Search Results (114)

Pancreatic cancer, specifically pancreatic ductal adenocarcinoma (PDAC), ranks among the most lethal malignancies, with a 5-year survival rate of under 10%. The most prevalent KRAS mutations occur in three hotspot residues: glycine-12 (G12), glycine-13 (G13), and glutamine-61 (Q61), leading to the constant activation of the Ras pathway, making them the primary focus in oncologic drug development. Selective KRAS G12C inhibitors (e.g., sotorasib, adagrasib) have demonstrated moderate efficacy in clinical trials; however, this mutation is infrequent in PDAC. Emerging therapies targeting KRAS G12D and G12V mutations, such as MRTX1133, PROTACs, and active-state inhibitors, show promise in preclinical studies. Pan-RAS inhibitors like ADT-007, RMC-9805, and RMC-6236 compounds provide broader coverage of mutations. Their efficacy and safety are currently being investigated in several clinical trials. A major challenge is the development of resistance mechanisms, including secondary mutations and pathway reactivation. Combination therapies targeting the RAS/MAPK axis, SHP2, mTOR, or SOS1 are under clinical investigation. Immunotherapy alone has demonstrated limited effectiveness, attributed to an immunosuppressive tumor microenvironment, although synergistic effects are noted when paired with KRAS-targeted agents. Furthermore, KRAS mutations reprogram cancer metabolism, enhancing glycolysis, macropinocytosis, and autophagy, which are being explored therapeutically. RNA interference technologies have also shown potential in silencing mutant KRAS and reducing tumorigenicity. Future strategies should emphasize the combination of targeted therapies with metabolic or immunomodulatory agents to overcome resistance and enhance survival in KRAS-mutated PDAC. Full article

Macropinocytosis is a non-selective, clathrin-independent endocytic process that facilitates bulk internalization of extracellular fluid and its dissolved components (including proteins, lipids, and nucleotides) through plasma membrane remodeling and the subsequent formation of macropinosomes. This evolutionarily conserved cellular process plays important roles in nutrient supply, immune response, and metabolism. Particularly, cancer cells exploit activated macropinocytosis to obtain nutrients for supporting proliferation and survival under nutritional stress. Thus, macropinocytosis emerges as an important target for cancer therapy. Furthermore, as activated macropinocytosis constitutively uptakes extracellular fluids into cancer cells, it has been utilized for delivering anti-tumor drugs in cancer therapy. In this review, we systematically addressed progress in cancer therapeutic strategies in both targeting macropinocytosis and utilizing macropinocytosis as an anti-cancer drug delivering tool, including therapeutic applications with macropinocytosis inhibitors; metabolic modulators; methuosis (the macropinocytosis-associated cell death) inducers; and macropinocytosis-mediated anti-cancer drug delivery strategies such as nanoparticles, viral vectors, extracellular vesicles, and targeted conjugates. We conclude that developing targeted macropinocytosis anti-cancer drugs and exploring macropinocytosis-dependent anti-cancer drug delivery systems open new avenues for cancer therapy. Full article

Mitochondrial transplantation (MTx) has emerged as a potential therapeutic approach for diseases associated with mitochondrial dysfunction, yet its scalability and cross-species feasibility remain underexplored. This study aimed to evaluate the dose-dependent uptake and molecular effects of xenogeneic mitochondrial transplantation (xeno-MTx) using rat-derived mitochondria in mouse neuronal systems. HT-22 hippocampal neuronal cells and a murine model of cardiac arrest-induced global cerebral ischemia were used to assess mitochondrial uptake, gene expression, and mitochondrial DNA presence. Donor mitochondria were isolated from rat pectoralis muscle and labeled with MitoTracker dyes. Flow cytometry and confocal microscopy revealed a dose-dependent increase in donor mitochondrial uptake in vitro. Quantitative PCR demonstrated a corresponding increase in rat-specific mitochondrial DNA and upregulation of Mfn2 and Bak1, with no changes in other fusion, fission, or apoptotic genes. Inhibitor studies indicated that mitochondrial internalization may involve actin-dependent macropinocytosis and cholesterol-sensitive endocytic pathways. In vivo, rat mitochondrial DNA was detected in mouse brains post–xeno-MTx, confirming donor mitochondrial delivery to ischemic tissue. These findings support the feasibility of xeno-MTx and its dose-responsive biological effects in neuronal systems while underscoring the need for further research to determine long-term functional outcomes and clinical applicability. Full article

