Biology subjects

Kanada, M.

Publications and source records attributed to Kanada, M..

7 recordsLinked to original sources

bioRxiv2025

Harnessing Extracellular Vesicles for Stabilized and Functional IL-10 Delivery in Macrophage Immunomodulation

Extracellular vesicles (EVs) are gaining recognition as promising therapeutic carriers for immune modulation. We investigated the potential of EVs derived from HEK293FT cells to stabilize and deliver interleukin-10 (IL-10), a key anti-inflammatory cytokine. Using minicircle (MC) DNA vectors, we achieved IL-10 overexpression and efficient incorporation into EVs, yielding superior stability compared to free, recombinant IL-10 protein. Detailed biophysical and functional analyses revealed that IL-10+ EVs contain both monomeric and oligomeric forms of IL-10 on their external surface and encapsulated within vesicles. IL-10+ EVs suppressed inflammatory cytokine expression in pro-inflammatory macrophages (from two to 14-fold compared to naive EVs) without inducing anti-inflammatory polarization, demonstrating a distinct immunosuppressive mechanism. Interestingly, naive EVs from non-transfected cells also exhibited significant anti-inflammatory effects, suggesting that the intrinsic bioactive cargo of EVs substantially contributes to their function, complicating the interpretation of IL-10-specific effects. Size-based fractionation analyses of IL-10+ large EVs (lEVs), small EVs (sEVs), and non-vesicular extracellular particles (NVEPs) revealed IL-10 presence across all fractions, predominantly in monomeric form, with anti-inflammatory activity distributed among subpopulations. Anion exchange chromatography successfully enriched IL-10+ exosomes while retaining immunomodulatory effects. However, the shared properties of naive and IL-10+ exosomes underscore the complexity of their immunomodulatory functions. These findings highlight the therapeutic potential of EVs while emphasizing the need to disentangle the contributions of engineered cytokines from endogenous vesicular components.

immunology↗

bioRxiv2022

Phosphatidylserine-exposing medium/large extracellular vesicles: potential cancer biomarkers

Under physiological conditions, phosphatidylserine (PS) predominantly localizes to the cytosolic leaflet of the plasma membrane of cells. During apoptosis, PS is exposed on the cell surface and serves as an "eat-me" signal for macrophages to prevent releasing self-immunogenic cellular components from dying cells which could potentially lead to autoimmunity. However, increasing evidence indicates that viable cells can also expose PS on their surface. Interestingly, tumor cell-derived extracellular vesicles (EVs) also externalize PS. Recent studies have proposed PS-exposing EVs as a potential biomarker for the early detection of cancer and other diseases. However, there are confounding results regarding subtypes of PS-positive EVs, and knowledge of PS exposure on the EV surface requires further elucidation. In this study, we enriched small EVs (sEVs) and medium/large EVs (m/lEVs) from conditioned media of breast cancer cells (MDA-MB-231, MDA-MB-468) and non-cancerous cells (keratinocytes, fibroblasts). Since several PS-binding molecules are available to date, we compared recombinant proteins of annexin A5 and the carboxylated glutamic acid domain of Protein S (GlaS), also specific for PS, to detect PS-exposing EVs. Firstly, PS externalization in each EV fraction was analyzed using a bead-based EV assay, which combines EV capture using microbeads and analysis of PS-exposing EVs by flow cytometry. The bulk EV assay showed higher PS externalization in m/lEVs derived from MDA-MB-468 cells but not from MDA-MB-231 cells, while higher binding of GlaS was also observed in m/lEVs from fibroblasts. Second, using single EV flow cytometry, PS externalization was also analyzed on individual sEVs and m/lEVs. Significantly higher PS externalization was detected in m/lEVs (annexin A1+) derived from cancer cells compared to m/lEVs (annexin A1+) from non-cancerous cells. These results emphasize the significance of PS-exposing m/lEVs as an undervalued EV subtype for early cancer detection and provide a better understanding of PS externalization in disease-associated EV subtypes.

cancer biology↗

bioRxiv2022

Gla-domain mediated targeting of externalized phosphatidylserine for intracellular delivery

Phosphatidylserine (PS) is a negatively charged phospholipid normally localized to the inner leaflet of the plasma membrane of cells but is externalized onto the cell surface during apoptosis as well as in malignant and infected cells. Consequently, PS may comprise an important molecular target in diagnostics, imaging and targeted delivery of therapeutic agents. While an array of PS binding-molecules exist, their utility has been limited by their inability to recognize PS and internalize diagnostic or therapeutic payloads. We describe the generation, isolation, characterization, and utility of a PS binding motif comprised of a carboxylated glutamic acid (GLA) residue domain, that both recognizes cell surface-exposed PS and is internalized into these cells after binding. Internalization is independent of the traditional endosomal-lysosomal pathway, directly entering the cytosol of the target cell in a rapid and energy-independent fashion. We demonstrate that this PS recognition extends to extracellular vesicles and stem cells and that GLA-domain conjugated probes can be detected upon intravenous administration in animal models of infectious disease and cancer. GLA domain binding and internalization offers new opportunities for targeting specific cells for imaging and delivery of therapeutics.

