Three-Dimensional Magnetic Chip with Extracorporeal Circulation for Circulating Tumor Cell In Vivo Detection and Tumor Growth Inhibition.

Liquid biopsy technology involves taking samples from body fluids in a minimally invasive way and analyzing tumor markers to achieve early diagnosis and efficacy evaluation of tumors. The development of real-time cancer diagnosis and treatment strategies based on liquid biopsy technology is of great significance to cancer management. This paper described an extracorporeal circulation based on a three-dimensional (3D) magnetic chip (3DMC-system) for in vivo detection and real-time monitoring of circulating tumor cells (CTCs). Utilizing biofunctionalized magnetic nanospheres (MNs) with CTC recognition function, this 3DMC-system could effectively achieve the real-time monitoring of CTCs in vivo with good stability and strong anti-interference. Compared with in vitro CTC detection, in vivo detection could not only detect more CTCs but also detect the presence of CTCs in the blood at an early stage of the tumor, when tumor metastasis is not observed in imaging. In addition, due to the flexibility of the chip design, the system can easily add a treatment module to integrate cancer diagnosis and treatment together. With good biocompatibility and high stability, this 3DMC-system is expected to provide a new personalized medical program for cancer patients.

p-AKT/VPS4B regulates the small extracellular vesicle size in venous malformation endothelial cells.

OBJECTIVE: Small extracellular vesicle (sEV)-mediated intercellular communication is increasingly the key for the understanding of venous malformations (VMs). This study aims to clarify the detailed changes of sEVs in VMs. SUBJECTS AND METHODS: Fifteen VM patients without treatment history and twelve healthy donors were enrolled in the study. sEVs were isolated from both fresh lesions and cell supernatant, and were examined by western blotting, nanoparticle tracking analysis and transmission electron microscopy. Western blot analysis, immunohistochemistry and immunofluorescence were adopted to screening candidate regulator of sEV size. Specific inhibitors and siRNA were employed to validate the role of dysregulated p-AKT/vacuolar protein sorting-associated protein 4B (VPS4B) signaling on the size of sEVs in endothelial cells. RESULTS: The size of sEVs derived from both VM lesion tissues and cell model was significantly increased. VPS4B, whose expression level was mostly significantly downregulated in VM endothelial cells, was responsible for the size change of sEVs. Targeting abnormal AKT activation corrected the size change of sEVs by recovering the expression level of VPS4B. CONCLUSION: Downregulated VPS4B in endothelial cells, resulted from abnormally activated AKT signaling, contributed to the increased size of sEVs in VMs.

Overexpression of RAB27A in Oral Squamous Cell Carcinoma Promotes Tumor Migration and Invasion via Modulation of EGFR Membrane Stability.

Oral squamous cell carcinoma (OSCC) is the most prevalent subtype of head and neck tumors, highly prone to lymph node metastasis. This study aims to examine the expression pattern of Ras-related protein Rab-27A (RAB27A) and explore its potential implications in OSCC. The expression of RAB27A was assessed through immunohistochemical analysis utilizing tissue microarrays. In vitro experiments were conducted using RAB27A-knockdown cells to investigate its impact on OSCC tumor cells. Additionally, transcriptome sequencing was performed to elucidate potential underlying mechanisms. RAB27A was significantly overexpressed in OSCC, and particularly in metastatic lymph nodes. It was positively correlated with the clinical progression and poor survival prognosis. Silencing RAB27A notably decreased the proliferation, migration, and invasion abilities of OSCC cells in vitro. A Gene Ontology (GO) enrichment analysis indicated a strong association between RAB27A and the epidermal growth factor receptor (EGFR) signaling pathway. Further investigations revealed that RAB27A regulated the palmitoylation of EGFR via zinc finger DHHC-type containing 13 (ZDHHC13). These findings provide insights into OSCC progression and highlight RAB27A as a potential therapeutic target for combating this aggressive cancer.

A histological study of vascular wall resident stem cells in venous malformations.

