This study aimed to elucidate the underlying mechanisms regarding microRNA-708-5p (miR-708-5p) in lung adenocarcinoma (LUAD). Here, the co-culture system of LUAD cells and macrophages, as well as a xenograft mouse model, were established. High levels of miR-708-5p were observed in LUAD. Exosomal miR-708-5p facilitated M2-like phenotype polarization, whereas miR-708-5p inhibition blocked the polarization. Exosomal miR-708-5p was identified as a pivotal signaling molecule for macrophages to mediate tumor cell proliferation, invasion, migration and IFN-γ production in T cells. In addition, miR708-5p was observed to induce PD-L1 expression, and PD-L1 silencing inhibited macrophage-induced tumor cell growth behavior and regulated CD8 T cell activity. In xenograft models, miR-708-5p inhibition and PD-L1 silencing attenuated macrophage-induced tumor growth, induced IFN-γ secretion and CD8 expression, and modulated the PTEN/AKT/mTOR pathway. In LUAD patients, there was an upregulation of both miR-708-5p and PD-L1 expression, accompanied by the activation of PTEN/AKT/mTOR. In conclusion, this study demonstrated the induction of M2 macrophage polarization and PD-L1 expression by exosomal miR-708-5p. We observed that exosomal miR-708-5p mediated the PTEN/AKT/mTOR pathway, diminished CD8 T cell activity and accelerated LUAD progression. The inhibition of specific exosomal miRNA secretion and anti-PD-L1 in the LUAD microenvironment may represent a promising avenue for LUAD immunotherapy.

Extracellular vesicles (EVs) are membrane-bound structures released by all cell types. They play a critical role in intercellular communication by transferring their cargo, comprising proteins, lipids, metabolites, RNAs, miRNAs, and DNA fragments, to recipient cells. This transfer influences gene expression, signaling pathways, and cellular behavior. Due to their ability to alter the physiology of recipient cells, EVs hold significant therapeutic potential. Additionally, EVs are implicated in various physiological and pathological processes, including immune regulation, cancer progression, and cardiovascular diseases. EVs have been detected in many biological fluids, such as peripheral blood, saliva, urine, cerebrospinal fluid, and breast milk. The cargo of EVs dynamically reflects the physiological and pathological state of their parent cells, making them promising candidates for liquid biopsies in various clinical conditions. Specifically, different EV subtypes in cardiovascular diseases have been studied, with both endothelial and platelet-derived EVs playing significant roles in cardiovascular pathologies. This review focuses on the diagnostic and prognostic potential of endothelial and platelet-derived EVs in cardiovascular diseases, highlighting the role of EV subpopulations.

Spinal cord injury (SCI) is a severe disorder characterized by regeneration challenges in the central nervous system (CNS), resulting in permanent paralysis, loss of sensation, and abnormal autonomic functions. The complex pathophysiology of SCI poses challenges to traditional treatments, highlighting the urgent need for novel treatment approaches. Exosomes have emerged as promising candidates for SCI therapy because of their ability to deliver a wide range of bioactive molecules, such as RNAs, proteins, and lipids, to target cells with minimal immunogenicity, which contribute to anti-inflammatory, anti-apoptotic, autophagic, angiogenic, neurogenic, and axon remodeling activities. In this study, we classified exosomes from different sources into four categories based on the characteristics of the donor cells (mesenchymal stem cells, neurogenic cells, immune cells, vascular-associated cells) and provided a detailed summary and discussion of the current research progress and future directions for each source. We also conducted an in-depth investigation into the applications of engineered exosomes in SCI therapy, focusing on their roles in drug delivery and combination with surface engineering technologies and tissue engineering strategies. Finally, the challenges and prospects of exosomal clinical applications in SCI repair are described.

Cisplatin (DDP) resistance presents a major challenge in bladder cancer (BLCA) treatment. Recent evidence suggests that Astragalus polysaccharide (APS), extracted from Astragalus membranaceus, may sensitize tumors to DDP. However, the precise mechanisms by which APS modulates DDP sensitivity in BLCA are not fully elucidated. The study employed computational biology, bioinformatics, and both in vitro and in vivo experiments to explore the role of APS in BLCA. The results demonstrate that APS inhibits BLCA cell proliferation, induces apoptosis in vitro, and suppresses tumor growth in vivo. Additionally, APS induces G0/G1 cell cycle arrest in BLCA cells by downregulating CCND1 expression. Moreover, APS further enhances DDP-induced apoptosis by downregulating PI3K-p110β and p-AKT expression, while upregulating FoxO1 expression. Bioinformatics analysis indicates that APS may remodel the tumor microenvironment (TME) and influence cell-cell interactions, specifically through modulation of macrophage M2 polarization and CD8+ T cell exhaustion, thereby overcoming DDP resistance. In conclusion, APS potentiates DDP-induced apoptosis in BLCA cells via the PI3K/AKT/FoxO1 axis and may act as an immunomodulator to remodel the TME, offering a potential strategy to combat DDP resistance in BLCA.

Radiotherapy (RT) remains a cornerstone in the treatment of prostate cancer (PCa). Extracellular vesicles (EVs), nano-sized particles secreted by cells, play important roles in intercellular communication within the tumour microenvironment (TME) and contribute to tumour growth, metastasis, and therapy resistance. Recent advancements demonstrate the potential of EVs as biomarkers for cancer diagnosis, prognosis, and treatment monitoring. Accumulating evidence supports the role of EVs in modulating RT outcomes by shaping the TME, mediating radioresistance, and influencing cancer metastasis. Despite substantial progress, challenges remain, including the heterogeneity of EV biogenesis, variability in cargo composition, and the absence of standardised methods for EV isolation and characterisation. While the therapeutic and diagnostic prospects of EVs in PCa management are promising, further research is needed to clarify the mechanisms through which EVs impact RT and to translate these findings into clinical practice. Incorporating EV research into PCa treatment paradigms could enhance diagnostic accuracy, enable real-time monitoring of RT responses, and support the development of new targeted therapeutic strategies. This review discusses recent progress in understanding EVs in the context of RT for PCa, focuses on their roles in modulating tumour growth, contributing to radioresistance within the TME, and facilitating the monitoring of RT efficacy and recurrence. In addition, the potential of EVs as biomarkers for liquid biopsy and their applications in enhancing radiosensitivity or overcoming radioresistance is also explored.

