Presence Of Vasculogenic Mimicry Research Articles

Abstract PO1-18-11: Imaging of tumour microvasculature using high-resolution contrast enhanced ultrasound together with markers of proliferation/angiogenesis/vascular mimicry to characterise response to NACT in TNBC

Abstract Background: In the UK, triple-negative breast cancer (TNBC) comprises 10-15% of breast cancer diagnoses annually. TNBC represents a clinical challenge due to a lack of expression of three treatable drug targets, namely oestrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER-2). The mainstay of treatment involves the use of neoadjuvant chemotherapy (NACT) followed by surgical excision where up to 50% of patients will achieve a pathological complete response (pCR). The addition of neo-adjuvant immunotherapy may increase the pCR rate to 60%. The presence of lymphatics and blood vessels within/around malignant tumours plays a role in cancer progression as does the formation of new blood vessels (angiogenesis). The vascular endothelial growth factor (VEGF) family and their receptors (VEGFRs) play an important role in angiogenesis as well as promoting the growth and survival of cancer cells. VEGFR is frequently overexpressed in TNBC and promotes changes in vascular endothelial cells, the basement membrane and the surrounding extracellular matrix. Co-expression of epidermal growth factor receptor (EGFR) and VEGFR enhances tumour growth and angiogenesis in an autocrine and paracrine manner. An alternate mechanism for tumour microcirculation in tumours is vasculogenic mimicry (VM). This is distinct from classical tumour angiogenesis. The presence of VM is associated with poor overall survival in breast cancer patients and anti-angiogenic treatment of TNBC may even promote tumour growth, proliferation and metastasis by stimulating VM. Conventional imaging techniques such as B-mode ultrasound and MRI are commonly used to monitor disease response in the breast but have limited accuracy in predicting pCR or residual disease. High-resolution contrast-enhanced ultrasound (HRCEUS) may offer a more accurate test to monitor TNBC response to NACT. This method utilises intravenously injected microbubbles, which consist of a gas core with an outer shell of lipid/albumin to image tumour microvasculature as well as gross tumour morphology. Trial Design: Single-centre study to evaluate HRCEUS to image the microvasculature of TNBC tumours in patients undergoing (NACT) and correlate imaging results with established markers of angiogenesis, proliferation and vasculogenic mimicry. Eligibility Criteria: Female, 18 to 60 years, histologically confirmed invasive TNBC with planned NACT, able to consent and in the Investigator’s opinion, adhering to the trial recommendations and governance. Aims: To investigate if imaging changes in the tumour microvasculature indicate a response to NACT and are reflected in the expression of proliferative markers. To determine whether changes in the tumour microvasculature and disease response are driven by angiogenesis +/- VM. To investigate whether overall disease response and changes in the microvasculature are influenced by the basal type phenotype or germline mutations in BRCA 1/2. Investigate patient satisfaction with the contrast ultrasound test. Statistical Methods: Quantify HRCEUS imaging characteristics of microvasculature at 3 treatment points. Compare this with the results of conventional imaging and the histopathological results of surgical excision at the end of NACT. Quantify immunohistochemical markers of angiogenesis, proliferation and VM at three treatment points and compare this with HRCEUS. Determine phenotype of all cases using immunohistochemistry to identify the basal Perform subtype analysis to assess if the basal phenotype is associated with imaging and immunohistochemical changes in the microvasculature during NACT. Test patients for germline BRCA1/2 gene mutations. Perform subtype analysis to assess if germline BRCA1/2 mutations are associated with imaging and immunohistochemical changes in the microvasculature during NACT. Present accrual (2 patients) and target accrual (5 patients) Contact details: j.rait@nhs.net Citation Format: Jaideep Rait, Michelle Garrett, Catherine Harper-Wynne, Sonia Saw, Mengxing Tang, Priya Palanisamy, Matthieu TOULEMONDE, Karina Cox. Imaging of tumour microvasculature using high-resolution contrast enhanced ultrasound together with markers of proliferation/angiogenesis/vascular mimicry to characterise response to NACT in TNBC [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-18-11.