Vascular wall resident stem cells (VW-SCs) play a key role in vascular formation and remodeling under both physiological and pathological situations. They not only serve as a reservoir to supply all types of vascular cells needed, but also regulate vascular homeostasis by paracrine effects. Venous malformations (VMs) are common congenital vascular malformations which are just characterized by the deficient quantity and abnormal function of vascular cells. However, the existence and role of VW-SCs in VMs is still unclear at present. In this study, the level and distribution of VW-SCs in 22 specimens of VMs were measured by immunochemistry, double-labeling immunofluorescence, and qPCR, followed by the Spearman rank correlation test. We found that both the protein and mRNA expression levels of CD34, vWF, VEGFR2, CD44, CD90, and CD105 were significantly downregulated in VMs compared with that in normal venules. VW-SCs were sporadically distributed or even absent within and outside the endothelium of VMs. The expression of the VW-SC-related markers was positively correlated with the density of both endothelial cells and perivascular cells. All those results and established evidence indicated that VW-SCs were more sporadically distributed with fewer amounts in VMs, which possibly contributing to the deficiency of vascular cells in VMs.

Endothelial cells induce degradation of ECM through enhanced secretion of MMP14 carried on extracellular vesicles in venous malformation.

Venous malformations (VMs), featuring localized dilated veins, are the most common developmental vascular anomalies. Aberrantly organized perivascular extracellular matrix (ECM) is one of the prominent pathological hallmarks of VMs, accounting for vascular dysfunction. Although previous studies have revealed various proteins involved in ECM remodeling, the detailed pattern and molecular mechanisms underlying the endothelium-ECM interplay have not been fully elucidated. Our previous studies revealed drastically elevated extracellular vesicle (EV) secretion in VM lesions. Here, we identified increased EV-carried MMP14 in lesion fluids of VMs and culture medium of TIE2-L914F mutant endothelial cells (ECs), along with stronger ECM degradation. Knockdown of RAB27A, a required regulator for vesicle docking and fusion, led to decreased secretion of EV-carried MMP14 in vitro. Histochemical analysis further demonstrated a highly positive correlation between RAB27A in the endothelium and MMP14 in the perivascular environment. Therefore, our results proved that RAB27A-regulated secretion of EV-MMP14, as a new pattern of endothelium-ECM interplay, contributed to the development of VMs by promoting ECM degradation.

In Situ Membrane Biotinylation Enables the Direct Labeling and Accurate Kinetic Analysis of Small Extracellular Vesicles in Circulation.

Circulating small extracellular vesicles (sEVs) are naturally occurring nanosized membrane vesicles that convey bioactive molecules between cells. Conventionally, to evaluate their behaviors in vivo, circulating sEVs have to be isolated from the bloodstream, then labeled with imaging materials in vitro, and finally injected back into the circulation of animals for subsequent detection. The tedious isolation-labeling-reinfusion procedures might have an undesirable influence on the natural properties of circulating sEVs, thereby changing their behaviors and the detected kinetics in vivo. Herein, we proposed an in situ biotinylation strategy to directly label circulating sEVs with intravenously injected DSPE-PEG-Biotin, aiming to evaluate the in vivo kinetics of circulating sEVs more biofriendly and accurately. Such an analysis strategy is free of isolation-labeling-reinfusion procedures and has no unfavorable influence on the natural behaviors of sEVs. The results showed that the lifetime of generic circulating sEVs in mice was around 3 days. Furthermore, we, for the first time, revealed the distinct in vivo kinetics of circulating sEV subpopulations with different cell sources, among which erythrocyte-derived sEVs showed the longest lifespan. Moreover, compared with circulating sEVs in situ or used as autograft, circulating sEVs used as allograft had the shortest lifetime. In addition, the in situ biotinylation strategy also provides a way for the enrichment of biotinylated circulating sEVs. In summary, this study provides a novel strategy for in situ labeling of circulating sEVs, which would facilitate the accurate characterization of their kinetics in vivo, thereby accelerating their future application as biomarkers and theranositic vectors.

