Recipient Cells Research Articles

Harnessing tissue-derived mitochondria-rich extracellular vesicles (Ti-mitoEVs) to boost mitochondrial biogenesis for regenerative medicine.

Mitochondrial damage is a critical pathological factor in various forms of tissue injury, and specific therapies with high biosafety are desirable. Inspired by the natural role of extracellular vesicles (EVs) in regulating mitochondrial metabolism, we report that healthy tissue-derived mitochondria-rich EVs (Ti-mitoEVs) can boost mitochondrial biogenesis for regenerative medicine. Ti-mitoEVs that contain abundant functional mitochondria can be highly efficiently isolated from muscles via an optimized method. In vitro, Ti-mitoEV treatment increased mitochondrial biogenesis and reduced mitochondrial damage in recipient cells, and these effects occurred at least partly via mitochondrial genome transfer. In vivo, Ti-mitoEV treatment attenuated diverse types of tissue injury (e.g., muscle and kidney) by rescuing mitochondrial injury and its associated inflammation. As natural nanovesicles, the therapeutic potency of mitoEVs can be further improved by integrating them with other engineering methods. This study highlights the promising role of Ti-mitoEVs in boosting mitochondrial biogenesis, positioning them as potential therapies for treating various types of tissue injury characterized by mitochondrial damage.

Three decades of mobile RNA-silencing movement within plants: what have we learnt?

In plant RNA-silencing, small interfering (si)RNAs and micro (mi)RNAs guide ARGONAUTE (AGO) effector proteins to silence sequence-complementary RNA/DNA. This helps regulate developmental patterning, adaptation to stress, antiviral defense or genome integrity-maintenance. Remarkably, these regulations not only occur intra-cellularly, but may also manifest in remote tissues. Here, I summarize the evidence that RNA-silencing moves from cell-to-cell and via the phloem, the long-distance extension of the symplasm-the cytosolic connection-network between cells through plasmodesmata (PDs). I then illustrate several biological functions linked to RNA-silencing movement. Besides a still largely putative role for mobile virus-derived (v)siRNAs in conferring immunity, several endogenous sRNAs act as systemic signals orchestrating organismal responses to abiotic stress or symbiosis. Other mobile sRNAs act as morphogens and generate gene expression gradients by moving from cell-to-cell. If RNA-silencing indeed moves symplasmically via PDs, then processes likely regulate its transport; discovering these processes was expected to illuminate macromolecular trafficking in general. In a final part of this perspective, I describe several forward genetic systems set in Arabidopsis to specifically tackle the above issue. Some were instrumental in revealing hitherto unknown AGO-mediated mechanisms that modulate silencing movement within silencing-incipient, traversed or recipient cells. Somewhat disappointingly, however, the systems fell short of identifying factors impacting the silencing cell-to-cell trafficking channels or their regulations. I discuss here plausible reasons for these shortcomings, what could be learnt from them, how they could be remedied, and how a better understanding of their physiological foundations might illuminate so far overlooked aspects of plant RNA- silencing movement.

Intercellular transfer of proteins and mitochondria - the contribution of proteomics.

Intercellular transfer of proteins and mitochondria - the contribution of proteomics.

Role of Kindlin-2-Expressing Extracellular Vesicles in the Invasiveness of Triple Negative Breast Cancer Tumor Cells.

Metastatic breast cancer (BC) is a major cause of cancer-related deaths among women. Its progression is influenced by extracellular vesicles (EVs) released by BC cells, which modulate distant tissue environments to promote metastasis. We previously identified the oncogenic protein Kindlin-2 (K2) as a key driver of BC metastasis, including its role in the nucleus in regulating cell senescence. Here, we investigated whether K2-containing EVs facilitate both autologous (cancer-to-cancer) and heterologous (cancer-to-stroma) communication to promote metastasis. We found that 10-15% of EVs from metastatic BC cells contained K2, while this subpopulation was nearly absent in the EVs from K2-knockout (KO) cells, indicating selective packaging. These EVs transferred K2 to recipient K2-KO cells, where they accumulated in the nucleus. Using a 3D tumorsphere assay, we showed that K2+ EVs enhanced cancer cell invasiveness. Moreover, K2+ EVs activated fibroblasts into a cancer-associated phenotype, increasing α-SMA and FAP expression. Conditioned media from these activated fibroblasts further boosted cancer cell invasion. These results show that EV-associated K2 is actively transferred to recipient cells and regulates metastasis through nuclear signaling, suggesting K2+ EVs are critical mediators of BC progression and potential targets for therapy.