The interaction between tumor-derived exosomes and stroma plays a crucial role in tumor progression. However, the mechanisms through which tumor cells influence stromal changes are not yet fully understood. In our study, through single-cell sequencing analysis of clear cell renal cell carcinoma tissues at varying stages of progression, we determined that the proportion of cancer-associated fibroblasts (CAFs) in advanced renal cell carcinoma tissues was notably higher compared to localized renal cell carcinoma tissues. Comparison of transcriptome sequencing and energy metabolism tests between CAFs primarily isolated from advanced renal cell carcinoma tissues and normal fibroblasts (NFs) revealed the occurrence of the Warburg effect during the fibroblast activation process. Additionally, we observed an increase in glucose transporter GLUT1 expression, total reactive oxygen species (ROS) levels, lactic acid production, and subsequent excretion of excess lactic acid through monocarboxylate transporter-4 (MCT4) in CAFs. Interestingly, renal cancer cells were found to uptake lactic acid via MCT1 upon interaction with CAFs, thereby enhancing their malignant phenotypes. Furthermore, the down-regulation of PANK3 induced by exosomes derived from renal cancer cells was identified as a crucial step in fibroblast activation. These findings indicate that exosomes play a role in facilitating intercellular communication between renal cancer cells and fibroblasts. Targeting this communication pathway could potentially offer new strategies for the prevention and treatment of advanced renal cell carcinoma.

Ovarian cancer (OC) is a highly aggressive malignancy characterized by early dissemination of cancer cells from the surface of the ovary to the peritoneum. To gain a deeper understanding of the mechanisms associated with this intraperitoneal spread, we aimed to characterize the role of extracellular vesicles (EVs) in metastatic colonization in OC.

To this purpose, a total of 150 samples of ascitic fluids, blood serum, tumor and normal tissues from 60 OC patients, were extensively analyzed to characterize the EVs released in blood and ascitic fluids of OC patients, in terms of size, expression of superficial epitopes and abundance of miRNAs biocargo.

A statistically significant difference in the size of EVs derived from ascitic fluid and serum was identified. Analysis of surface protein expression highlighted twenty epitopes with a significant difference between the two biological matrices, of which 18 were over- and two were under-expressed in ascitic fluid. With regard to miRNA levels, Principal Component Analysis (PCA) assessed four distinct clusters representing tumor tissue, normal tissue, ascitic fluid, and serum. A prominent difference in circulating miRNAs was observed in serum and ascitic fluid highlighting 98 miRNAs significantly deregulated (P-adj < 0.05) between the two bodily fluids. Deregulated miRNAs and epitopes underline an enrichment in ascites in components contributing to the metastatic spread.

The results highlight a clear difference between the two biological fluids, suggesting that tumor selectively releases specific EVs populations in serum or ascites. In this context, it seems that ascites-derived EVs play a major role in modulating EMT and metastatic cascade, which is a key feature of OC.

Multiple reports have shown an effect of keratinocyte-derived extracellular vesicles (EVs) on keratinocytes and other cell types. However, the contribution of keratinocyte-derived EVs under physiological and pathological conditions is not fully elucidated. Therefore, whether there is an effect of EVs on macrophages in cervical cancer (CC) is also unknown. Here, we evaluated the effect of tumor and non-tumor keratinocyte-derived EVs on the polarization of peripheral blood mononuclear cells (PBMCs)-derived macrophages and THP-1 cell line. Flow cytometric evaluation of macrophages cultured in the presence of keratinocyte-derived EVs mainly indicated an increase in classical activation markers CD80 and CD86 (M1 phenotype) and little or no modification of alternative activation markers (M2 phenotype). ELISA evaluation of macrophage supernatants revealed an increase in the secretion of proinflammatory cytokines such as IL-1β and IL-6. On the other hand, TGF-β was not significantly modified and only EVs derived from non-cancerous keratinocytes induced a significant increase in IL-10. The expression levels of transcripts associated with the M1 phenotype were also evaluated by qRT-PCR with similar results to ELISA for TGF-β and IL-10; but also an increase in the expression of HLA-DRα and TNF-α was observed, and no statistically significant changes in ARG1. The ROS production was also evaluated and this increase mainly in macrophages treated with CC keratinocytes-derived EVs. So, our results suggest that the uptake of EVs derived from released by non-tumor and cervical cancer keratinocytes promotes in macrophages their polarization to an M1-like phenotype.

Cell-to-cell communication plays a crucial role in many processes, both in physiological and pathological assets [...].

Extracellular vesicles (EVs), tiny packages of information released by cells, are well established as being involved in unwanted cell-to-cell communication in cancer. EVs from cancer cells have been associated with the spread of drug resistance, immune suppression, and metastasis. Additional to cancer cells, the tumour microenvironment (TME) involves many cell types -including immune cells, fibroblasts, and endothelial cells, each of which has a potential role in how tumours grow, spread, and respond (or otherwise) to therapy. This review collates and distils research developments regarding the role of EVs in multi-way communication between cells in the TME. Further research including tailored clinical studies are now warranted to determine how best to prevent this extensive adverse communication occurring and/or how best to exploit it for biomarker discovery and as a therapeutic approach, in the interest of patients and also for economic benefit.