Transformation of Viral Light Particles into Near-Infrared Fluorescence Quantum Dot-Labeled Active Tumor-Targeting Nanovectors for Drug Delivery.

Nanosized oncolytic viral light particles (L-particles), separated from progeny virions, are composed of envelopes and several tegument proteins of viruses, free of nucleocapsids. The noninfectious L-particles experience the same internalization process as mature oncolytic virions, which exhibits great potential to act as targeted therapeutic platforms. However, the clinical applications of L-particle-based theranostic platforms are rare due to the lack of effective methods to transform L-particles into nanovectors. Herein, a convenient and mild strategy has been developed to transform L-particles into near-infrared (NIR) fluorescence Ag2Se quantum dot (QD)-labeled active tumor-targeting nanovectors for real-time in situ imaging and drug delivery. Utilizing the electroporation technique, L-particles can be labeled with ultrasmall water-dispersible NIR fluorescence Ag2Se QDs with a labeling efficiency of ca. 85% and loaded with antitumor drug with a loading efficiency of ca. 87%. Meanwhile, by harnessing the infection mechanism of viruses, viral L-particles are able to recognize and enter tumor cells without further modification. In sum, a trackable and actively tumor-targeted theranostics nanovector can be obtained efficiently and simultaneously. Such multifunctional nanovectors transformed from viral L-particles have exhibited excellent properties of active tumor-targeting, in vivo tumor imaging, and antitumor efficacy, which opens a new window for the development of natural therapeutic nanoplatforms.

Lymphotoxin-α promotes tumor angiogenesis in HNSCC by modulating glycolysis in a PFKFB3-dependent manner.

Tumor angiogenesis is critical for tumor progression as the new blood vessels supply nutrients and facilitate metastasis. Previous studies indicate tumor associated lymphocytes, including B cells and T cells, contribute to tumor angiogenesis and tumor progression. The present study aims to identify the function of Lymphotoxin-α (LT-α), which is secreted by the activated lymphocytes, in the tumor angiogenesis of head and neck squamous cell carcinoma (HNSCC). The coculture system between HNSCC cell line Cal27 and primary lymphocytes revealed that tumor cells promoted the LT-α secretion in the cocultured lymphocytes. In vitro data further demonstrated that LT-α promoted the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) by enhancing the PFKFB3-mediated glycolytic flux. Genetic and pharmacological inhibition of PFKFB3 suppressed the enhanced proliferation and migration of HUVECs. We further identified that LT-α induced PFKFB3 expression was dependent on the TNFR/NF-κB signaling pathway. In addition, we proved that PFKFB3 blockade decreased the density of CD31 positive blood vessels in HNSCC xenografts. Finally, the results from the human HNSCC tissue array revealed that the expression of LT-α in HNSCC samples positively correlated with microvessel density, lymphocytes infiltration and endothelial PFKFB3 expression. In conclusion, infiltrated lymphocyte secreted LT-α enhances the glycolysis of ECs in a PFKFB3-dependent manner through the classical NF-κB pathway and promotes the proliferation and migration of ECs, which may contribute to the aberrant angiogenesis in HNSCCs. Our study suggests that PFKFB3 blockade is a promising therapeutic approach for HNSCCs by targeting tumor angiogenesis.

Magnetic Chip Based Extracorporeal Circulation: A New Tool for Circulating Tumor Cell in Vivo Detection.

In vivo detection of circulating tumor cells (CTCs) which inspect all of the circulating blood in body seems to have more advantages on cell capture, especially in earlier cancer diagnosis. Herein, based on in vivo microfluidic chip detection system (IV-chip-system), an extracorporeal circulation was constructed to effectively detect and monitor CTCs in vivo. Combined with microfluidic chip and immunomagnetic nanosphere (IMN), this system not only acts as a window for CTC monitoring but also serves as a collector for further cancer diagnosis and research on CTCs. Compared with the current in vivo detection method, this system can capture and detect CTCs in the bloodstream without any pretreatments, and it also has a higher CTC capture efficiency. It is worth mentioning that this system is stable and biocompatible without any irreversible damage to living animals. Taking use of this system, the mimicked CTC cleanup process in the blood vessel is monitored, which may open new insights in cancer research and early cancer diagnosis.

