Consensus statement on extracellular vesicles in liquid biopsy for advancing laboratory medicine.

Extracellular vesicles (EVs) represent a diverse class of nanoscale membrane vesicles actively released by cells. These EVs can be further subdivided into categories like exosomes and microvesicles, based on their origins, sizes, and physical attributes. Significantly, disease-derived EVs have been detected in virtually all types of body fluids, providing a comprehensive molecular profile of their cellular origins. As a result, EVs are emerging as a valuable addition to liquid biopsy techniques. In this collective statement, the authors share their current perspectives on EV-related research and product development, with a shared commitment to translating this newfound knowledge into clinical applications for cancer and other diseases, particularly as disease biomarkers. The consensus within this document revolves around the overarching recognition of the merits, unresolved questions, and existing challenges surrounding EVs. This consensus manuscript is a collaborative effort led by the Committee of Exosomes, Society of Tumor Markers, Chinese anti-Cancer Association, aimed at expediting the cultivation of robust scientific and clinically applicable breakthroughs and propelling the field forward with greater swiftness and efficacy.

Milk-derived small extracellular vesicles: nanomaterials to promote bone formation.

Small extracellular vesicles (sEVs) are an important component in the paracrine pathway. They can be used as a substitute for seed cells and have shown good application prospects in promoting bone regeneration. Cow's milk could be used as a source of sEVs with good biocompatibility and cost-effectiveness, with easy availability, low cost and low toxicity. This study focused on the role and mechanism of small extracellular vesicles derived from milk in bone repair. In order to explore the mechanism via which Milk-sEVs promote bone repair, we screened the differential gene GJA1 in Milk-sEV-treated osteoblasts through transcriptome chips, and verified the transcript AP3B1 of GJA1 through chromatin immunoprecipitation (CHIP). We have proved by in vivo and in vitro experiments that milk-derived sEVs (Milk-sEVs) increase the repair ability of bone tissue, and promote expression of the osteogenic gene GJA1 through the transcript AP3B1.

Multiplexed profiling of single-cell extracellular vesicles secretion.

Extracellular vesicles (EVs) are important intercellular mediators regulating health and diseases. Conventional methods for EV surface marker profiling, which was based on population measurements, masked the cell-to-cell heterogeneity in the quantity and phenotypes of EV secretion. Herein, by using spatially patterned antibody barcodes, we realized multiplexed profiling of single-cell EV secretion from more than 1,000 single cells simultaneously. Applying this platform to profile human oral squamous cell carcinoma (OSCC) cell lines led to a deep understanding of previously undifferentiated single-cell heterogeneity underlying EV secretion. Notably, we observed that the decrement of certain EV phenotypes (e.g., CD63+EV) was associated with the invasive feature of both OSCC cell lines and primary OSCC cells. We also realized multiplexed detection of EV secretion and cytokines secretion simultaneously from the same single cells to investigate the multidimensional spectrum of cellular communications, from which we resolved tiered functional subgroups with distinct secretion profiles by visualized clustering and principal component analysis. In particular, we found that different cell subgroups dominated EV secretion and cytokine secretion. The technology introduced here enables a comprehensive evaluation of EV secretion heterogeneity at single-cell level, which may become an indispensable tool to complement current single-cell analysis and EV research.

High-Throughput Single-Cell Extracellular Vesicle Secretion Analysis on a Desktop Scanner without Cell Counting.

Single-cell EV (extracellular vesicle) secretion analysis is emerging for a better understanding of non-genetic cellular heterogeneity regulating human health and diseases through intercellular mediators. However, the requirements of expensive and bulky instrumentations hinder its widespread use. Herein, by combining gold nanoparticle-enhanced silver staining and the Poisson distribution, we reported the use of a home-use scanner to realize high-throughput single-cell EV secretion analysis without cell counting. We applied the platform to analyze the secretions of different EV phenotypes with the human oral squamous cell carcinoma cell line and primary cells from patients, which generated single-cell results comparable with those of the immunofluorescence approach. Notably, we also realized the quantification of the number of EVs secreted from every single cell using their respective titration curves obtained from population samples, making it possible to directly compare different EV phonotypes in regard to their secretion number, secretion rate, and so forth. The technology introduced here is simple, easy to operate, and of low cost, which make it a potential, easily accessible, and affordable tool for widespread use in both basic and clinical research.