Melanoma is the main cause of death from skin cancer. The current treatment methods have prominent toxic side effects. In order to more effectively inhibit melanoma and reduce the toxic side effects during treatment, this paper constructs an engineering system using DSPE-PEG2000-pYEEIE(pYEEIE) molecules to modify exosome-like nanovesicles vesicles of Rhodiola rosea (RELNs) and load Doxorubicin (DOX). As a drug system, the aim is to achieve better targeting activity of the system towards melanoma cell A375. The results showed that the morphology and particle size of the prepared RELNs met the defined criteria for evaluating extracellular vesicles. The pYEEIE-RELNs-DOX drug delivery system has a better inhibitory effect on cell proliferation compared to DOX and RELNs-DOX. At the same time, the pYEEIE-RELN-DOX drug delivery system also showed better targeting towards tumor cells. In summary, this study proposes for the first time RELNs as a new generation of drug delivery carriers and uses them for drug delivery and inhibition of melanoma cell toxicity. This lays the foundation for subsequent animal and clinical experiments, and provides new ideas for the treatment of skin cancer caused by melanoma.

Colorectal cancer (CRC) ranks as the second most prevalent malignancy globally, highlighting the urgent need for more effective diagnostic and therapeutic strategies, as well as a deeper understanding of its molecular basis. Extensive research has demonstrated that cells actively secrete extracellular vesicles (EVs) to mediate intercellular communication at both proximal and distal sites. In this study, we conducted a comprehensive analysis of the RNA content of small extracellular vesicles (sEVs) secreted into the culture media of five frequently utilised CRC cell lines (RKO, HCT116, HCT15, HT29, and DLD1). RNA sequencing data revealed significant insights into the RNA profiles of these sEVs, identifying nine protein-coding genes and fourteen long non-coding RNA (lncRNA) genes that consistently ranked among the top 30 most abundant across all cell lines. Notably, the genes found in sEVs were highly similar among the cell lines, indicating a conserved molecular signature. Several of these genes have been previously documented in the context of cancer biology, while others represent novel discoveries. These findings provide valuable insights into the molecular cargo of sEVs in CRC, potentially unveiling novel biomarkers and therapeutic targets.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease. Lung cancer (LC) is among the most crucial comorbidity factors in patients with IPF. IPF patients that are diagnosed with LC have a reduced mean survival time. Therapeutic strategies for LC in patients with IPF need to be adapted according to the individual treatment risk. Life-threatening acute exacerbation (AE) of IPF may occur in association with cancer treatment, thereby severely restricting the therapeutic options for IPF-associated LC. Because LC and anticancer treatments can worsen the prognosis of IPF, the prevention of LC is as critical as managing patients with IPF.

Radioresistance presents a major challenge in the treatment of cervical cancer (CC). Apoptotic tumor cells can create an "onco-regenerative niche," contributing to radioresistance. However, the intercellular signaling mechanisms mediating the transfer of radioresistance from apoptotic to surviving cancer cells remain unclear.

The role of apoptotic tumor cell-derived extracellular vesicles (apoEVs) in mediating radioresistance was investigated through integrated bioinformatics and experimental approaches. The GSE236738 dataset was analyzed to identify potential regulators, with subsequent validation of apoEV-MTA1 function using in vitro and in vivo models. Mechanistic studies focused on caspase-3 activation, p-STAT1 signaling pathway, and dormancy-associated protein networks. Furthermore, therapeutic strategies targeting MTA1 and its downstream signaling were evaluated for radiosensitization potential.

MTA1 was identified as a critical factor enriched in and transferred by apoEVs from apoptotic tumor cells to neighboring CC cells. Caspase-3 activation facilitated the nuclear export and encapsulation of MTA1 in apoEVs. Transferred MTA1 retained transcriptional activity, activated the p-STAT1 signaling pathway, and induced cellular dormancy via NR2F1, a key dormancy regulator, resulting in increased radioresistance. Knockdown of MTA1 in apoEVs or inhibition of p-STAT1 in recipient cells enhanced radiosensitivity. Furthermore, apoEV-MTA1 promoted tumor radioresistance and reduced survival rates in irradiated cervical cancer mouse model.

This study demonstrates that apoEV-MTA1 confers radioresistance in CC by promoting cellular dormancy via the p-STAT1/NR2F1 signaling axis. Targeting this pathway could improve radiosensitivity and provide a promising therapeutic strategy for CC patients.

Oral squamous cell carcinoma (OSCC) represents a heterogeneous group of malignancies originating from the mucosal lining of the oral cavity. Current treatment modalities primarily involve surgery, chemotherapy, and radiotherapy. Despite the use of multimodal therapy, the 5 year overall survival rate for OSCC remains around 50%, underscoring the need for the development of nontoxic agents with potent antitumor activity. Extracellular vesicles (EVs) are nanoscale, membrane-bound structures that can selectively deliver small molecules, nucleic acids, and proteins to target cells, making them a promising platform for drug delivery in cancer therapy. Strategies to improve the uptake of EVs and enhance the delivery of therapeutic molecules to target cells are critical for advancing precision medicine. Tetrahedral DNA nanostructures (TDNs) have shown significant potential in facilitating drug endocytosis and delivery, as well as improving tissue penetration. In this study, TDN@EVs were conducted by modifying the membrane surface of M1-EVs with TDNs, which demonstrated improved biological stability and drug delivery efficiency compared to unmodified EVs. In vitro and in vivo experiments showed that TDN@EVs significantly inhibited OSCC cell proliferation and migration while promoting apoptosis. TDN@EVs exhibited superior drug penetration properties, further amplifying their antitumor effects. Proteomic analysis identified Hsc70 as the key protein responsible for the antitumor activity of the TDN@EVs. The efficient delivery of Hsc70 into tumor cells by TDN@EVs led to the degradation of GPX4, inducing ferroptosis, mitochondrial stress, and DNA damage in tumor cells. These findings highlight the potential of TDN@EVs as an effective and safe approach for cancer therapy. In conclusion, TDN@EVs present as a promising effective strategy for the targeted delivery of therapeutic agents in OSCC treatment, offering enhanced biological stability, efficient drug delivery, and significant antitumor effects.