Spatiotemporal characteristics of tissue derived small extracellular vesicles is associated with tumor relapse and anti-PD-1 response.

Small extracellular vesicles (sEVs) residing at tumor tissues are valuable specimens for biopsy. Tumor heterogeneity is common across all cancer types, but the heterogeneity of tumor tissue-derived sEVs (Ti-sEVs) is undefined. This study aims to discover the spatial distributions of Ti-sEVs in oral squamous cell carcinoma (OSCC) tissues and explore how these vesicle distributions affect the patients' prognosis. Multi-regional sampling enabled us to uncover that Ti-sEVs' accumulation at peritumoral sites correlates with a higher disease-free survival rate, and conversely, sparse peritumoral Ti-sEVs tend to forecast a higher risk of relapse. Of those relapsed patients, Ti-sEVs strongly bind to extracellular matrix and subsequently degrade it for allowing themselves enter the bloodstream rather than staying in situ. In advanced OSCC patients, the quantity and spatial distribution of Ti-sEVs prior to anti-PD-1 treatment, as well as the temporal variance of Ti-sEVs before and after immunotherapy, strongly map the clinical response and can help to distinguish the patients with shrinking tumors from those with growing tumors. Our work elucidates the correlation of spatiotemporal features of Ti-sEVs with patients' therapeutic outcomes and exhibit the potential for using Ti-sEVs as a predictor to forecast prognosis and screen the responders to anti-PD-1 therapy.

PD-1/CD80+ small extracellular vesicles from immunocytes induce cold tumours featured with enhanced adaptive immunosuppression.

Only a minority of cancer patients benefit from immune checkpoint blockade therapy. Sophisticated cross-talk among different immune checkpoint pathways as well as interaction pattern of immune checkpoint molecules carried on circulating small extracellular vesicles (sEV) might contribute to the low response rate. Here we demonstrate that PD-1 and CD80 carried on immunocyte-derived sEVs (I-sEV) induce an adaptive redistribution of PD-L1 in tumour cells. The resulting decreased cell membrane PD-L1 expression and increased sEV PD-L1 secretion into the circulation contribute to systemic immunosuppression. PD-1/CD80+ I-sEVs also induce downregulation of adhesion- and antigen presentation-related molecules on tumour cells and impaired immune cell infiltration, thereby converting tumours to an immunologically cold phenotype. Moreover, synchronous analysis of multiple checkpoint molecules, including PD-1, CD80 and PD-L1, on circulating sEVs distinguishes clinical responders from those patients who poorly respond to anti-PD-1 treatment. Altogether, our study shows that sEVs carry multiple inhibitory immune checkpoints proteins, which form a potentially targetable adaptive loop to suppress antitumour immunity.

Red Blood Cell-Derived Extracellular Vesicles: An Overview of Current Research Progress, Challenges, and Opportunities.

Red blood cell-derived extracellular vesicles (RBC EVs) are small, spherical fragments released from red blood cells. These vesicles, similar to EVs derived from other cell types, are crucial for intercellular communication processes and have been implicated in various physiological and pathological processes. The diagnostic and therapeutic potential of RBC EVs has garnered increasing attention in recent years, revealing their valuable role in the field of medicine. In this review, we aim to provide a comprehensive analysis of the current research status of RBC EVs. We summarize existing studies and highlight the progress made in understanding the characteristics and functions of RBC EVs, with a particular focus on their biological roles in different diseases. We also discuss their potential utility as diagnostic and prognostic biomarkers in diseases and as vectors for drug delivery. Furthermore, we emphasize the need for further research to achieve selective purification of RBC EVs and unravel their heterogeneity, which will allow for a deeper understanding of their diverse functions and exploration of their potential applications in diagnostics and therapeutics.

Continuous magnetic separation microfluidic chip for tumor cell in vivo detection.