Increased abundance of Segatella copri (S. copri) within the gut microbiota is associated with systemic inflammatory diseases, including rheumatoid arthritis. Although outer-membrane vesicles (OMVs) of Gram-negative bacteria are important players in microbiota–host communication, the effect of S. copri-derived OMVs on immune cells is unknown. Macrophages engulf and eliminate foreign material and are conditioned by environmental signals to promote either homeostasis or inflammation. Thus, we aimed to explore the impact of S. copri-OMVs on human macrophages in vitro, employing THP-1 and monocyte-derived macrophage models. The uptake of DiO-labeled S. copri-OMVs into macrophages was monitored by confocal microscopy and flow cytometry. Furthermore, the effect of S. copri and S. copri-OMVs on the phenotype and cytokine secretion of naïve (M0), pro-inflammatory (M1), and anti-inflammatory (M2) macrophages was analyzed by flow cytometry and ELISA, respectively. We show that S. copri-OMVs enter human macrophages through macropinocytosis and clathrin-dependent mechanisms. S. copri-OMVs, but not the parental bacterium, induced a dose-dependent increase in the expression of M1-related surface markers in M0 and M2 macrophages and activated the secretion of large amounts of pro-inflammatory cytokines in M1 macrophages. These results highlight an important role of S. copri-OMVs in promoting pro-inflammatory macrophage responses, which might contribute to systemic inflammatory diseases. Full article

During sexual reproduction, the freshwater ciliate Tetrahymena thermophila sheds membrane-bound vesicles into the extracellular environment (cEMVs: ciliary extracellular micro-vesicles). We provide evidence that 100 nm vesicles shed from the cilia of starved cells promote mating between cells of complementary mating types. A proteomic analysis revealed that these EMVs are decorated with mating-type proteins expressed from the MAT locus, proteins that define a cell’s sex (one of seven). Once the mating junction is established between cells, smaller 60 nm vesicles (junction vesicles) appear within the extracellular gap that separates mating partners. Junction vesicles (jEMVs) may play a role in remodeling the mating junction through which gametic pronuclei are exchanged. Evidence is presented demonstrating that cells must be able to internalize extracellular signals via some form of endocytosis in order to trigger conjugation. Finally, an evolutionarily conserved fusogen (Hap2) implicated in pore formation also appears necessary for jEMV processing. This system offers an excellent opportunity for studies on ectosome shedding, intercellular signaling and shed vesicle uptake by macro-pinocytosis, as they relate to sexual reproduction in the ciliate Tetrahymena thermophila. Full article

African swine fever (ASF) is an acute and highly contagious disease that has caused great losses in the past years. It is caused by African swine fever virus (ASFV), which is a large DNA virus encoding about 165 genes. It has been shown that the purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis, and the virus utilizes apoptotic bodies for infection and cell cell transmission. The ASFV-encoded RNA polymerase subunit C315R is thought to play an important role in ASFV replication and transcription. However, its involvement in ASFV infection, particularly in host response, remains only partially understood. In this study, the role of C315R in enhancing ASFV replication was investigated through RNA-Seq transcriptomic analysis, which was based on 3D4/21 cells transfected the plasmid expressing HA-tagged C315R or the empty vector. Our findings revealed that C315R significantly upregulates the expression of inflammatory mediators with a particular emphasis on IL-6. The most differentially expressed genes (DEGs) were predominantly associated with the TNF, IL-17, MAPK, and JAK STAT signaling pathways. RNA-seq results were validated through RT-PCR. Subsequently, we observed that ASFV infection increases IL-6 expression and STAT3 phosphorylation, which is regulated by the ASFV C315R protein. Notably, inhibiting STAT3 phosphorylation with specific inhibitors suppressed ASFV replication. In conclusion, our study demonstrates that the ASFV C315R protein actives STAT3 phosphorylation through promoting the transcription of IL-6 to facilitate virus replication. These findings highlight C315R as a positive regulator in the IL-6 STAT3 signaling axis during ASFV infection. Full article