The phenotypic profiling of extracellular vesicles (EVs) within the tumor microenvironment (TME) provides critical insights into the intercellular communication mechanisms of EVs underlying tumor physiology. However, conventional methods typically isolate EVs from the extracellular space through tissue fragmentation, which compromises tissue viability, and neglects the spatial organization of the tissue and the dynamic nature of EV secretion. Herein, we introduce an innovative microfluidic platform to cultivate intact tumor tissues while preserving their spatial architecture and facilitating natural EV secretion. This system enables the direct replication of EVs onto the chip for high-fidelity phenotypic analysis. Utilizing a combinatorial-aptamer-induced dual-switch logic gate methodology, this approach allows for the precise subtyping of EVs derived from both tumor cells and immune cells within the TME. Specifically, aptamers targeting EpCAM and PD-L1, along with the connector probe, were employed to induce a dual-switch signal to identify distinct EV populations. This strategy enables noninvasive, real-time capture and phenotypic profiling of EVs directly within the microfluidic environment. Furthermore, our findings indicate that immunotherapy with PD-1 antibodies significantly enhances the secretion of EVs by immune cells within the TME, underscoring the potential role of EVs as mediators of therapeutic responses. Overall, we have developed a robust, noninvasive method for the phenotypic profiling of EVs in the TME, offering a powerful tool for investigating the biological functions and implications of EVs in tumor pathophysiology.

Gastric cancer (GC) is one of the most common gastrointestinal cancers worldwide, with consistently high morbidity and mortality rates and poor prognosis. Most patients are diagnosed at an advanced stage due to the lack of specific presentation in the early stages. Exosomes are a class of extracellular vesicles (EVs) widely found in body fluids and can release genetic material or multiple proteins to facilitate intercellular communication. In recent years, exosomal miRNAs have gained attention for their role in various cancers. These exosomal miRNAs can impact GC development and progression by targeting specific genes or influencing signaling pathways and cytokines involved in Angiogenesis, epithelial-mesenchymal transition (EMT), drug resistance, and immune regulation. They show great potential in terms of diagnosis, prognosis, and treatment of GC. Notably, the gastrointestinal tract has the largest number of macrophages, which play a significant role in GC progression. Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment (TME) and can influence macrophage programming through various mediators, including macrophage polarization. Macrophage polarization is involved in inflammatory responses and significantly impacts the GC process.

Breastfeeding is widely recognized for its essential nutritional benefits and broader biological impacts. Beyond providing infants with a balanced mix of vitamins, proteins, and fats critical for growth and development, breast milk contains bioactive extracellular vesicles (BMEVs). These membrane-bound particles, rich in proteins, lipids, and nucleic acids, play a pivotal role in immune modulation, intercellular communication, and the overall development of the infant's immune system. This review explores the emerging therapeutic potential of BMEVs, highlighting their capacity to modulate recipient cell functions, influence immune responses, and contribute to overall infant health. Preclinical evidence suggests that these vesicles can prevent and manage conditions such as necrotizing enterocolitis, allergies, and viral infections, which are common in early childhood. Furthermore, BMEVs offer promise as vehicles for targeted drug delivery, enhancing the efficacy of therapeutic interventions. Despite the growing body of evidence, challenges such as the need for standardized isolation methods, characterization techniques, and larger-scale clinical studies persist, hindering the translation of this research into clinical practice. This review addresses these challenges and discusses future directions, emphasizing the need for comprehensive mechanistic studies to fully realize the potential of BMEVs as novel therapeutic agents and biomarkers of health. Ultimately, these vesicles represent a promising frontier in maternal and child health, with potential applications extending far beyond traditional nutrition. By harnessing their unique properties, BMEVs could revolutionize infant care, offering new strategies for disease prevention and innovative therapeutic interventions that enhance infant health outcomes.

Cancer cells compensate with increasing mitochondria-derived vesicles (MDVs) to maintain mitochondrial homeostasis, when canonical MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta)-mediated mitophagy is lacking. MDVs promote the transport of mitochondrial components into extracellular vesicles (EVs) and induce tumor metastasis. Although HSP90 (heat shock protein 90) chaperones hundreds of client proteins and its inhibitors suppress tumors, HSP90 inhibitors-related chemotherapy is associated with unexpected metastasis. Herein, we find that HSP90 inhibitor causes mitochondrial damage but stimulates the low LC3-induced MDVs and the release of MDVs-derived EVs. However, why LC3 decreases and what is the transcriptional regulatory mechanism of MDVs formation under HSP90 inhibition remain unknown. Because TFEB (transcription factor EB) is the most important mitophagy transcription factor, and the HSP90 client HCFC1 (host cell factor C1) regulates TFEB transcription, there should be a hidden connection between TFEB, HCFC1 and HSP90 in MDVs formation. Our results support the idea that HSP90 N-terminal inhibition reduces TFEB transcription via decreased HSP90AA1-HCFC1 interaction, which prevents HCFC1 from binding to the TFEB proximal promoter region. Decreased TFEB transcription and consequently reduced LC3, ultimately promoted MDVs formation. Blocking MDVs formation with the microtubule inhibitor nocodazole (NOC) activates the HCFC1-TFEB-LC3 axis, weakens HSP90 inhibitors-induced MDVs and the release of MDVs-derived EVs, inhibits the growth of tumor cell spheres and primary liver tumors, and reduces the extravasation of cancer cells to secondary metastatic sites. Taken together, these data suggest that combination therapy should be used to reduce the metastatic risk of low TFEB-triggered-MDVs formation caused by HSP90 inhibitors.Abbreviation: ACIs: ATP-competitive inhibitors; BaFA1: bafilomycin A1; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ChIP: chromatin immunoprecipitation; CHX: cycloheximide; CTD: C-terminal domain; EVs: extracellular vesicles; HCFC1: host cell factor C1; HSP90: heat shock protein 90; ILVs: intralumenal vesicles; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MD: middle domain; MDVs: mitochondria-derived vesicles; MQC: mitochondrial quality control; ΔΨm: mitochondrial membrane potential; MVBs: multivesicular bodies; NB: novobiocin; TEM: transmission electron microscopy; TFEB: transcription factor EB; TFs: transcription factors. NOC: nocodazole; NTD: N-terminal nucleotide binding domain; OCR: oxygen consumption rate; RFP: red fluorescent protein; ROS: reactive oxygen species; STA9090: Ganetespib; VPS35: VPS35 retromer complex component.