Co-Production of KPC-2 and NDM-5 in a Carbapenem-Resistant Klebsiella Pneumoniae Clinical Isolate: Genetic Insights and Risks

Background Carbapenem-resistant Klebsiella pneumoniae (CRKP), particularly strains co-producing KPC and NDM carbapenemases, poses a severe global health threat due to limited treatment options. Understanding the genetic drivers of resistance and transmission is critical.Methods A multidrug-resistant CRKP strain, KP3T58, co-harboring blaKPC-2 and blaNDM-5, was isolated from an ICU patient. Whole-genome sequencing and comparative genomic analyses were performed. Plasmid transferability was assessed via conjugation assays using E. coli EC600 as the recipient. Virulence phenotypes were evaluated through siderophore production (CAS assay), capsule quantification (uronic acid), serum resistance, and Galleria mellonella infection models. Antimicrobial susceptibility was determined (Vitek-2; CLSI/EUCAST standards).Results KP3T58 exhibited resistance to nearly all antibiotics except polymyxin. The strain’s multidrug-resistant (MDR) phenotype resulted from a combination of chromosomal mutations and the multiple plasmid-borne resistance genes. Whole-genome analysis identified three key plasmids: a conjugative plasmid pKP3T58_1 (IncFIB/FII/R type, carrying a large resistance gene cluster); a conjugative plasmid pKP3T58_2 (IncI1-I type, high-frequency transfer, carrying blaNDM-5); and a non-conjugative plasmid pKP3T58_3 (IncFII type, carrying blaKPC-2). Conjugation assays confirmed that pKP3T58_1 and pKP3T58_2 could transfer individually or jointly to E. coli EC600, while pKP3T58_3 (retaining only a partial T4SS) could co-transfer to recipient cells with the assistance of pKP3T58_2. MLST confirmed clonal persistence of the high-risk ST11 lineage within the patient. Phylogenetic analysis revealed KP3T58’s clustering within a dominant epidemic ST11-KL64 subclade prevalent in China. Multiple virulence assays (including siderophore production, capsule quantification, serum resistance, and Galleria mellonella infection models) demonstrated that KP3T58 lacks a typical hypervirulent phenotype but retains a certain level of pathogenicity.Conclusion This study demonstrates clonal evolution of ST11 CRKP to co-produce KPC-2 and NDM-5 within a host, clustering within a prevalent epidemic lineage. Critically, we provide experimental evidence that mobilizable plasmids retaining only a partial T4SS can undergo horizontal transfer when assisted by conjugative plasmids, fundamentally expanding our understanding of resistance dissemination. The convergence of high-risk epidemiology, MDR, and novel plasmid transfer mechanisms in strains like KP3T58 necessitates enhanced surveillance and urgent molecular investigation into the transmission dynamics of these threats.

Distinguishing Protein and Gene Delivery Enables Characterization and Bioengineering of Extracellular Vesicle-Adeno-Associated Virus Vectors.

Adeno-associated virus (AAV) gene therapies have achieved clinical success, with multiple products reaching regulatory approval. Encapsulation of AAV vectors within engineered extracellular vesicles (EVs) is an emerging strategy which could help overcome challenges including pre-existing anti- capsid immunity and the need for controlling targeting and tropism. To guide the development of EV-AAV technologies, we developed an assay for quantifying and controlling for the contribution of pseudotransduction to evaluations of EV-AAV-mediated gene delivery. We developed an AAV vector that switches its transgene output from one reporter to another when acted upon by Cre recombinase expressed in a recipient cell. Using this platform, we investigated EV-AAV transduction as a function of various engineered EV surface modifications. For actively endocytic cells (HEK293FTs), modifications that enhance EV uptake and membrane fusion influence protein delivery but not gene delivery. Conversely, in less endocytic Jurkat T cells, modifications enhancing EV uptake improved both protein and gene delivery. These conclusions held across multiple AAV serotypes. Our results resolve apparent conflicts in prior reports and suggest that effects of enhancing uptake and membrane fusion of EV-AAV vectors are recipient cell type specific. The methods developed here unambiguously dissect EV-AAV transduction mechanisms and can guide future bioengineering of EV-AAV vectors.