Background/Objectives: Colorectal cancer (CRC) remains a leading cause of cancer mortality globally, with KRAS mutations occurring in 30–40% of cases, contributing to poor prognosis and resistance to anti-EGFR therapy. This review explores the biological significance, clinical implications, and therapeutic targeting of KRAS mutations in CRC. Methods: A comprehensive analysis of the existing literature and clinical trials was performed, highlighting the role of KRAS mutations in CRC pathogenesis, their impact on prognosis, and recent advancements in targeted therapies. Specific attention was given to emerging therapeutic strategies and resistance mechanisms. Results: KRAS mutations drive tumor progression through persistent activation of MAPK/ERK and PI3K/AKT signaling pathways. These mutations influence the tumor microenvironment, cancer stem cell formation, macropinocytosis, and cell competition. KRAS-mutant CRC exhibits poor responsiveness to anti-EGFR monoclonal antibodies and demonstrates primary and acquired resistance to KRAS inhibitors. Recent breakthroughs include the development of KRAS G12C inhibitors (sotorasib and adagrasib) and promising agents targeting G12D mutations. However, response rates in CRC remain suboptimal compared to other cancers, necessitating combination therapies and novel approaches, such as vaccines, nucleic acid-based therapeutics, and macropinocytosis inhibitors. Conclusions: KRAS mutations are central to CRC pathogenesis and present a significant therapeutic challenge. Advances in KRAS-targeted therapies offer hope for improved outcomes, but resistance mechanisms and organ-specific differences limit efficacy. Continued efforts in personalized treatment strategies and translational research are critical for overcoming these challenges and improving patient survival. Full article

Our group has recently demonstrated that exercise intervention affects the release and function of bone marrow endothelial progenitor cell-derived extracellular vesicles (EVs) in transgenic hypertensive mice. Whether such an exercise regimen can impact circulating EVs (cEVs) remains unknown. In this study, we investigated the influence of exercise on cEV level and function. Transgenic hypertensive mice (Alb1-Ren) underwent 8-week treadmill exercise (10 m/min for 1 h, 5 days per week). Age- and sex-matched sedentary Alb1-Ren mice served as controls. cEVs were isolated from the blood of exercised and sedentary mice and are denoted as ET-cEV and nET-cEV, respectively. cEVs were labeled to determine their uptake efficiency and pathways. The functions of cEVs were assessed in an Angiotensin II (Ang II) plus hypoxia-injured cerebral microvascular endothelial cell (mBMEC) injury model. Cellular migration ability and oxidative stress were evaluated. We found that treadmill exercise stimulated cEV release, and ET-cEVs were more prone to be internalized by mBMECs. The ET-cEV internalization was mediated by macropinocytosis and endocytosis pathways. Functional studies showed that ET-cEVs can improve the compromised migration capability of mBMECs challenged by Ang II plus hypoxia. Additionally, ET-cEV treatment upregulated the expression of p-Akt/Akt in mBMECs. Compared to nET-cEVs, ET-cEVs significantly reduced ROS overproduction in Ang II plus hypoxia-injured mBMECs, associated with decreased Nox2 expression. All these findings suggest that exercise-intervened cEVs can protect cerebral microvascular endothelial cells against hypertensive and hypoxic injury. Full article

Microplastics (MPs) in fish can cross the intestinal barrier and are often bioaccumulated in several tissues, causing adverse effects. While the impacts of MPs on fish are well documented, the mechanisms of their cellular internalization remain unclear. A rainbow-trout (Oncorhynchus mykiss) intestinal platform, comprising proximal and distal intestinal epithelial cells cultured on an Alvetex scaffold, was exposed to 50 mg/L of MPs (size 1–5 µm) for 2, 4, and 6 h. MP uptake was faster in RTpi-MI compared to RTdi-MI. Exposure to microplastics compromised the cellular barrier integrity by disrupting the tight-junction protein zonula occludens-1, inducing significant decreases in the transepithelial-electrical-resistance (TEER) values. Consequently, MPs were internalized by cultured epithelial cells and fibroblasts. The expression of genes related to endocytosis (cltca, cav1), macropinocytosis (rac1), and tight junctions’ formation (oclna, cldn3a, ZO-1) was analyzed. No significant differences were observed in cltca, oclna, and cldn3a expression, while an upregulation of cav1, rac1, and ZO-1 genes was detected, suggesting macropinocytosis as the route of internalization, since also cav1 and ZO-1 are indirectly related to this mechanism. The obtained results are consistent with data previously reported in vivo, confirming its validity for identifying MP internalization pathways. This could help to develop strategies to mitigate MP absorption through ingestion. Full article