Extracellular vesicles (EVs) are heterogeneous vesicles released by donor cells that can be taken up by recipient cells, thus inducing cellular phenotype changes. Since their discovery decades ago, roles of EVs in modulating initiation, growth, survival and metastasis of cancer have been revealed. Recent studies from multifaceted perspectives have further detailed the contribution of EVs to cancer drug resistance; however, the role of EV uptake in conferring drug resistance seems to be overlooked. In this comprehensive review, we update the EV subtypes and approaches for determining EV uptake. The biological basis of EV uptake is systematically summarized. Moreover, we focus on the diverse uptake mechanisms by which EVs carry out the intracellular delivery of functional molecules and drug resistance signaling. Furthermore, we highlight how EV uptake confers drug resistance and identify potential strategies for targeting EV uptake to overcome drug resistance. Finally, we discuss the research gap on the role of EV uptake in promoting drug resistance. This updated knowledge provides a new avenue to overcome cancer drug resistance by targeting EV uptake.

Extracellular vesicles (EVs), as cell-derived small vesicles, facilitate intercellular communication within the tumor microenvironment (TME) by transporting biomolecules. EVs from different sources have varied contents, demonstrating differentiated functions that can either promote or inhibit cancer progression. Thus, regulating the formation, secretion, and intake of EVs becomes a new strategy for cancer intervention. Advancements in EV isolation techniques have spurred interest in EV-based therapies, particularly for tumor immunotherapy. This review explores the multifaceted functions of EVs from various sources in tumor immunotherapy, highlighting their potential in cancer vaccines and adoptive cell therapy. Furthermore, we explore the potential of EVs as nanoparticle delivery systems in tumor immunotherapy. Finally, we discuss the current state of EVs in clinical settings and future directions, aiming to provide crucial information to advance the development and clinical application of EVs for cancer treatment.

Accidents caused by Loxosceles spiders, commonly known as brown spiders, are frequent in warm and temperate regions worldwide, with a higher prevalence in South America and the southern United States. In the venoms of species clinically associated with accidents, phospholipases D (PLDs) are the most expressed toxins. This classification is based on the toxins' ability to cleave various phospholipids, with a preference for sphingomyelin. Studies using purified PLDs have demonstrated that these enzymes cleave phospholipids from cells, producing derivatives that can activate leukocytes. A dysregulated inflammatory response is the primary effect following envenomation, leading to dermonecrosis, which is histopathologically characterized by aseptic coagulative necrosis-a key feature of envenomation. Although advances in understanding the structure-function relationship of enzymes have been achieved through molecular biology, heterologous expression, site-directed mutations, crystallography, and bioinformatic analyses-describing PLDs in the venoms of various species and highlighting the conservation of amino acid residues involved in catalysis, substrate binding, and magnesium stabilization-little is known about the cellular biology of these PLDs. Studies have shown that the treatment of various cells with recombinant PLDs stimulates the formation of ectosomes and ectocytosis, events that initiate a cascade of intracellular signaling in PLD-binding cells and lead to the release of extracellular microvesicles. These microvesicles may act as signalosomes for other target cells, thereby triggering an inflammatory response and dermonecrosis. In this review, we will discuss the biochemical properties of PLDs, the target cells that bind to them, and the ectocytosis-dependent pathophysiology of envenoming.

Docetaxel resistance is a significant obstacle in the treatment of prostate cancer (PCa), resulting in unfavorable patient prognoses. Intratumoral heterogeneity, often associated with epithelial-to-mesenchymal transition (EMT), has previously emerged as a phenomenon that facilitates adaptation to various stimuli, thus promoting cancer cell diversity and eventually resistance to chemotherapy, including docetaxel. Hence, understanding intratumoral heterogeneity is essential for better patient prognosis and the development of personalized treatment strategies.

To address this, we employed a high-throughput single-cell flow cytometry approach to identify a specific surface fingerprint associated with docetaxel-resistance in PCa cells and complemented it with proteomic analysis of extracellular vesicles. We further validated selected antigens using docetaxel-resistant patient-derived xenografts in vivo and probed primary PCa specimens to interrogate of their surface fingerprint.

Our approaches revealed a 6-molecule surface fingerprint linked to docetaxel resistance in primary PCa specimens. We observed consistent overexpression of CD95 (FAS/APO-1), and SSEA-4 surface antigens in both in vitro and in vivo docetaxel-resistant models, which was also observed in a cell subpopulation of primary PCa tumors exhibiting EMT features. Furthermore, CD95, along with the essential enzymes involved in SSEA-4 synthesis, ST3GAL1, and ST3GAL2, displayed a significant increase in patients with PCa undergoing docetaxel-based therapy, correlating with poor survival outcomes.