Conjugative delivery of toxin genes ccdB and kil confers synergistic killing of bacterial recipients.

The bacterial type IV secretion systems (T4SS) are medically problematic for their roles in the dissemination of mobile genetic elements or effector proteins, but they also have great potential for new antimicrobial therapies. Recent studies have deployed the T4SS subfamily of conjugation systems to deliver gene editing CRISPR/Cas systems to disrupt drug resistance genes or kill targeted bacterial recipients. However, the therapeutic potential of conjugative CRISPR/Cas delivery is compromised by mutations or host repair systems that diminish the efficiency with which CRISPR/Cas induces double-strand breaks in new transconjugants. Here, we compared the efficiencies of conjugation-based killing systems based on the delivery of CRISPR-Cas9 elements or toxin genes encoding the bacteriophage lambda Kil peptide or the F plasmid-encoded CcdB. Escherichia coli equipped with one of two efficient conjugation systems, pKM101 (IncN) or F (IncF), served as donors to mobilize plasmids carrying the cognate oriT sequence and one or more toxic elements. Overall, toxin gene delivery proved significantly more effective than CRISPR-Cas9 in killing of transconjugant population, but the highest levels of growth suppression of both E. coli and Klebsiella pneumoniae recipients were achieved by a combination of CRISPR-Cas9 plus one or two toxin genes. By contrast, capsule production conferred no or very slight protective effects on plasmid acquisition and killing of either species. We propose that the conjugative co-transfer of two or more toxic elements with distinct mechanisms of action has strong potential for growth suppression of targeted species in environmental or clinical settings.IMPORTANCEThe prevalence of antibiotic resistance emphasizes the need for alternative antimicrobial intervention strategies. We engineered Escherichia coli for conjugative transmission of plasmids encoding CRISPR-Cas9 elements or genes encoding the cell division inhibitor Kil or gyrase poisoner CcdB. Delivery of toxin genes more effectively suppressed the growth of E. coli recipients than CRISPR-Cas9, but the combinatorial delivery of CRISPR-Cas9 and a toxin gene or two toxin genes elicited the strongest killing effects. Capsule production by E. coli or Klebsiella pneumoniae recipient cells had no or little protective effect on plasmid acquisition or growth suppression. Our findings suggest that probiotic donor strains equipped for conjugative delivery of two or more toxic elements may prove effective as an alternative or adjunct to traditional antimicrobials.

Irradiation of prostate cancer alters circulating small extracellular vesicle functions

It is known that β1 integrins and a downstream signaling molecule c-Src are upregulated in prostate cancer (PrCa) tissues, are co-expressed in circulating small extracellular vesicles (sEVs) and contribute to cancer progression. Here, we demonstrate that sEVs from PrCa patients show robust expression of both β1 integrins and c-Src. The impact of irradiation, a widely used therapy for the treatment of PrCa, on circulating sEVs is however not fully understood. We show that sEVs isolated from the plasma of transgenic adenocarcinoma of mouse prostate (TRAMP) mice, stimulate migration and anchorage-independent growth of recipient cancer cells, but sEVs are not active when isolated from mice that had undergone pelvic irradiation of PrCa. In addition, circulating sEVs from irradiated mice do not show co-sedimentation of β1 integrins and either c-Src or sEV markers, previously observed in sEVs from non-irradiated mice, but show reduced expression of c-Src. To rule out that the observed effect of irradiation on sEVs is not due to reduced tumor size, we demonstrate that irradiation of PrCa cells in vitro diminishes sEV capability to stimulate recipient cell anchorage-independent growth. Irradiation-induced changes in the composition of circulating sEVs illustrate a tumor-suppressive effect of radiation, which may have potential therapeutic implications.