Mycoplasma bovis (M. bovis) is capable of causing pneumonia, arthritis, mastitis, and various other ailments in cattle of all age groups, posing a significant threat to the healthy progression of the worldwide cattle industry. The invasion of non-phagocytic host cells serves as a pivotal mechanism enabling M. bovis to evade the immune system and penetrate mucosal barriers, thereby promoting its spread. To investigate the differences in M. bovis invasion into four types of non-phagocytic cells (Madin–Darby bovine kidney (MDBK) cells, embryonic bovine lung (EBL) cells, bovine embryo tracheal (EBTr) cells and bovine turbinate (BT) cells) and further elucidate its invasion mechanism, this study first optimized the experimental methods for M. bovis invasion into cells. Utilizing laser scanning confocal microscopy, transmission electron microscopy, and high-content live-cell imaging systems, the invasion process of M. bovis into four types of non-phagocytic cells was observed. The invasion rates of three different strains of M. bovis (PG45, 07801, 08M) were quantified through the plate counting method. In order to clarify the specific pathway of M. bovis invasion into cells, chlorpromazine (CPZ), amiloride (AMI), and methyl-β-cyclodextrin (M-β-CD) were used to inhibit CLR-mediated clathrin-dependent endocytosis (CDE) pathway, macropinocytosis, and lipid raft pathway, respectively. Subsequently, the invasion rates of PG45 into these four types of cells were measured. Using siRNA technology, the expression of clathrin (CLR) in EBL cells was knocked down to further verify the role of CLR in the invasion process of M. bovis. The results showed that the optimal conditions for M. bovis to invade non-phagocytic cells were a multiplicity of infection (MOI) of 1000 and an optimal invasion time of 4 h. All three strains of M. bovis have the ability to invade the four types of non-phagocytic cells, yet their invasion abilities vary significantly. Observations from transmission electron microscopy further confirmed that at 120 min post-infection, PG45 had successfully invaded EBL cells and was present within endocytic vesicles. It is noteworthy that almost all PG45 successfully escaped from the endocytic vesicles after 240 min of infection had passed. Through chemical inhibition experiments and CLR protein knockdown experiments, it was found that when the CDE and lipid raft pathways were blocked or CLR protein expression was reduced, the invasion rates of PG45, 07801, and 08M in MDBK, EBL, EBTr, and BT cells were significantly decreased (p < 0.05). The above results indicate that M. bovis can invade all types of non-phagocytic cells through endocytic pathways involving CDE (clathrin-dependent endocytosis) or lipid raft-mediated endocytosis, and possesses the ability to escape from phagosomes. Full article

Arsenic trioxide (ATO) has been shown to inhibit pancreatic cancer (PC) cell growth in vitro and to promote the inhibitory effects of gemcitabine (Gem) on PC in vivo. However, the high toxicity of ATO associated with the required high doses and indiscriminate targeting has limited its clinical application. This study aimed to determine whether coupling arsenic to a tumor homing peptide would increase the inhibitory potency against PC cells. The effects of this peptide-linked arsenic compound (PhAs-LHP), the analogous non-targeting arsenic compound (phenylarsine oxide, PAO), and marketed ATO on PC growth were tested in vitro and in a mouse model. The data demonstrated that PhAs-LHP inhibited PC cell growth in vitro more potently, with IC₅₀ values 10 times lower than ATO. Like ATO, PhAs-LHP induced cell death and cell cycle arrest. This cytotoxic effect of PhAs-LHP was mediated via a macropinocytosis-linked uptake pathway as amiloride (a macropinocytosis inhibitor) reduced the inhibitory effect of PhAs-LHP. More importantly, PhAs-LHP inhibited PC growth in mice and enhanced the inhibitory effect of Gem on PC growth at 2 times lower molar concentration than PAO. These results indicate that PhAs-LHP inhibited PC more potently than ATO/PAO and suggest a potential clinical use for the combination of Gem with the peptide-linked arsenic compound for the treatment of pancreatic cancer. Full article

Membrane ruffles are cell actin-based membrane protrusions that have distinct structural characteristics. Linear ruffles with columnar spike-like and veil-like structures assemble at the leading edge of cell membranes. Circular dorsal ruffles (CDRs) have no supporting columnar structures but their veil-like structures, connecting from end to end, present an enclosed ring-shaped circular outline. Membrane ruffles are involved in multiple cell functions such as cell motility, macropinocytosis, receptor internalization, fluid viscosity sensing in a two-dimensional culture environment, and protecting cells from death in response to physiologically compressive loads. Herein, we review the state-of-the-art knowledge on membrane ruffle structure and function, the growth factor-induced membrane ruffling process, and the growth factor-independent ruffling mode triggered by calcium and other stimulating factors, together with the respective underlying mechanisms. We also summarize the inhibitors used in ruffle formation studies and their specificity. In the last part, an overview is given of the various techniques in which the membrane ruffles have been visualized up to now. Full article