In summary, we demonstrate that the identified 6-molecule surface fingerprint associated with docetaxel resistance pre-exists in a subpopulation of primary PCa tumors before docetaxel treatment. Thus, this fingerprint warrants further validation as a promising predictive tool for docetaxel resistance in PCa patients prior to therapy initiation.

The management and treatment of tumor complications pose continuous challenges due to the inherent complexity. However, the advent of drug delivery systems (DDSs) brings promising opportunities to address the tumor complications using innovative technological approaches. This review focuses on common oncological complications, including cancer thrombosis, malignant serous effusion, tumor-associated infections, cancer pain, and treatment-related complications. Emphasis was placed on the application and potential of DDSs in mitigating and treating these tumor complications, and we delved into the underlying mechanisms of common cancer-associated complications, discussed the limitations of conventional treatments, and outlined the current status and potential development of DDSs for various complications in this review. Moreover, we have discussed the existing challenges in DDSs research, underscoring the need for addressing issues related to biocompatibility and targeting of DDSs, optimizing drug delivery routes, and enhancing delivery efficiency and precision. In conclusion, DDSs offer promising avenues for treating cancer complications, offering the potential for the development of more effective and safer drug delivery strategies, thereby improving the quality of life and survival rates of cancer patients.

Liver cancer exhibits diverse molecular characteristics and distinct immune cell infiltration patterns, which significantly influence patient outcomes. In this study, we thoroughly examined the liver cancer tumor environment by analyzing data from 419,866 individual cells across nine datasets involving 99 patients. By categorizing patients into different groups based on their immune cell profiles, including immune deficiency, B cells-enriched, T cells-enriched and macrophages-enriched, we better understood how these cells change in various patient subgroups. Our investigation of liver metastases from intestinal cancer uncovered a group of mast cells that might promote metastasis through pathways like inositol phosphate metabolism. Using genomic and clinical data from The Cancer Genome Atlas, we identified specific cell components linked to tumor characteristics and genetics. Our detailed study of cancer-associated fibroblasts (CAFs) revealed how they adapt and acquire new functions in the tissue environment, highlighting their flexibility. Additionally, we found a significant connection between CAF-related genes and the prognosis of hepatocellular carcinoma patients. This research provides valuable insights into the makeup of the liver cancer tumor environment and its profound impact on patient outcomes, offering fresh perspectives for managing this challenging disease.

The immunopeptidome, a diverse set of peptides presented by Major Histocompatibility Complex (MHC) molecules, is a critical component of immune recognition and response. This review article delves into the mechanisms of peptide presentation by MHC molecules, particularly emphasizing the roles of ncRNA-derived peptides and extracellular vesicles (EVs) in shaping the immunopeptidome landscape. We explore established and emerging insights into MHC molecule interactions with peptides, including the dynamics of peptide loading, transport, and the influence of cellular and genetic variations. The article highlights novel research on non-coding RNA (ncRNA)-derived peptides, which challenge conventional views of antigen processing and presentation and the role of EVs in transporting these peptides, thereby modulating immune responses at remote body sites. This novel research not only challenges conventional views but also opens up new avenues for understanding immune responses. Furthermore, we discuss the implications of these mechanisms in developing therapeutic strategies, particularly for cancer immunotherapy. By conducting a comprehensive analysis of current literature and advanced methodologies in immunopeptidomics, this review aims to deepen the understanding of the complex interplay between MHC peptide presentation and the immune system, offering new perspectives on potential diagnostic and therapeutic applications. Additionally, the interactions between ncRNA-derived peptides and EVs provide a mechanism for the enhanced surface presentation of these peptides and highlight a novel pathway for their systemic distribution, potentially altering immune surveillance and therapeutic landscapes.

Neuropilin-1 (NRP1) is a transmembrane protein involved in surface receptor complexes for a variety of extracellular signals. NRP1 expression in human cancers is associated with prominent angiogenesis and advanced progression stage. However, the molecular mechanisms underlying NRP1 activity in the tumor microenvironment remain unclear. Notably, diffusible forms of NRP1 in the extracellular space have been reported, but their functional role is poorly understood.

Extracellular vesicles (EV) were isolated from conditioned media of diverse cancer cells. The quality of exosome-enriched preparations was validated by the presence of specific markers in western blotting, as well as by light scattering and nanoparticle tracking analysis. Wound healing, transwell, and digital real-time migration assays were carried out to assess the activity of cancer cell-derived exosomes in the regulation of endothelial cells. RNA interference was applied to obtain NRP1 knock-down, and cDNA transfer to achieve its overexpression, in exosome-releasing cells. The micro-RNA profile carried by exosomes was investigated by Next Generation Sequencing. miRNA-Scope in situ hybridization was used to assess the transfer of miRNA exosome cargo to target cells, and immunofluorescence analysis revealed expression regulation of targeted proteins. miRNA activity was blocked by the use of specific antago-miRs.

In this study, we show that diverse human cancer cells release NRP1 embedded in exosome-like small extracellular vesicles, which mediate a previously unknown NRP1-dependent paracrine signaling mechanism regulating endothelial cell migration. By transcriptomic analysis of the cargo of NRP1-loaded exosomes, we found a significant enrichment of miR-210-3p, known to promote tumor angiogenesis. Gene knock-down and overexpression experiments demonstrated that the loading of miR-210-3p into exosomes is dependent on NRP1. Data furthermore indicate that the exosomes released through this NRP1-driven mechanism effectively transfer miR-210-3p to human endothelial cells, causing paracrine downregulation of the regulatory cue ephrin-A3 and promotion of cell migration. The mechanistic involvement of miR-210-3p in this pathway was confirmed by applying a specific antago-miR.