There is no transfer of mitochondria from donor hematopoietic cells to recipient mesenchymal stromal cells after allogeneic hematopoietic stem cells transplantation in humans.

There is no transfer of mitochondria from donor hematopoietic cells to recipient mesenchymal stromal cells after allogeneic hematopoietic stem cells transplantation in humans.

Cancer-associated fibroblast-derived extracellular vesicles facilitate metastasis in hepatocellular carcinoma by delivering CTGF.

The tumor microenvironment (TME) plays a crucial role in cancer progression. Cancer-associated fibroblasts (CAFs) are key components of the TME and play critical roles in tumor development and metastasis. However, the mechanisms by which CAFs influence hepatocellular carcinoma (HCC) metastasis are not fully understood. Extracellular vesicles (EVs) from CAFs and normal fibroblasts (NFs) were characterized via western blotting, transmission electron microscopy, and nanoparticle tracking analysis. An iTRAQ-based proteomic sequencing analysis was conducted to quantify proteins in the EVs from these cells. Colony formation assays and Transwell assays were used to assess tumor cell proliferation and migration. Xenograft tumor models were established in nude mice to evaluate tumor progression in vivo. Coimmunoprecipitation and molecular docking were performed to explore the interactions between CTGF and Notch1. A high CAF abundance is associated with poor prognosis in HCC patients. EVs from CAFs significantly enhanced the proliferative and invasive abilities of HCC cells in vitro and in vivo. Connective tissue growth factor (CTGF) was found to be highly upregulated in CAF-derived EVs, and CTGF knockdown in CAF-derived EVs attenuated their tumor-promoting capacities. Mechanistically, CTGF derived from CAF-EVs activated the Notch1/Snail1 signaling pathway in recipient cells via interaction with the Notch1 receptor, enhancing HCC cell proliferation and invasion. Furthermore, high CTGF expression was significantly correlated with poor clinicopathological features in HCC patients. Our findings revealed that CTGF derived from CAF-EVs promoted the proliferation and invasion of HCC cells via activation of the Notch1/Snail1 pathway, highlighting CTGF derived from CAF-EVs as a prognostic biomarker and therapeutic target in HCC.

Aquaporin 5 transfer via MDA-MB-231cell extracellular vesicles increases recipient endothelial cell invasion.

Aquaporin 5 transfer via MDA-MB-231cell extracellular vesicles increases recipient endothelial cell invasion.

Mitochondrial transplantation: adaptive bio-enhancement

Mitochondria, often referred to the powerhouse of the cell, are essential for cellular energy production, and their dysfunction can profoundly affect various organs. Transplantation of healthy mitochondria can restore the bioenergetics in diseased cells and address multiple conditions, but more potentials of this approach remain unclear. In this study, I demonstrated that the source of transplanted mitochondria is not limited by species, as exhibit no significant responses to mitochondria derived from different germlines. Moreover, I identified that metabolic compatibility between the recipient and exogenous mitochondria as a crucial factor in mitochondrial transplantation, which confers unique metabolic properties to recipient cells, enabling them to combat different diseases. Additionally, my findings indicated competitive interactions among mitochondria with varying functions, with more bioenergetic-active mitochondria yielded superior therapeutic benefits. Notably, no upper limit for the bioenhancement provided by exogenous mitochondria has been identified. Based on these insights, I proposes a novel therapeutic approach—adaptive bioenhancement through mitochondrial transplantation.

Defining the Parameters for Sorting of RNA Cargo Into Extracellular Vesicles.

Extracellular vesicles (EVs) are small particles that are released by cells and mediate cell-cell communication by transferring bioactive molecules such as RNA. RNA cargo of EVs, including coding and non-coding RNAs, can change the behaviour of recipient cells, affecting processes including gene expression, proliferation, and Fapoptosis. CircRNAs are stable and resistant to degradation and have been shown to be enriched in EVs. They play key roles in gene regulation and are also emerging as promising biomarkers for disease diagnosis due to their stability and disease-specific expression. Although microRNAs (miRNAs) are the most well studied RNA cargo of EVs, very little is known about the mechanisms of enrichment of circular RNAs (circRNAs) as well as long linear RNAs. Here, we take a comprehensive genome-wide approach to investigate the role of structuredness and shape along with GC%, size, exon count and coding potential, in the sorting and enrichment of circular and long linear RNAs into EVs. We developed a model using these parameters to predict the likelihood of EV packaging of RNA and it was validated by using single molecule RNA imaging of EV bound RNAs. Furthermore, we found that structuredness could explain the relative enrichment of circRNAs over their linear counterparts. These results were validated on existing public databases of circular and linear RNAs in EVs. By identifying and analysing these factors, we aim to better understand the complex mechanisms behind EV-mediated RNA transfer and its impact on cell communication in both health and disease. This mechanistic understanding of RNA enrichment in EVs is crucial for engineering EVs with selective RNA cargo.