Extracellular vesicles (EVs) have attracted great attention as promising intracellular drug delivery carriers. While the endocytic pathways of small EVs (sEVs, <200 nm) have been reported, there is limited understanding of large EVs (lEVs, >200 nm), despite their potential applications for drug delivery. Additionally, the low yield of EVs during isolation remains a major challenge in their application. Herein, we aimed to compare the endocytic pathways of sEVs and lEVs using MIA PaCa-2 pancreatic cancer cell-derived EVs as models and to explore the efficiency of their production. The cellular uptake of EVs by MIA PaCa-2 cells was assessed and the pathways were investigated with the aid of endocytic inhibitors. The yield and protein content of sEVs and lEVs from the Integra CELLine culture system and the conventional flasks were compared. Our findings revealed that both sEVs and lEVs produced by the Integra CELLine system entered their parental cells via multiple routes, including caveolin-mediated endocytosis, clathrin-mediated endocytosis, and actin-dependent phagocytosis or macropinocytosis. Notably, caveolin- and clathrin-mediated endocytosis were more prominent in the uptake of sEVs, while actin-dependent phagocytosis and macropinocytosis were significant for both sEVs and lEVs. Compared with conventional flasks, the Integra CELLine system demonstrated a 9-fold increase in sEVs yield and a 6.5-fold increase in lEVs yield, along with 3- to 4-fold higher protein content per 10¹⁰ EVs. Given that different endocytic pathways led to distinct intracellular trafficking routes, this study highlights the unique potentials of sEVs and lEVs for intracellular cargo delivery. The Integra CELLine proves to be a highly productive and cost-effective system for generating EVs with favourable properties for drug delivery. Full article

The bioavailability of a monoclonal antibody (mAb) or another therapeutic protein after subcutaneous (SC) dosing is challenging to predict from first principles, even if the impact of injection site physiology and drug properties on mAb bioavailability is generally understood. We used a physiologically based pharmacokinetic model to predict pre-systemic clearance after SC administration mechanistically by incorporating the FcRn salvage pathway in antigen-presenting cells (APCs) in peripheral lymph nodes, draining the injection site. Clinically observed data of the removal rate of IgG from the arm as well as its plasma concentration after SC dosing were mostly predicted within the 95% confidence interval. The bioavailability of IgG was predicted to be 70%, which mechanistically relates to macropinocytosis in the draining lymph nodes and transient local dose-dependent partial saturation of the FcRn receptor in the APCs, resulting in higher catabolism and consequently less drug reaching the systemic circulation. The predicted free FcRn concentration was reduced to 40–45%, reaching the minimum 1–2 days after the SC administration of IgG, and returned to baseline after 8–12 days, depending on the site of injection. The model predicted the uptake into APCs, the binding affinity to FcRn, and the dose to be important factors impacting the bioavailability of a mAb. Full article

Virus-like nanoparticles (VNP) are regarded as efficient vaccination platforms and have proven to be useful for the non-anaphylactogenic delivery of allergen-specific immunotherapy in preclinical models previously. Herein, we sought to determine the mode of VNP uptake by antigen presenting cells (APC). Accordingly, we screened a collection of substances known to inhibit different uptake pathways by APC. The human leukemia monocytic cell line THP-1 and the murine dendritic cell line DC 2.4 were examined for the uptake of fluorescently labelled VNP in the presence or absence of inhibitors. The inhibitory effect of candidate substances that blocked VNP uptake in APC lines was subsequently evaluated in studies with primary APC present in splenocyte and lung cell homogenates in vitro and upon intratracheal application of VNP in vivo. The uptake of allergen-specific VNP in vitro and in vivo was mainly observed by macrophages and CD103⁺ dendritic cells and was sensitive to inhibitors that block macropinocytosis, such as hyperosmolarity induced by sucrose or the polyphenol compound Rottlerin at low micromolar concentrations but not by other inhibitors. Also, T-cell proliferation induced by allergen-specific VNP was significantly reduced by both substances. In contrast, substances that stimulate macropinocytosis, such as Heparin and phorbol myristate acetate (PMA), increased VNP-uptake and may, thus, help modulate allergen-specific T-cell responses. We have identified macropinocytosis as the principal uptake mechanism of APC for allergen-specific VNP in vitro and in vivo, paving the way for further improvement of VNP-based therapies, especially those that can be used for tolerance induction in allergy, in the future. Full article