Continuously recording the dynamic changes of circulating tumor cells (CTCs) is crucial for tumor metastasis. This paper creates a continuous magnetic separation microfluidic chip that enables rapid and continuous in vivo cell detection. The chip shows its potential to study tumor cell circulation in the blood, offering a new platform for studying the cellular mechanism of tumor metastasis.

HRS Regulates Small Extracellular Vesicle PD-L1 Secretion and Is Associated with Anti-PD-1 Treatment Efficacy.

PD-L1 localized to immunosuppressive small extracellular vesicles (sEV PD-L1) contributes to tumor progression and is associated with resistance to immune-checkpoint blockade (ICB) therapy. Here, by establishing a screening strategy with a combination of tissue microarray (TMA), IHC staining, and measurement of circulating sEV PD-L1, we found that the endosomal sorting complex required for transport (ESCRT) member protein hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was the key regulator of circulating sEV PD-L1 in head and neck squamous cell carcinoma (HNSCC) patients. Increased HRS expression was found in tumor tissues and positively correlated with elevated circulating sEV PD-L1 in patients with HNSCC. The expression of HRS was also negatively correlated to the infiltration of CD8+ T cells. Knockdown of HRS markedly reduced PD-L1 expression in HNSCC cell-derived sEVs, and these sEVs from HRS knockdown cells showed decreased immunosuppressive effects on CD8+ T cells. Knockout of HRS inhibited tumor growth in immunocompetent mice together with PD-1 blockade. Moreover, a higher HRS expression was associated with a lower response rate to anti-PD-1 therapy in patients with HNSCC. In summary, our study reveals HRS, the core component of ESCRT-0, regulates sEV PD-L1 secretion, and is associated with the response to ICB therapy in patients with HNSCC, suggesting HRS is a promising target to improve cancer immunotherapy.

ALIX promotes cell migration and invasion of head and neck squamous cell carcinoma by regulating the expression of MMP9, MMP14, VEGF-C.

OBJECTIVE: The poor survival rate of head and neck squamous cell carcinoma (HNSCC), one of the most prevalent human cancer, is attributed to frequent locoregional recurrence and lymph node metastases. Though it is reported that the expression of ALG-2 interacting protein X (ALIX) closely correlates with the progression of various tumors, its role in HNSCC remains unclear. The present study aims to investigate the role of ALIX in the development of HNSCC. DESIGN: With immunohistochemical staining, the expression levels of ALIX and series of related functional proteins were compared in normal mucosal (n = 18), HNSCC tissues (n = 54), and metastatic lymph nodes (n = 11). Further, the correlation analysis was performed among the proteins detected. By knocking down ALIX in HNSCC cell lines, the correlation of ALIX with the proteins was verified in vitro. The role of ALIX in proliferation, migration, and invasion of HNSCC cells was further studied by flow cytometry, wounding healing, and transwell assays, respectively. RESULTS: Higher expression level of ALIX was revealed in HNSCC samples, especially in metastatic lymph nodes, than in normal mucosal tissues. Accordingly, increasing levels of MMP9, MMP14, and VEGF-C were also discovered in metastatic lymph nodes and significantly correlated with the expression of ALIX. In vitro assays demonstrated that the knockdown of ALIX reduced both the transcriptional and protein levels of MMP9, MMP14, and VEGF-C, together with suppressed migration and weakened invasion of HNSCC cell lines. CONCLUSIONS: ALIX up-regulated the expression of MMP9, MMP14 and VEGF-C, and promoted migration and invasion of HNSCC cells.

Real-Time Dissection of the Transportation and miRNA-Release Dynamics of Small Extracellular Vesicles.