Theoretical Understanding of Target Search Dynamics in Horizontal Gene Transfer in Bacteria.

Horizontal gene transfer (HGT) is a fundamental process of increasing genetic diversity in microbial species. It allows bacterial cells to acquire new beneficial traits quickly by incorporating new genetic material into existing genomes. Despite the critical importance of HGT phenomena, the underlying molecular mechanisms are still poorly understood. Recent experiments investigated the dynamics of conjugation HGT processes in which DNA is transmitted directly from the donor to the recipient bacterial cell. It is accomplished by special mobile genetic particles known as integrative and conjugative elements (ICE). However, the molecular picture of how ICE can efficiently find the unique integration sites in a new genome is not yet clear. We present a novel theoretical model to explain the dynamic processes in HGT after ICE reaches the recipient cell. It is shown that the target search for integration sites can be viewed as a set of stochastic transitions between discrete states, allowing us to obtain an explicit description of the dynamic properties using analytical calculations supported by Monte Carlo computer simulations. Search times are found to depend on the location of integration sites, the size of the genome, the effective diffusion rate of mobile genetic elements, and the binding/unbinding transitions between ICE and DNA. Theoretical estimates for search times agree well with experimental observations for integration in Bacillus subtilis bacterial species. Physical-chemical arguments are presented to explain the dynamics of the ICE target search. This study clarifies some important mechanistic aspects of HGT phenomena.

Powering Up the Brain: Intercellular Mitochondrial Quality Control and Its Implication in Stroke.

Mitochondria are critical for neuronal survival and function, and their dysregulation is closely related to the incidence and prevalence of various neurological disorders, including stroke. Mitochondrial quality control (MQC) is vital for maintaining mitochondrial integrity, particularly in neurons. Under ischemic conditions, neurons evolve a range of adaptive strategies to preserve mitochondria function dynamically, either by generating functional mitochondria or by eliminating dysfunctional ones via autophagy, both of which play key roles in keeping neuronal survival under the conditions of stroke. Besides these intracellular strategies, the intercellular mechanisms underlying MQC have been observed in the nervous system. Functional mitochondria from healthy cells can be supplemented to ischemic neurons in distinct manners and thus restore the mitochondrial network of recipient cells. Conversely, injured neurons release dysfunctional mitochondria, which can be further degraded by adjacent glial cells. Alternatively, the discarded mitochondria act as a threat to surrounding cells and can disrupt the homeostasis of the nervous system. In this review, the key discoveries in intercellular MQC in the nervous system were summarized, and further discussed the implications of intercellular MQC strategies for stroke therapy.

Reduction of cellular toxicity with hybrid nanoparticles for mRNA delivery.

Building on the success of COVID-19 vaccine development, lipid nanoparticles (LNPs) have emerged as leading vehicles for mRNA delivery in a range of therapeutic applications. Naturally-occurring extracellular vesicles (EVs), which share similar physical properties with LNPs, present a promising alternative platform because of their relative stability and lower immunogenicity. A key challenge common to both EVs and LNPs is enabling efficient vesicle - cell interactions and establishing a polarized permeability pathway required for effective cargo transfer. Membrane recognition and intercalation are essential for the function and delivery capacity of both systems, regardless of their complexity. In this study, we leveraged recent advances to create hybrid extracellular vesicles (HEVs) by using LNPs to load mRNA into EVs. We characterized HEV formation using Forster resonance energy transfer (FRET), cryo-electron microscopy (Cryo-EM), and super-resolution microscopy, and demonstrated their ability to deliver mRNA to recipient cells. In both, in vitro and in vivo models, HEVs exhibited superior transfection efficiency compared to conventional LNPs composed of synthetic lipids, while significantly reducing LNPs cytotoxicity - a not-well-recognized limitation of synthetic lipid-based systems. These results highlight HEVs as a safer and more effective alternative for mRNA and small molecule delivery. Future therapeutic strategies could involve isolating EVs from patients, hybridizing them with synthetic lipid carriers loaded with therapeutic cargo, and reintroducing them for personalized treatment.

