Objectives: Carcinoma-associated fibroblasts (CAFs) have been known to promote cancer progression by modifying the primary tumor microenvironment. We aimed to elucidate the intercellular communication between CAFs and secondary organs in salivary adenoid cystic carcinoma (SACC) metastasis. Methods: Pre-metastatic and metastatic animal models of SACC were established using extracellular vesicles (EVs) from CAFs and SACC cells. Lung fibroblasts (LFs) were treated with EVs and their transcriptomic alterations were identified by RNA sequencing. ITRAQ were performed to analyze EV cargos. TC I-15 was used to inhibit EV uptake by LFs and SACC lung metastasis in vivo. Results: Here, we show that CAF EVs induced lung pre-metastatic niche formation in mice and consequently increased SACC lung metastasis. The pre-metastatic niche induced by CAF EVs was different from that induced by SACC EVs. CAF EVs presented a great ability for matrix remodeling and periostin is a potential biomarker characterizing the CAF EV-induced pre-metastatic niche. We found that lung fibroblast activation promoted by CAF EVs was a critical event at the pre-metastatic niche. Integrin α2β1 mediated CAF EV uptake by lung fibroblasts, and its blockage by TC I-15 prevented lung pre-metastatic niche formation and subsequent metastasis. Plasma EV integrin β1 was considerably upregulated in the mice bearing xenografts with high risk of lung metastasis. Conclusions: We demonstrated that CAF EVs participated in the pre-metastatic niche formation in the lung. Plasma EV integrin β1 might be a promising biomarker to predict SACC metastasis at an early stage. An integrated strategy targeting both tumor and stromal cells is necessary to prevent SACC metastasis.

Increasing attention has been attracted by exosomes in blood-based diagnosis because cancer cells release more exosomes in serum than normal cells and these exosomes overexpress a certain number of cancer-related biomarkers. However, capture and biomarker analysis of exosomes for clinical application are technically challenging. In this study, we developed a microfluidic chip for immunocapture and quantification of circulating exosomes from small sample volume and applied this device in clinical study. Circulating EpCAM-positive exosomes were measured in 6 cases breast cancer patients and 3 healthy controls to assist diagnosis. A significant increase in the EpCAM-positive exosome level in these patients was detected, compared to healthy controls. Furthermore, we quantified circulating HER2-positive exosomes in 19 cases of breast cancer patients for molecular classification. We demonstrated that the exosomal HER2 expression levels were almost consistent with that in tumor tissues assessed by immunohistochemical staining. The microfluidic chip might provide a new platform to assist breast cancer diagnosis and molecular classification.

Circulating tumor aggregates exhibit a high metastatic potential and could potentially serve as an important target for cancer therapies. In this study, we developed a microfluidic model that reconstitutes and is representative of the principal components of biological blood vessels, including vessel cavity, endothelium, and perivascular matrix containing chemokines. Using this model, the transendothelial invasion of tumor aggregates can be observed and recorded in realtime. In this study we analyzed the extravasation process of salivary gland adenoid cystic carcinoma (ACC) cell aggregates. ACC aggregates transmigrated across the endothelium under the stimulation of chemokine CXCL12. The endothelial integrity was irreversibly damaged at the site of transendothelial invasion. The transendothelial invasion of ACC aggregates was inhibited by AMD3100, but the adhesion of ACC aggregates to the endothelium was not affected by the CXCR4 antagonist. This model allows for detailed study of the attachment and transendothelial invasion of tumor aggregates; thus, it would be a useful tool for analysis of the underlying mechanisms of metastasis and for testing novel anti-metastasis agents.

Carcinoma-associated fibroblasts (CAFs) are the main cellular components of the tumor microenvironment and promote cancer progression by modifying the extracellular matrix (ECM). The tumor-associated ECM is characterized by collagen crosslinking catalyzed by lysyl oxidase (LOX). Small extracellular vesicles (sEVs) mediate cell-cell communication. However, the interactions between sEVs and the ECM remain unclear. Here, we demonstrated that sEVs released from oral squamous cell carcinoma (OSCC)-derived CAFs induce collagen crosslinking, thereby promoting epithelial-mesenchymal transition (EMT). CAF sEVs preferably bound to the ECM rather than being taken up by fibroblasts and induced collagen crosslinking, and a LOX inhibitor or blocking antibody suppressed this effect. Active LOX (αLOX), but not the LOX precursor, was enriched in CAF sEVs and interacted with periostin, fibronectin, and bone morphogenetic protein-1 on the surface of sEVs. CAF sEV-associated integrin α2β1 mediated the binding of CAF sEVs to collagen I, and blocking integrin α2β1 inhibited collagen crosslinking by interfering with CAF sEV binding to collagen I. CAF sEV-induced collagen crosslinking promoted the EMT of OSCC through FAK/paxillin/YAP pathway. Taken together, these findings reveal a novel role of CAF sEVs in tumor ECM remodeling, suggesting a critical mechanism for CAF-induced EMT of cancer cells.

Primary tumors selectively modify the microenvironment of distant organs such as the lung, liver, brain, bone marrow, and lymph nodes to facilitate metastasis. This supportive metastatic microenvironment in distant organs was termed the pre-metastatic niche (PMN) that is characterized by increased vascular permeability, extracellular matrix remodeling, bone marrow-derived cells recruitment, angiogenesis, and immunosuppression. Extracellular vesicles (EVs) are a group of cell-derived membranous structures that carry various functional molecules. EVs play a critical role in PMN formation by delivering their cargos to recipient cells in target organs. We provide an overview of the characteristics of the PMN in different organs promoted by cancer EVs and the underlying mechanisms in this review.

Liposomes are the earliest and most widely used nanoparticles for targeted drug delivery. Exosomes are nanosized membrane-bound particles and important mediators of intercellular communication. Combining liposomes and exosomes using various membrane fusion methods gives rise to a novel potential drug delivery system called membrane fusion-based hybrid exosomes (MFHE). These novel MFHEs not only exhibit potential advantageous features, such as high drug loading rate and targeted cellular uptake via surface modification, but are also endowed with high biocompatibility and low immunogenicity. Here, we provide an overview of MFHEs’ various preparation methods, characterization strategies, and their applications for disease treatment and scientific research.