Single-Cell Secretion Analysis in the Engineered Tumor Microenvironment Reveals Differential Modulation of Macrophage Immune Responses.

It is increasingly recognized that the cellular microenvironment plays critical roles in regulating the fate and physiology of cells. Despite recent advancements in single-cell analysis technologies, engineering and integration of the microenvironment for single-cell analysis platforms remain limited. Here, we report a single-cell cytokine secretion analysis platform that integrated both the three-dimensional cell culture and the primary oral squamous cell carcinoma tumor cell co-culture to provide both physical and physiological cues for single cells to be analyzed. We apply the platform to investigate the immune responses of human macrophages stimulated with the ligand of toll-like receptor 4 lipopolysaccharide. Notably, we observe the differential modulation effect in cytokine secretions by the tumor microenvironment, in which antitumor cytokine TNF-a secretion was attenuated, and protumor cytokine IL-6 would increase. The differential modulation effect is conserved from cell line-derived macrophages to primary macrophages derived from healthy donors. Immunofluorescence staining further reveals that ∼50% of macrophage cells could be polarized from M1 to the M2 phenotype within 12 h in the engineered tumor microenvironment. This work demonstrates the significance of the cell microenvironment toward single-cell analysis, which could help to evaluate how immune cells will respond in the complex microenvironment more accurately.

CNTs-CaP/chitosan-coated AZ91D magnesium alloy extract promoted rat dorsal root ganglia neuron growth via activating ERK signalling pathway.

Increasing attention has been paid on the application of biodegradable materials such as magnesium and its alloys in neuron repair. AZ91D magnesium alloy coated with carbon nanotubes (CNTs) and/or calcium phosphate (CaP)/chitosan (CS) was fabricated in this study. To evaluate the bioactivity of these AZ91D-based composites, the extracts were prepared by immersing samples in modified simulated body fluid (m-SBF) for 0, 2, 8, 16, 24, 34, 44, 60, or 90 days. Immunofluorescence staining for neuronal class III ß-tubulin (TUJ1) revealed that both CNTs-CaP/CS-AZ91D and CaP/CS-AZ91D extracts promoted axon outgrowth of dorsal root ganglia (DRG) neurons, accompanied with increased expression of phosphorylated focal adhesion kinase (p-FAK) and growth associated protein-43 (GAP-43). Besides, the extracts increased the expression and the release of neurotrophic factors including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). ERK signalling was activated in DRG neurons after treating with either CNTs-CaP/CS-AZ91D or CaP/CS-AZ91D extracts, and its inhibition with U0126 counteracted the beneficial effects of these extracts on DRG neuron. Overall, the extracts from these AZ91D-based composites might promote DRG neuron growth via activating ERK signalling pathway. Notably, CNTs-CaP/CS-AZ91D extracts showed a better promoting effect on neuron growth than CaP/CS-AZ91D. Assessment of ion elements showed that the addition of CNTs coating enhanced magnesium corrosion resistance and reduced the deposition of calcium and phosphorus on the surface of CaP/CS-AZ91D alloy. These findings demonstrate that CNTs-CaP/CS-AZ91D likely provide a more suitable environment for neuron growth, which suggests a potential implantable biomaterial for the treatment of nerve injury. SIGNIFICANCE: AZ91D magnesium alloy coated with carbon nanotubes (CNTs) and/or calcium phosphate (CaP)/chitosan (CS) was fabricated and their immersion extracts were prepared using modified simulated body fluid in this study. Both extracts from CNTs-CaP/CS and CaP/CS-coated AZ91D magnesium alloy promotes rat dorsal root ganglia (DRG) neuron growth via activating ERK signalling pathway. Notably, the addition of CNTs improves the performance of CaP/CS-AZ91D. For the first time, our research demonstrates that CNTs-CaP/CS-AZ91D likely provide a suitable environment for neuron growth, suggesting these AZ91D-based composites as potential implantable biomaterials for the treatment of nerve injury.

LncRNA MALAT1 aggravates oxygen-glucose deprivation/reoxygenation-induced neuronal endoplasmic reticulum stress and apoptosis via the miR-195a-5p/HMGA1 axis.