Circ_0084927 promotes progression of intrahepatic cholangiocarcinoma by sponging miR-4725-5p to activate the PDPK1/AKT/mTOR signaling pathway.

Circ_0084927 promotes progression of intrahepatic cholangiocarcinoma by sponging miR-4725-5p to activate the PDPK1/AKT/mTOR signaling pathway.

The RNA binding protein hnRNP A/B controls exosomal miR-27a-5p loading during Salmonella infection.

The RNA binding protein hnRNP A/B controls exosomal miR-27a-5p loading during Salmonella infection.

Nerve-to-cancer transfer of mitochondria during cancer metastasis.

The nervous system has a pivotal role in cancer biology, and pathological investigations have linked intratumoural nerve density to metastasis1. However, the precise impact of cancer-associated neurons and the communication channels at the nerve-cancer interface remain poorly understood. Previous cancer denervation models in rodents and humans have highlighted robust cancer dependency on nerves, but the underlying mechanisms that drive nerve-mediated cancer aggressivity remain unknown2,3. Here we show that cancer-associated neurons enhance cancer metabolic plasticity by transferring mitochondria to cancer cells. Breast cancer denervation and nerve-cancer coculture models confirmed that neurons significantly improve tumour energetics. Neurons cocultured with cancer cells undergo metabolic reprogramming, resulting in increased mitochondrial mass and subsequent transfer of mitochondria to adjacent cancer cells. To precisely track the fate of recipient cells, we developed MitoTRACER, a reporter of cell-to-cell mitochondrial transfer that permanently labels recipient cancer cells and their progeny. Lineage tracing and fate mapping of cancer cells acquiring neuronal mitochondria in primary tumours revealed their selective enrichment at metastatic sites following dissemination. Collectively, our data highlight the enhanced metastatic capabilities of cancer cells that receive mitochondria from neurons in primary tumours, shedding new light on how the nervous system supports cancer metabolism and metastatic dissemination.

Exosome encapsulation of miR-205-5p suppresses neuroblastoma progression by targeting RUNX2

ObjectiveThis study investigates the tumor-suppressive role of microRNA (miR)-205-5p in neuroblastoma (NB) and evaluates exosome-mediated delivery of miR-205-5p as a therapeutic strategy.MethodsmiR-205-5p expression in NB cells was quantified via quantitative reverse transcription PCR. Functional assays (CCK-8, colony formation, wound healing, Transwell) assessed proliferation, migration, and invasion. Bioinformatic tools and dual-luciferase assays identified miR-205-5p/Runt-related transcription factor 2 (RUNX2) binding. RUNX2 rescue experiments reversed miR-205-5p effects. Exosomes from SH-SY5Y cells transfected with miR-205-5p mimics/NC (negative control) lentiviruses were isolated, characterized, and co-cultured with recipient cells. In vivo, subcutaneous NB xenografts in nude mice were established using OE-miR-205-5p, sh-miR-205-5p, or NC lentiviral cells, followed by exosome injections to evaluate tumor growth.ResultsmiR-205-5p was downregulated in NB cells. Its overexpression suppressed proliferation, migration, invasion, and tumor growth in vitro and in vivo. RUNX2 was confirmed as a direct target; its restoration reversed miR-205-5p-mediated inhibition. Exosomes efficiently delivered miR-205-5p to recipient cells, downregulating RUNX2 and impairing malignant behaviors. In mice, miR-205-5p-enriched exosomes significantly inhibited tumor progression.ConclusionsExosome-encapsulated miR-205-5p inhibits NB progression by targeting RUNX2, highlighting its potential as a novel therapeutic modality.