cell biology↗

bioRxiv2022

Listeria monocytogenes Infection Alters Extracellular Vesicles Produced by Trophoblast Stem Cells to Confer a Pro-Inflammatory State

Placental immunity is critical for fetal health during pregnancy, as invading pathogens can be transmitted from the maternal blood to the fetus through this organ. However, inflammatory responses in the placenta can adversely affect both the fetus and the pregnant mother, and the balance between protective placental immune response and detrimental inflammation is poorly understood. Extracellular vesicles (EVs) are membrane-enclosed vesicles that play a critical role in placental immunity. EVs produced by placental trophoblasts mediate immune tolerance to the fetus and to the placenta itself, but these EVs can also activate detrimental inflammatory responses. The regulation of these effects is not well-characterized, and the role of trophoblast EVs (tEVs) in the response to infection has yet to be defined. The Gram-positive bacterial pathogen Listeria monocytogenes (Lm) infects the placenta, serving as a model to study tEV function in this context. We investigated the effect of Lm infection on the production and function of tEVs, using a trophoblast stem cell (TSCs) model. We found that tEVs from infected TSCs were immunostimulatory, activating macrophages to a pro-inflammatory state. Surprisingly, this activation made RAW 264.7 macrophages more susceptible to subsequent Lm infection. Increased susceptibility to infection has not previously been reported as an effect of EVs. Proteomic analysis and RNA sequencing revealed that tEVs from infected TSCs had altered cargo compared to those from uninfected TSCs. Together, these results suggest an immunomodulatory role for tEVs during prenatal infection.

immunology↗

bioRxiv2021

A Dual-reporter Platform for Screening Tumor-targeted Extracellular Vesicles

Extracellular vesicle (EV)-mediated transfer of biomolecules plays an essential role in intercellular communication and may improve targeted drug delivery. In the past decade, various approaches to EV surface modification for targeting specific cells or tissues have been proposed, including genetic engineering of parental cells or postproduction EV engineering. However, due to technical limitations, targeting moieties of engineered EVs have not been thoroughly characterized. Here, we report the bioluminescence resonance energy transfer (BRET) EV reporter, PalmReNL-based dual-reporter platform for characterizing the cellular uptake of tumor homing peptide (THP)-engineered EVs, targeting PDL1, uPAR, or EGFR proteins expressed in MDA-MB-231 breast cancer cells, simultaneously by bioluminescence measurement and fluorescence microscopy. Bioluminescence analysis of cellular EV uptake revealed the highest binding efficiency of uPAR-targeted EVs, whereas PDL1-targeted EVs showed slower cellular uptake. EVs engineered with two known EGFR-binding peptides via lipid nanoprobes did not increase cellular uptake, indicating that designs of EGFR-binding peptide conjugation to the EV surface are critical for functional EV engineering. Fluorescence analysis of cellular EV uptake allowed us to track individual PalmReNL-EVs bearing THPs in recipient cells. These results demonstrate that the PalmReNL-based EV assay platform can be a foundation for high-throughput screening of tumor-targeted EVs.

bioengineering↗

bioRxiv2021

In vitro and in vivo analysis of microvesicle-mediated metastasis using a bright, red-shifted bioluminescent reporter protein of extracellular vesicles

Cancer cells produce heterogeneous extracellular vesicles (EVs) as mediators of intercellular communication. Our study focused on a novel method to image EV subtypes and their biodistribution in vivo. Regardless of injection routes, we established that reporter EVs isolated from murine mammary carcinoma cells expressing PalmReNL, which utilizes bioluminescence resonance energy transfer (BRET), localized to the lungs. This new EV reporter allowed highly sensitive EV tracking in vitro and in vivo and enabled us to begin studies to understand the commonalities and functional differences of the EV subtypes. We demonstrated the early appearance of metastatic foci in the lungs of mammary tumor-bearing mice following multiple injections of the microvesicle (MV)-enriched fraction derived from mammary carcinoma cells. In addition, the results we present here show that tumor cell-derived MVs act on distant tissues through upregulating LC3 expression within the lung.

cancer biology↗

bioRxiv2020

Longitudinal Saliva Omics Responses to Immune Perturbation: A Case Study

Saliva omics, a rapidly developing field for non-invasive diagnostics, may be utilized for monitoring very young or elderly populations, as well as individuals in remote locations. In this study, multiple saliva omics from an individual were monitored over 100 timepoints, over three periods involving: (i) hourly sampling over 24 hours without intervention, (ii) hourly sampling over 24 hours including immune system activation using the standard 23-valent pneumococcal polysaccharide vaccine, (iii) daily sampling for 33 days profiling the post-vaccination response. At each timepoint total saliva transcriptome and proteome were profiled, and salivary extracellular vesicles were derived, from which small-RNA sequencing was used to determine RNA, miRNA, piRNA and bacterial RNA components. The two 24-hour periods were used in a paired analysis to reveal vaccination responses. Temporal trends were classified and collective behavior revealed broad immune-responses captured in saliva, both at the innate as well as the adaptive response time frames.

genomics↗