BACKGROUND: This study aimed to investigate the potential role and molecular mechanism of lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in cerebral ischemia/reperfusion injury. RESULTS: Using an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model, we determined that the expression of MALAT1 was significantly increased during OGD/R. MALAT1 knockdown reversed OGD/R-induced apoptosis and ER stress. Mechanistically, MALAT1 promoted OGD/R-induced neuronal injury through sponging miR-195a-5p to upregulating high mobility group AT-hook1 (HMGA1). CONCLUSIONS: Collectively, these data demonstrate the mechanism underlying the invovlvement of MALAT1 in cerebral ischemia/reperfusion injury, thus providing translational evidence that MALAT1 may serve as a novel biomarker and therapeutic target for ischemic stroke.

PDMS Microwell Stencil Based Multiplexed Single-Cell Secretion Analysis.

Multiplexed single-cell protein secretion analysis provides an in-depth understanding of cellular heterogeneity in intercellular communications mediated by secreted proteins in both fundamental and clinical research. However, it has been challenging to increase the proteomic parameters co-profiled from every single cell in a facile way. Herein, a simple method to improve the multiplexed proteomic parameters of PDMS microwell based single-cell secretion analysis platform by sandwiching PDMS stencil in between two antibody-coated glass slides is introduced. Two different antibody panels can be immobilized easily by static coating, without using sophisticated fluid handling or bulky equipment. 5-plexed, 3-fluorescence color single-cell secretion assay is demonstrated with this platform to investigate human monocytic U937 cells in response to lipopolysaccharide and phorbol myristate acetate stimulation, which identified the existence of functional subsets dictated by different cytokine profiles. The technology introduced here is simple, easy to operate, which holds great potential to become a powerful tool for profiling multiplexed single-cell cytokine secretion at high throughput to dissect cellular heterogeneity in secretome signatures.

MicroRNA-29b-3p suppresses oral squamous cell carcinoma cell migration and invasion via IL32/AKT signalling pathway.

Oral squamous cell carcinoma (OSCC) is aggressive accompanied with poor prognosis. We previously isolated the most invasive cells resembling the invasive tumour front by microfluidic technology and explored their differentially expressed microRNAs (miRNAs) in our previous work. Here, we verified the miR-29b-3p as a guarder that suppressed migration and invasion of OSCC cells and was down-regulated in the most invasive cells. Besides that, the invasion suppression role of miR-29b-3p was achieved through the IL32/AKT pathway. Thus, miR-29b-3p and IL32 might serve as therapeutic targets for blocking the progression and improving the outcome of OSCC.

Hypoxia modulates stem cell properties and induces EMT through N-glycosylation of EpCAM in breast cancer cells.

Epithelial cell adhesion molecule (EpCAM), which is a transmembrane glycoprotein, is related to tumor progression. We demonstrated that EpCAM plays important roles in proliferation, apoptosis, and metastasis during breast cancer (BC) progression. But the role of N-glycosylation in EpCAM in tumor aggressiveness is not clear. Here, we evaluated the role of N-glycosylation of EpCAM in stemness and epithelial-mesenchymal transition (EMT) characteristics. EpCAM overexpression increases the expression of stemness markers (NANOG，SOX2, and OCT4) and EMT markers (N-cadherin and vimentin) under the condition of hypoxia in BC. Knockdown of EpCAM and mutation of N-glycosylation of EpCAM maintained in severe hypoxia lead to a significant reduction of stemness/EMT markers. In addition, we found that N-glycosylation of EpCAM is a crucial factor during this process. This demonstrates that EpCAM has a novel regulatory role in stemness/EMT dependence of hypoxia-inducible factor 1-alpha via regulating nuclear factor kappa B in BC cells. Hence, our study reveals EpCAM glycosylation modification as a new regulator of stemness/EMT under hypoxic in BC and points out EpCAM as a potential therapeutic target.

Deglycosylation of epithelial cell adhesion molecule affects epithelial to mesenchymal transition in breast cancer cells.