RBMS3-induced circHECTD1 encoded a novel protein to suppress the vasculogenic mimicry formation in glioblastoma multiforme

Glioblastoma multiforme (GBM) is a highly vascularized malignant cancer of the central nervous system, and the presence of vasculogenic mimicry (VM) severely limits the effectiveness of anti-vascular therapy. In this study, we identified downregulated circHECTD1, which acted as a key VM-suppressed factor in GBM. circHECTD1 elevation significantly inhibited cell proliferation, migration, invasion and tube-like structure formation in GBM. RIP assay was used to demonstrate that the flanking intron sequence of circHECTD1 can be specifically bound by RBMS3, thereby inducing circHECTD1 formation to regulate VM formation in GBM. circHECTD1 was confirmed to possess a strong protein-encoding capacity and the encoded functional peptide 463aa was identified by LC-MS/MS. Both circHECTD1 and 463aa significantly inhibited GBM VM formation in vivo and in vitro. Analysis of the 463aa protein sequence revealed that it contained a ubiquitination-related domain and promoted NR2F1 degradation by regulating the ubiquitination of the NR2F1 at K396. ChIP assay verified that NR2F1 could directly bind to the promoter region of MMP2, MMP9 and VE-cadherin, transcriptionally promoting the expression of VM-related proteins, which in turn enhanced VM formation in GBM. In summary, we clarified a novel pathway for RBMS3-induced circHECTD1 encoding functional peptide 463aa to mediate the ubiquitination of NR2F1, which inhibited VM formation in GBM. This study aimed to reveal new mechanisms of GBM progression in order to provide novel approaches and strategies for the anti-vascular therapy of GBM.The schematic illustration showed the inhibitory effect of circHECTD1-463aa in the VM formation in GBM.

A novel peptide P1-121aa encoded by STK24P1 regulates vasculogenic mimicry via ELF2 phosphorylation in glioblastoma

Glioblastoma (GBM) is the most common malignant tumor of the central nervous system. Vasculogenic mimicry (VM) is a hematological system composed of tumor cells that exert blood perfusion without relying on vascular endothelial cells. The current poor results of anti-vascular therapy for clinical GBM are associated with the presence of VM; therefore, it is important to investigate VM formation in GBM. Our results demonstrate that STK24P1 encodes P1-121aa with a kinase structural domain, and in vitro kinase assays demonstrated that P1-121aa mediates modification of ELF2 phosphorylation. ChIP and dual luciferase reporter gene assays demonstrated that the transcription factor ELF2 binds to VE-cadherin and the VEGFR2 promoter region, thereby promoting VM formation in glioma cells. P1-121aa, encoded by the pseudogene STK24P1, phosphorylates ELF2 at S107, increasing the stability of the ELF2 protein. ELF2 promotes VEGFR2 and VE-cadherin expression at the transcriptional level, which in turn promotes VM in GBM. This study demonstrates the important roles of STK24P1, P1-121aa, and ELF2 in regulating VM in GBM, which could provide potential targets for GBM treatment.

Metastatic breast tumors downregulate miR-145 regulating the hypoxia-induced vasculogenic mimicry

Tumor cells grow in three-dimensional (3D) channels-like structures denoted as vasculogenic mimicry (VM), which provides a route for nutrients and oxygen acquisition. VM is activated by hypoxia and associated with metastasis and poor prognosis. MetastamiRs are microRNAs regulating metastasis, however, if they control VM in breast cancer remains poorly understood. The aim of this study was to evaluate the expression of VM-associated microRNAs in tumors of metastatic breast cancer patients. Firstly, we constructed microRNAs/mRNAs coregulation networks using expression data from TCGA databases. Dozens of microRNAs regulating genes involved in VM and metastasis were found. Of these, we selected 10 microRNAs for further characterization. The presence of VM in histological samples from patients with or without metastasis was evaluated using CD31-/PAS+ immunophenotyping. Remarkably, data showed that VM was significantly increased in tumors from patients with metastasis in comparison with no-metastatic group. Gene expression analysis indicated that miR-145, miR-142-3p, miR-31, miR-148a, miR-200b-3p and miR-526b were downregulated in primary tumors from patients with metastatic disease and positive for VM. Moreover, modulated microRNAs showed a predictive clinical value in overall survival in a cohort (n=1262) of breast cancer patients. Of these, we evaluated the role of miR-145 in formation of hypoxia-induced 3D channels-like using an in vitro model that recapitulates the early stages of VM. Data showed that miR-145 mimics was able to abolish the VM development in both metastatic Hs578t and MDA-MB-231 breast cancer cells. In conclusion, manipulation of miR-145 levels may represent a therapeutic approach in metastatic breast cancer patients that developed VM.