In sum, we unveiled a previously unknown NRP1-dependent paracrine signaling mechanism, mediated by the loading of pro-angiogenic miR-210-3p in exosomes released by cancer cells, which underscores the relevance of NRP1 in controlling the tumor microenvironment.

Extracellular vesicles (EVs) are key mediators in the communication between cancer cells and their microenvironment, significantly influencing drug resistance. This review provides a comprehensive analysis of the roles of EVs in promoting drug resistance through mechanisms such as drug efflux, apoptosis resistance, autophagy imbalance, and tumor microenvironment modulation. Despite extensive research, details of EVs biogenesis, cargo selection, and specific pathways in EVs-mediated drug resistance are not fully understood. This review critically examines recent advancements, highlighting key studies that elucidate the molecular mechanisms of EVs functions. Additionally, innovative therapeutic strategies targeting EVs are explored, including inhibiting EVs biogenesis, engineering EVs for drug delivery, and identifying resistance-inhibiting molecules within EVs. By integrating insights from primary research and proposing new directions for future studies, this review aims to advance the understanding of EVs in cancer biology and foster effective interventions to mitigate drug resistance in cancer therapy.

Small extracellular vesicles (sEV) released by tumor cells (tumor-derived sEV; TEX) mediate intercellular communication between tumor and non-malignant cells and were shown to impact disease progression. This study investigates the relationship between the expression levels of the vesiculation-related genes linked to sEV production and the tumor microenvironment (TME).

Two independent gene sets were analyzed, both previously linked to sEV production in various non-malignant or malignant cells. Expression profiles were compared among 28 tumor types listed in the Cancer Genome Atlas (TCGA). Gene expression and survival analysis (GEPIA2), immunogenomic analysis (TISIDB), and genomic analysis (GSCA) were performed.

Vesiculation-related genes were overexpressed in tissues of most tumor types compared to healthy tissues, and high expression levels were associated with worse overall survival in cervical squamous cell carcinoma, kidney chromophobe, lower grade glioma, hepatocellular carcinoma, lung squamous cell carcinoma, and pancreatic adenocarcinoma but with improved overall survival in kidney renal clear cell carcinoma. Expression of these signatures correlated with an increased abundance of infiltrating CD4( +) T cells and dendritic cells, a decreased abundance of B cells and eosinophils, and activation of tumor cell apoptosis and epithelial-mesenchymal transition pathways in all tumor types. 17-AAG was identified as a potential drug candidate to target tumors with elevated expression of vesiculation-related genes.

Vesiculation-related genes were associated with distinct immunological and genomic landscapes further emphasizing the important role of TEX in cancer progression.

Double C-2 Like Domain Beta (DOC2B) located at 17q13.3 prevents metastasis by senescence induction and epithelial to mesenchymal transition inhibition in cervical cancer (CC). The extracellular vesicle (EV) mediated trafficking of DOC2B and its impact on tumor suppressive activity are not investigated in CC. Using a retroviral method, we first ectopically expressed DOC2B in SiHa, which do not normally express DOC2B. DOC2B-SiHa and vector-SiHa EVs were co-incubated separately with recipient cell and subjected to various cellular and biochemical experiments. For the first time, we demonstrated that DOC2B localizes to EVs, and its transfer to EV may require intracellular calcium. Co-culture of SiHa and HeLa cells with DOC2B-SiHa derived EVs induced morphological changes and suppressed their growth and migration, possibly by induction of G0/G1 to S phase arrest and anoikis. DOC2B-SiHa EVs elevated intracellular reactive oxygen species (ROS) and calcium levels and promoted lipid droplet accumulation and lipid peroxidation rate in recipient cells. DOC2B-SiHa EVs reduced active AKT1 and ERK1/2 levels and EMT marker expression and enhanced cellular senescence and cytotoxic effects of cisplatin. Re-expression of DOC2B significantly altered the global metabolite profile of EVs.  Finally, we demonstrated that intracellular calcium chelation significantly reduces DOC2B localization to EVs and impacts its tumor-suppressive properties. Altogether, EV-mediated DOC2B transfer may reduce the aggressive behavior of CC cells.

In precision cancer therapy, addressing intra-tumor heterogeneity poses a significant obstacle. Due to the heterogeneity of each cell subtype and between cells within the tumor, the sensitivity and resistance of different patients to targeted drugs, chemotherapy, etc., are inconsistent. Concerning a specific tumor type, many feasible treatments or combinations can be used by specifically targeting the tumor microenvironment. To solve this problem, it is necessary to further study the tumor microenvironment. Single-cell sequencing techniques can dissect distinct tumor cell populations by isolating cells and using statistical computational methods. This technology may assist in the selection of targeted combination therapy, and the obtained cell subset information is crucial for the rational application of targeted therapy. In this review, we summarized the research and application advances of single-cell sequencing technology in the tumor microenvironment, including the most commonly used single-cell genomic and transcriptomic sequencing, and their future development direction was proposed. The application of single-cell sequencing technology has been expanded to include epigenomics, proteomics, metabolomics, and microbiome analysis. The integration of these different omics approaches has significantly advanced the development of single-cell multiomics sequencing technology. This innovative approach holds immense potential for various fields, such as biological research and medical investigations. Finally, we discussed the advantages and disadvantages of using single-cell sequencing to explore the tumor microenvironment.

Nanopore long-read RNA sequencing is reshaping extracellular vesicle (EV) research by providing the capacity to analyze full-length RNA molecules. EVs are crucial for intercellular communication, carrying a diverse range of RNA cargo that can regulate recipient cell behavior. However, traditional short-read sequencing methods involve transcript fragmentation, limiting our understanding of the EV transcriptomic landscape. Furthermore, it has been generally assumed that EV RNAs are likely to be fragmentation products of cellular RNAs, and the extent to which full length RNAs are present within EVs remains to be clarified. Recent advancements in sequencing technology, particularly long-read sequencing by Oxford Nanopore Technologies (ONT), offer a solution to this limitation. Hence, long-read sequencing allows for the analysis of full-length EV RNA molecules, providing deeper insights into their integrity and isoform diversity. Here, we present a comprehensive protocol for EV RNA purification, cDNA library preparation, and sequencing using ONT's MinION platform.