The transmembrane glycoprotein epithelial cell adhesion molecule (EpCAM) is overexpressed in most epithelial cancers including breast cancer, where it plays an important role in cancer progression. Previous study has demonstrated that knockdown of EpCAM inhibits breast cancer cell growth and metastasis via inhibition of the Ras/Raf/ERK signaling pathway and matrix metallopeptidase-9 (MMP-9). Although glycosylation is believed to be associated with the function of EpCAM, the contribution of N-glycosylation to this function remains unclear. We constructed the N-glycosylation mutation plasmid of EpCAM and used it to treat breast cancer cells. Loss of N-glycosylation at all three sites EpCAM had no effect on its level of expression or membrane localization. However, mutation at glycosylation sites significantly reduced the ability of EpCAM to promote epithelial to mesenchymal transition in breast cancer. N-glycosylation mutation of EpCAM led to decrease phosphorylation of Raf, ERK, and Akt, and inhibited the Ras/Raf/ERK and PI3K/Akt signaling pathways. Furthermore, we demonstrated that N-glycosylation mutation of EpCAM-mediated invasion and metastasis of breast carcinoma cells required the downregulation of MMP-9 via inhibition of these two signaling pathways. Our results identified the characteristics and function of EpCAM glycosylation. These data could illuminate molecular regulation of EpCAM by glycosylation and promote our understanding of the application of glycosylated EpCAM as a target for breast cancer therapy.

Extracellular vesicles of carcinoma-associated fibroblasts creates a pre-metastatic niche in the lung through activating fibroblasts.

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 α2ß1 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 ß1 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 ß1 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.

A liver-chip-based alcoholic liver disease model featuring multi-non-parenchymal cells.

Non-parenchymal cells play a key role in the occurrence and development of alcoholic liver disease. However, this cellular behaviour has not been fully characterized, and it is inconvenient to observe in traditional in vitro alcoholic liver disease (ALD) models and animal models. Herein we developed a demountable liver-on-chip device for investigation of pathophysiological process of individual non-parenchymal cells in alcohol induced ALD. This liver-device comprised of HepG2, LX-2, EAhy926 and U937 cells, which were ordered in a physiological distribution under perfuse. This device allows improved HepG2 cells activities and maintained high liver functions which including albumin synthesis and urea secretion. This novel liver-device is able to recreate the damage process of hepatic non-parenchymal cell lines induced by alcohol, and to understand the intercellular communication between different types of hepatic cells during ALD by measuring multiple biomarkers of each types of hepatic non-parenchymal cell lines, including Ve-cadherin, eNOS, VEGF and α-SMA. The proposed liver-device is able to further studies of pathological analysis and drug- and toxicity-screening.

The association between health professionals' international experience and the academic output of their students in Harbin, China.

BACKGROUND: It is common practice for health professionals in China to have international experience. However, the association between such experience and these professionals' students' scientific research ability has not previously been evaluated. Our study aimed to quantify this association among the students of health professionals in China. METHODS: We constructed a self-administered questionnaire and distributed it to all students at Harbin Medical University and its affiliated hospitals, including 257 students (Group A) of health professionals who had studied overseas ("returning" professionals) and 257 age-, enrollment year-, and specialty-matched students (Group B) of health professionals who had not studied overseas ("resident" professionals). SPSS software was used for the data entry and analysis. RESULTS: The total impact factor (IF) for articles published during their PhD study was 1031.68 in Group A and 727.65 in Group B (P = 0.001), and the number of articles was 297 in Group A and 228 in Group B (P = 0.040). The total IF for articles published by the 151 clinical medicine students of returning professionals during their PhD study was positively correlated with their advisor's total IF for articles published while abroad (P = 0.019). CONCLUSIONS: This study indicates that medical students may benefit from their advisors' international experience. Medical education administrators and the government could encourage clinical professionals to study overseas and to prolong the duration of their study abroad. Medical students should consider potential advisors' overseas experience when choosing a mentor.

Paper-Based 3D Scaffold for Multiplexed Single Cell Secretomic Analysis.