Targets Related to Vasculogenic Mimicry in Breast Cancer

Vasculogenic mimicry (VM) is the release of angiogenic factors from tumor cells, leading to morphological changes, migration and proliferation of vascular endothelial cells, ultimately leading to neovascularization. The presence of VM has been identified in breast cancer, the formation of VM also gives breast cancer a poor prognosis such as drug resistance and metastasis. The authors focus on the role of the PI3K/Akt signaling pathway, hypoxia-inducible factorα(HIFα), the P38/MAPK signaling pathway, cyclooxygenase (COX2), the epithelial-mesenchymal transition (EMT) pathway, non-coding RNAs, and the tumor phenotype in the development of VM in breast cancer, thus offering new ideas for the future treatment of breast cancer.

CircMAN1A2 promotes vasculogenic mimicry of nasopharyngeal carcinoma cells through upregulating ERBB2 via sponging miR-940.

Nasopharyngeal carcinoma (NPC) is the most prevalent human primary malignancy of the head and neck, and the presence of vasculogenic mimicry (VM) renders anti-angiogenic therapy ineffective and poorly prognostic. However, the underlying mechanisms are unclear. In the present study, we used miR-940 silencing and overexpression for in vitro NPC cell EdU staining, wound healing assay and 3D cell culture assay, and in vivo xenograft mouse model and VM formation to assess miR-940 function. We found that ectopic miR-940 expression reduced NPC cell proliferation, migration and VM, as well as tumorigenesis in vivo. By bioinformatic analysis, circMAN1A2 was identified as a circRNA that binds to miR-940. Mechanistically, we confirmed that circMAN1A2 acts as a sponge for miR-940, impairs the inhibitory effect of miR-940 on target ERBB2, and then activates the PI3K/AKT/mTOR signaling pathway using RNA-FISH, dual luciferase reporter gene and rescue analysis assays. In addition, upregulation of ERBB2 expression is associated with clinical staging and poor prognosis of NPC. Taken together, the present findings suggest that circMAN1A2 promotes VM formation and progression of NPC through miR-940/ERBB2 axis and further activates the PI3K/AKT/mTOR pathway. Therefore, circMAN1A2 may become a biomarker and therapeutic target for anti-angiogenic therapy in patients with nasopharyngeal carcinoma.

Vasculogenic mimicry correlates to presenting symptoms and mortality in uveal melanoma

PurposeFluid-conducting extracellular matrix patterns known as vasculogenic mimicry (VM) have been associated with poor prognosis in uveal melanoma and other cancers. We investigate the correlations between VM, presenting symptoms, mortality, and the area density of periodic acid-Schiff positive histological patterns (PAS density).MethodsSixty-nine patients that underwent enucleation for uveal melanoma between 2000 and 2007 were included. Clinicopathological parameters presenting symptoms and outcomes were collected. Histological tumor sections were evaluated for VM and PAS density was quantified with digital image analysis.ResultsThirty-four patients (49%) presented with blurred vision. 18 (26%) with a shadow in the visual field, 7 (10%) with photopsia and/or floaters, and 2 (3%) with metamorphopsia. Nine patients (13%) had no symptoms at all. Median follow-up was 16.7 years (SD 2.6). A shadow in the visual field, but no other symptom, was positively correlated with the presence of VM (φ 0.70, p < 0.001) and greater PAS density (p < 0.001). In multivariate regression, retinal detachment (RD), presence of VM, and PAS density ≥ median were independent predictors of a shadow, but not tumor distance to the macula, tumor apical thickness, tumor diameter, or ciliary body engagement. The presence of VM was associated with significantly shorter cumulative disease-specific survival (Wilcoxon p = 0.04), but not PAS density ≥ median, presenting symptoms or RD (p > 0.28).ConclusionTumors from uveal melanoma patients that report a visual field shadow are likely to display VM and greater PAS density, likely explaining the previously reported association between this symptom and poor prognosis.

An Update on Molecular Pathways Regulating Vasculogenic Mimicry in Human Osteosarcoma and Their Role in Canine Oncology.