Enzalutamide (ENZ) has emerged as a major treatment advance in castration-resistant prostate cancer (CRPC) patients; however the development of resistance remains a key challenge. The extracellular vesicles (VEs)-derived miRNAs play crucial roles tumor microenvironment cell communication, thereby influencing resistance mechanisms. Considering the urgent need for molecular biomarkers to monitor ENZ response and predict resistance, we intend to identify an EV-derived miRNA profile associated with ENZ resistance using an innovative 3D-spheroid in vitro model. Through the generation of this model, we provide a comprehensive platform for elucidating the molecular alterations involved in the process. An in vitro model of ENZ resistance was established through continuous exposure of LNCaP to increasing ENZ concentrations. A screening of 799 miRNAs from resistant and normal LNCaP cells were quantified. A bioinformatic analysis was performed using miRTarbase and Cytoscape and top 5 overexpressed miRNAs were selected, that will be analyzed in extracellular vesicles derived from ENZ resistance 3D spheroid models. We identified 12 up- and 13 downregulated miRNAs in LNCaP 30 μM ENZ cells compare to LNCaP·In silico analysis led to the construction of a 76 proteins cluster and functional enrichment revealed terms like PI3K/AKT, TFG-β and FOXO. hsa-miR-22-3p was significantly decreased at 5 and 20 μM ENZ concentration intracellularly, but significantly increased at 20 μM ENZ in EVs. hsa-miR-221-3p and miR-222-3p were upregulated in all concentrations both intracellularly and in EVs. The developed 3D-spheroid model effectively replicated the ENZ resistance to ENZ in an AR-independent manner, underscoring the importance of EVs-derived miRNAs in this adaptive process.

Hepatocellular carcinoma (HCC) mainly depends on liver fibrosis/cirrhosis, which is regulated by tumor cells and the tumor microenvironment (TME), and is a crucial factor in tumor progression. This study aimed to identify abnormally expressed miR-500a-3p in the hepatitis-cirrhosis-HCC pathway and explored the roles of miR-500a-3p in HCC progression. A clinical cohort of patients with HCC is studied retrospectively. Subsequently, the role of miR-500a-3p transported by HCC exosomes in hepatic stellate cell (HSC) activation, hepatoma growth and invasion, and immune cell differentiation is determined by in vitro and in vivo experiments. In clinical tissues, miR-500a-3p is significantly enriched in HCC and cirrhosis tissues, and co-expression of the immune marker CD4 or PD-L1 significantly correlates with low survival rates in patients. Extracellular miR-500a-3p is taken up by HSC and PBMC, which promotes the secretion of the cytokines TGF-β1 and IL-10, increases PD-L1 expression in HSC, and stabilizes PD-1 expression in PBMC to affect the TME. Moreover, miR-500a-3p is associated with CD4+ T-cell exhaustion and Treg differentiation and is significantly associated with increased tumorigenicity in in situ mouse HCC models. Mechanistically, HCC-derived exosomal miR-500a-3p directly influences SOCS2 to regulate the JAK3/STAT5A/STAT5B signaling pathway. MiR-500a-3p promotes the growth and migration of HCC through the SOCS2/JAK3/STAT5A/STAT5B axis.

Extracellular vesicles (EVs) play a crucial role in the complex process of cancer metastasis by facilitating cellular communication and influencing the microenvironment to promote the spread and establishment of cancer cells in distant locations. This paper explores the process of EV biogenesis, explaining their various sources that range from endosomal compartments to plasma membrane shedding. It also discusses the complex mechanisms that control the sorting of cargo within EVs, determining their chemical makeup. We investigate the several functions of EVs in promoting the spread of cancer to other parts of the body. These functions include influencing the immune system, creating environments that support the formation of metastases before they occur, and aiding in the transformation of cells from an epithelial to a mesenchymal state. Moreover, we explore the practical consequences of EV cargo, such as nucleic acids, proteins, and lipids, in influencing the spread of cancer cells, from the beginning of invasion to the creation of secondary tumor sites. Examining recent progress in the field of EV-based diagnostics and treatments, we explore the potential of EVs as highly promising biomarkers for predicting the course of cancer and as targets for therapeutic intervention. This review aims to provide a complete understanding of the biology of EVs in the context of cancer metastasis. By unravelling the nuances of EV biology, it seeks to pave the way for new tactics in cancer detection, treatment, and management.

The proliferation of tumors is not merely self-regulated by the cancer cells but is also intrinsically connected to the tumor microenvironment (TME). Within this complex TME, cancer-associated fibroblasts (CAFs) are pivotal in the modulation of tumor onset and progression. Rich signaling interactions exist between CAFs and tumor cells, which are crucial for tumor regulation. Long non-coding RNAs (LncRNAs) emerge from cellular transcription as a class of functionally diverse RNA molecules. Recent studies have revealed that LncRNAs are integral to the crosstalk between CAFs and tumor cells, with the capacity to modify cellular transcriptional activity and secretion profiles, thus facilitating CAFs activation, tumor proliferation, metastasis, drug resistance, and other related functionalities. This comprehensive review revisits the latest research on LncRNA-mediated interactions between CAFs and tumor cells, encapsulates the biological roles of LncRNAs, and delves into the molecular pathways from a broader perspective, aspiring to offer novel perspectives for a deeper comprehension of the etiology of tumors and the enhancement of therapeutic approaches.