Despite rapid progresses in single-cell analysis technologies, efforts to control the three-dimensional microenvironment for single cell measurements have been lacking. Here, we report a simple method to incorporate three-dimensional scaffolds, including polyvinylidene fluoride (PVDF) membranes and PVDF membrane replicated analog polydimethylsiloxane, into multiplexed single cell secretomic analysis platforms (including a microwell array and a single cell barcode microchip) to mimic the extracellular physical matrix and mechanical support for single cells. Applying this platform to brain tumor cell line U87 to investigate single cell protein secretion behavior on different substrates, we revealed that single cell protein secretions were regulated differently in three-dimensional (3D) microenvironments. This finding was further verified with intracellular cytokine staining, highlighting the significance of 3D single cell microenvironments. This new single cell biomimetic platform can be easily adaptable to other three-dimensional cell culture scaffolds or other single cell assays and may become a broadly applicable three-dimensional single cell analysis system to study the effect of microenvironment conditions on cellular functional heterogeneity in vitro.

Mutation of N-linked glycosylation in EpCAM affected cell adhesion in breast cancer cells.

Epithelial cell adhesion molecule (EpCAM) expression is elevated in breast cancer tissue, and correlates with the cancer metastasis and cell adhesion. Although EpCAM glycosylation is supposed to be associated with its function, the contribution of N-glycosylation to its function remains unclear. Here we analyzed cell adhesion ability of EpCAM in breast cancer cells. The results showed that EpCAM expression was associated with cell adhesion and N-glycosylation mutation of EpCAM decreased adhesion capacity. N-glycosylation mutation of EpCAM was correlated with lower levels of integrin ß1 and fibronectin. We also found that effect of N-glycosylation of EpCAM on cell adhesion was regulated via FAK/Akt/Gsk-3ß/ß-catenin signaling pathway, which further adjusted MMP2/9 expression and activities. Our studies identified the characteristics and function of EpCAM glycosylation sites on breast cancer cell adhesion. These data could potentially clarify molecular regulation of EpCAM by N-glycosylation and intensify our understanding of the utility of glycosylated EpCAM as a target for breast cancer therapy.

The role of epithelial cell adhesion molecule N-glycosylation on apoptosis in breast cancer cells.

Glycosylation of cell surface proteins plays an important role in the regulation of apoptosis. It has been demonstrated that knockdown of epithelial cell adhesion molecule promoted apoptosis, inhibited cell proliferation, and caused cell-cycle arrest. In this study, we investigated whether and how N-glycosylation of epithelial cell adhesion molecule influenced the apoptosis in breast cancer cells. We applied the N-glycosylation mutation epithelial cell adhesion molecule plasmid to express deglycosylation of epithelial cell adhesion molecule and then to study its function. Our results showed that deglycosylation of epithelial cell adhesion molecule promoted apoptosis and inhibited cell proliferation. Deglycosylation of epithelial cell adhesion molecule enhanced the cytotoxic effect of 5-fluorouracil, promoting apoptosis by downregulating the expression of the anti-apoptotic protein Bcl-2 and upregulating the expression of the pro-apoptotic proteins Bax and Caspase 3 via the extracellular-signal-regulated kinase 1/2 and c-Jun N-terminal kinase mitogen-activated protein kinase signaling pathways in MCF-7 and MDA-MB-231 cells. These findings are important for a better understanding of epithelial cell adhesion molecule apoptosis regulation and suggest epithelial cell adhesion molecule as a potential target for the treatment of breast cancer.

Streptozotocin Inhibits Electrophysiological Determinants of Excitatory and Inhibitory Synaptic Transmission in CA1 Pyramidal Neurons of Rat Hippocampal Slices: Reduction of These Effects by Edaravone.