Extracellular vesicles (EVs) are cell-derived membrane-enclosed structures that deliver biomolecules for intercellular communication. Developing visualization methods to elucidate the spatiotemporal dynamics of EVs' behaviors will facilitate their understanding and translation. With a quantum dot (QD) labeling strategy, a single particle tracking (SPT) platform is proposed here for dissecting the dynamic behaviors of EVs. The interplays between tumor cell-derived small EVs (T-sEVs) and endothelial cells (ECs) are specifically investigated based on this platform. It is revealed that, following a clathrin-mediated endocytosis by ECs, T-sEVs are transported to the perinuclear region in a typical three-stage pattern. Importantly, T-sEVs frequently interact with and finally enter lysosomes, followed by quick release of their carried miRNAs. This study, for the first time, reports the entire process and detailed dynamics of T-sEV transportation and cargo-release in ECs, leading to better understanding of their proangiogenic functions. Additionally, the QD-based SPT technique will help uncover more secrets of sEV-mediated cell-cell communication.

A liquid biopsy-guided drug release system for cancer theranostics: integrating rapid circulating tumor cell detection and precision tumor therapy.

Theranostics combining precision diagnosis and concurrent therapy has attracted significant attention as a promising strategy against life-threatening cancer. Liquid biopsy provides a real-time assessment of cancer by the analysis of tumor biomarkers, among which circulating tumor cells (CTCs) have been widely used to monitor disease progression and therapeutic response. In this study, a liquid biopsy-guided drug release system (LBDR system) integrating cancer diagnostic and therapeutic functions on a magnetically controlled microfluidic platform is presented. Two kinds of magnetic nanospheres (MNs), recognition MNs and drug-loaded MNs, are loaded onto the microfluidic chip to integrate the rapid detection of CTCs and controlled drug release. When CTCs bind to aptamers on the recognition MNs, complementary strands (cDNAs) hybridized with the aptamers are released and then conjugated with drug-loaded MNs to further trigger the release of anti-cancer drugs. The amount of drug released is controlled according to the number of detected CTCs, which can provide effective treatment for individual patients according to the diagnostic results. This LBDR system provides a novel strategy for cancer therapy and may facilitate the development of personalized cancer therapy.

Effects of bleomycin on tooth eruption: a novel potential application.

There are many kinds of potentially undesirable teeth. At present, surgical extraction is the most efficient way to eliminate these teeth, but it's very complex and invasive. In this study, we investigated the effects of bleomycin (BLM) on dental follicle and tooth eruption as a potential conservative therapy for undesirable teeth. Our data showed that local injection of 0.2 U/kg BLM had no significant effects on tooth eruption compared to the control group in Wistar rats. With higher dose of BLM (0.5 or 2 U/kg), the eruption of treated teeth was interrupted and their root formation failed until 4 weeks postnatal without significant systemic toxicity. Additionally, those effects were not depending on the toxicity of overdose evidenced by TUNEL assay. In summary, injecting BLM into dental follicle at an early stage could interrupt tooth development and eruption, and may prevent the potentially clinical problems resulting from undesirable teeth instead of surgical removal.

Association of ATF4 Expression With Tissue Hypoxia and M2 Macrophage Infiltration in Infantile Hemangioma.

Accumulating studies have revealed the hypoxic condition and its crucial role in the distinctive progression of infantile hemangioma (IH), the most common benign tumor in infancy. Activating transcription factor 4 (ATF4), an important gene mediating cellular adaptation to various stress signals, could confer a survival advantage for tumor cells under hypoxia and regulate tumor progression. However, the potential role of ATF4 in IH was still unknown. In this study, the expression of hypoxia inducible factor (HIF)-1α, ATF4, and macrophage colony-stimulating factor (M-CSF) in 27 specimens of IH was measured by immunochemistry and double-labeling immunofluorescence, followed by the Spearman rank correlation test. Our results showed that the expression of HIF-1α, ATF4, and M-CSF was significantly upregulated in proliferating IH compared with involuting IH. Meanwhile, HIF-1α and ATF4, in parallel with ATF4 and M-CSF, exhibited positive correlation and synchronous expression. In addition, our in vitro studies demonstrated that hypoxia obviously upregulated the expression of HIF-1α, ATF4, and M-CSF in hemangioma stem cells. Most importantly, their expression was uniformly correlated with the percentage of M2-polarized macrophages in IH. All those results and established evidence indicated that hypoxia-induced ATF4 expression may promote progression of proliferating IH through M-CSF-induced M2-polarized macrophages infiltration.