhR-responsive operator sites in a pyruvate dose-dependent and reversible manner. We observed that the activity of transcription units on the DNA was strongly attenuated following recruitment to the coacervates. However, the addition of pyruvate resulted in a reversible and dose-dependent reconstitution of transcriptional activity. The coacervate-based synthetic organelles linking metabolic cues to transcriptional signals represent a materials approach to confer stimulus-responsiveness to minimal bottom-up synthetic biological systems and open opportunities in materials for sensor applications
Actin-templated Structures: Nature's Way to Hierarchical Surface Patterns (Gecko's Setae as Case Study)
Get PDFThe hierarchical design of the toe pad surface in geckos and its reversible adhesiveness have inspired material scientists for many years. Micro- and nano-patterned surfaces with impressive adhesive performance have been developed to mimic gecko's properties. While the adhesive performance achieved in some examples has surpassed living counterparts, the durability of the fabricated surfaces is limited and the capability to self-renew and restore function—inherent to biological systems—is unimaginable. Here the morphogenesis of gecko setae using skin samples from the Bibron´s gecko (Chondrodactylus bibronii) is studied. Gecko setae develop as specialized apical differentiation structures at a distinct cell–cell layer interface within the skin epidermis. A primary role for F-actin and microtubules as templating structural elements is necessary for the development of setae's hierarchical morphology, and a stabilization role of keratins and corneus beta proteins is identified. Setae grow from single cells in a bottom layer protruding into four neighboring cells in the upper layer. The resulting multicellular junction can play a role during shedding by facilitating fracture of the cell–cell interface and release of the high aspect ratio setae. The results contribute to the understanding of setae regeneration and may inspire future concepts to bioengineer self-renewable patterned adhesive surfaces
Design of a Biohybrid Materials Circuit with Binary Decoder Functionality
Get PDFSynthetic biology applies concepts from electrical engineering and information processing to endow cells with computational functionality. Transferring the underlying molecular components into materials and wiring them according to topologies inspired by electronic circuit boards has yielded materials systems that perform selected computational operations. However, the limited functionality of available building blocks is restricting the implementation of advanced information-processing circuits into materials. Here, a set of protease-based biohybrid modules the bioactivity of which can either be induced or inhibited is engineered. Guided by a quantitative mathematical model and following a design-build-test-learn (DBTL) cycle, the modules are wired according to circuit topologies inspired by electronic signal decoders, a fundamental motif in information processing. A 2-input/4-output binary decoder for the detection of two small molecules in a material framework that can perform regulated outputs in form of distinct protease activities is designed. The here demonstrated smart material system is strongly modular and can be used for biomolecular information processing for example in advanced biosensing or drug delivery applications
The RNA binding protein IGF2BP2/IMP2 alters the cargo of cancer cell-derived extracellular vesicles supporting tumor-associated macrophages
Get PDFBackground: Tumor cells release extracellular vesicles (EVs) that contribute to the polarization of macrophages towards tumor-associated macrophages (TAMs). High expression levels of the RNA binding protein IGF2BP2/IMP2 are correlated with increased tumor cell proliferation, invasion, and poor prognosis in the clinic. However, there is a lack of understanding of whether IMP2 affects the cargo of cancer cell-derived EVs, thereby modulating macrophage polarization.
Methods: EVs were isolated from IMP2-expressing HCT116 parental cells (WT) and CRISPR/Cas9 IMP2 knockout (KO) cells. EVs were characterized according to MISEV guidelines, microRNA cargo was assessed by microRNA-Seq, and the protein cargo was analyzed by proteomics. Primary human monocyte-derived macrophages (HMDMs) were polarized by EVs, and the expression of genes and surface markers was assessed using qPCR and flow cytometry, respectively. Morphological changes of macrophages, as well as the migratory potential of cancer cells, were assessed by the Incucyte® system and macrophage matrix degradation potential by zymography. Changes in the metabolic activity of macrophages were quantified using a Seahorse® analyzer. For in vivo studies, EVs were injected into the yolk sac of zebrafish larvae, and macrophages were isolated by fluorescence-activated cell sorting.
Results: EVs from WT and KO cells had a similar size and concentration and were positive for 25 vesicle markers. The expression of tumor-promoting genes was higher in macrophages polarized with WT EVs than KO EVs, while the expression of TNF and IL6 was reduced. A similar pattern was observed in macrophages from zebrafish larvae treated in vivo. WT EV-polarized macrophages showed a higher abundance of TAM-like surface markers, higher matrix degrading activity, as well as a higher promotion of cancer cell migration. MicroRNA-Seq revealed a significant difference in the microRNA composition of WT and KO EVs, particularly a high abundance of miR-181a-5p in WT EVs, which was absent in KO EVs. Inhibitors of macropinocytosis and phagocytosis antagonized the delivery of miR-181a-5p into macrophages and the downregulation of the miR-181a-5p target DUSP6. Proteomics data showed differences in protein cargo in KO vs. WT EVs, with the differentially abundant proteins mainly involved in metabolic pathways. WT EV-treated macrophages exhibited a higher basal oxygen consumption rate and a lower extracellular acidification rate than KO EV-treated cells.
Conclusion: Our results show that IMP2 determines the cargo of EVs released by cancer cells, thereby modulating the EVs' actions on macrophages. Expression of IMP2 is linked to the secretion of EVs that polarize macrophages towards a tumor-promoting phenotype