BACKGROUND: Streptozotocin (STZ) has served as an agent to generate an Alzheimer's disease (AD) model in rats, while edaravone (EDA), a novel free radical scavenger, has recently emerged as an effective treatment for use in vivo and vitro AD models. However, to date, these beneficial effects of EDA have only been clearly demonstrated within STZ-induced animal models of AD and in cell models of AD. A better understanding of the mechanisms of EDA may provide the opportunity for their clinical application in the treatment of AD. Therefore, the purpose of this study was to investigate the underlying mechanisms of STZ and EDA as assessed upon electrophysiological alterations in CA1 pyramidal neurons of rat hippocampal slices. METHODS: Through measures of evoked excitatory postsynaptic currents (eEPSCs), AMPAR-mediated eEPSCs (eEPSCsAMPA), evoked inhibitory postsynaptic currents (eIPSCs), evoked excitatory postsynaptic current paired pulse ratio (eEPSC PPR) and evoked inhibitory postsynaptic current paired pulse ratio (eIPSC PPR), it was possible to investigate mechanisms as related to the neurotoxicity of STZ and reductions in these effects by EDA. RESULTS: Our results showed that STZ (1000 µM) significantly inhibited peak amplitudes of eEPSCs, eEPSCsAMPA and eIPSCs, while EDA (1000 µM) attenuated these STZ-induced changes at holding potentials ranging from -60mV to +40 mV for EPSCs and -60mV to +20 mV for IPSCs. Our work also indicated that mean eEPSC PPR were substantially altered by STZ, effects which were partially restored by EDA. In contrast, no significant effects upon eIPSC PPR were obtained in response to STZ and EDA. CONCLUSION: Our data suggest that STZ inhibits glutamatergic transmission involving pre-synaptic mechanisms and AMPAR, and that STZ inhibits GABAergic transmission by post-synaptic mechanisms within CA1 pyramidal neurons. These effects are attenuated by EDA.

Genistein suppresses smooth muscle cell-derived foam cell formation through tyrosine kinase pathway.

PURPOSE: Genistein, as a protein tyrosine kinase inhibitor, has been shown to possess anti-atherosclerotic effects. Since the smooth muscle cell-derived foam cells are key components of atherosclerotic plaques. The aim of this study is to investigate the influence of genistein on foam cell transformation from vascular smooth muscle cells and possible mechanisms contributing to these effects. METHODS AND RESULTS: Vascular smooth muscle cells exposed to ox-LDL developed into foam cell, as demonstrated by Oil Red O staining and cholesterol content analysis. Ox-LDL induced phenotype transformation of smooth muscle cells, decreased expression of α-actin and increased expression of CD68 (a specific marker for monocytes, can also function as a subtype of scavenger receptors). The expression of scavenger receptors CD36 and LOX-1 was measured, and their role in foam cell formation in the presence of genistein, daidzein (a structurally similar analogue of genistein) and herbimycin A (a commonly tyrosine kinase inhibitor). The results showed that foam cell formation was markedly reduced by genistein and herbimycin A, as well as the expression of CD68, CD36 and LOX-1. However, daidzein had no such effect. In addition, genistein-induced down-regulation of CD68, CD36 and LOX-1 could be reversed by sodium orthovanadate (a membrane-permeable protein tyrosine phosphatase inhibitor). CONCLUSION: The results showed that ox-LDL induce smooth muscle cell-derived foam cell formation and transform the phenotype of smooth muscle cell. While tyrosine kinase inhibitor, genistein could suppress smooth muscle cell-derived foam cell formation through inhibiting the protein expressions of CD68, CD36 and LOX-1.

Myofibroblasts from salivary gland adenoid cystic carcinomas promote cancer invasion by expressing MMP2 and CXCL12.

OBJECTIVES: Salivary gland adenoid cystic carcinoma (ACC) is one of the most common malignant tumours in the oral and maxillofacial region, and has high aggressive potential. Tumour and stroma interactions are critical in determining the biological characteristics of malignancy. The aim of this study was to investigate the presence of myofibroblasts and their roles in the invasive characteristics of ACC. METHODS AND RESULTS: Immunohistochemistry was used to detect the expression of vimentin (VIM), α-smooth muscle actin (α-SMA), matrix metalloproteinase 2 (MMP2) and CD34 in ACCs and normal salivary gland controls. A significant difference in α-SMA expression was found between normal controls and ACCs, suggesting the presence of myofibroblasts in ACCs. Immunohistochemical staining also demonstrated higher MMP2 expression in the stroma of ACCs than in the controls (P < 0.001). Primary culture of myofibroblasts from one ACC showed great invasive activity, with high expression of MMP2 and C-X-C motif chemokine 12 (CXCL12) by reverse transcription polymerase chain reaction (RT-PCR) analysis. CONCLUSIONS: This study demonstrated the presence of myofibroblasts in ACC. Myofibroblasts might be related to the aggressive growth behaviour of ACC, owing to their high levels of expression of MMP2 and CXCL12.