Canine tumors are valuable comparative models for human counterparts, especially to explore novel biomarkers and to understand pathways and processes involved in metastasis. Vasculogenic mimicry (VM) is a unique property of malignant cancer cells which promote metastasis. Thus, it represents an opportunity to investigate both the molecular mechanisms and the therapeutic targets of a crucial phenotypic malignant switch. Although this biological process has been largely investigated in different human cancer types, including osteosarcoma, it is still largely unknown in veterinary pathology, where it has been mainly explored in canine mammary tumors. The presence of VM in human osteosarcoma is associated with poor clinical outcome, reduced patient survival, and increased risk of metastasis and it shares the main pathways involved in other type of human tumors. This review illustrates the main findings concerning the VM process in human osteosarcoma, search for the related current knowledge in canine pathology and oncology, and potential involvement of multiple pathways in VM formation, in order to provide a basis for future investigations on VM in canine tumors.

The effect of interleukin-17F on vasculogenic mimicry in oral tongue squamous cell carcinoma.

Oral tongue squamous cell carcinoma (OTSCC) is one of the most common cancers worldwide and is characterized by early metastasis and poor prognosis. Recently, we reported that extracellular interleukin‐17F (IL‐17F) correlates with better disease‐specific survival in OTSCC patients and has promising anticancer effects in vitro. Vasculogenic mimicry (VM) is the formation of an alternative vasculogenic system by aggressive tumor cells, which is implicated in treatment failure and poor survival of cancer patients. We sought to confirm the formation of VM in OTSCC and to investigate the effect of IL‐17F on VM formation. Here, we showed that highly invasive OTSCC cells (HSC‐3 and SAS) form tube‐like VM on Matrigel similar to those formed by human umbilical vein endothelial cells. Interestingly, the less invasive cells (SCC‐25) did not form any VM structures. Droplet‐digital PCR, FACS, and immunofluorescence staining revealed the presence of CD31 mRNA and protein in OTSCC cells. Additionally, in a mouse orthotopic model, HSC‐3 cells expressed VE‐cadherin (CD144) but lacked Von Willebrand Factor. We identified different patterns of VM structures in patient samples and in an orthotopic OTSCC mouse model. Similar to the effect produced by the antiangiogenic drug sorafenib, IL‐17F inhibited the formation of VM structures in vitro by HSC‐3 and reduced almost all VM‐related parameters. In conclusion, our findings indicate the presence of VM in OTSCC and the antitumorigenic effect of IL‐17F through its effect on the VM. Therefore, targeting IL‐17F or its regulatory pathways may lead to promising therapeutic strategies in patients with OTSCC.

HIF-1\u03b1 promoted vasculogenic mimicry formation in lung adenocarcinoma through NRP1 upregulation in the hypoxic tumor microenvironment

Neovascularization is a key factor that contributes to tumor metastasis, and vasculogenic mimicry (VM) is an important form of neovascularization found in highly invasive tumors, including lung cancer. Despite the increasing number of studies focusing on VM, the mechanisms underlying VM formation remain unclear. Herein, our study explored the role of the HIF-1α/NRP1 axis in mediating lung adenocarcinoma metastasis and VM formation. HIF-1α, NRP1 expression, and VM in lung adenocarcinoma (LUAD) patient samples were examined by immunohistochemical staining. Quantitative real-time (qRT-PCR), western blot, transwell assay, wound healing assay, and tube formation assay were performed to verify the role of HIF-1α/NRP1 axis in LUAD metastasis and VM formation. ChIP and luciferase reporter assay were used to confirm whether NRP1 is a direct target of HIF-1α. In LUAD tissues, we confirmed a positive relationship between HIF-1α and NRP1 expression. Importantly, high HIF-1α and NRP1 expression and the presence of VM were correlated with poor prognosis. We also found that HIF-1α could induce LUAD cell migration, invasion, and VM formation by regulating NRP1. Moreover, we demonstrated that HIF-1α can directly bind to the NRP1 promoter located between −2009 and −2017 of the promoter. Mechanistically, MMP2, VE-cadherin, and Vimentin expression were affected. HIF-1α plays an important role in inducing lung adenocarcinoma cell metastasis and VM formation via upregulation of NRP1. This study highlights the potential therapeutic value of targeting NRP1 for suppressing lung adenocarcinoma metastasis and progression.

Regulation Networks Driving Vasculogenic Mimicry in Solid Tumors.

Vasculogenic mimicry (VM) is a mechanism whereby cancer cells form microvascular structures similar to three-dimensional channels to provide nutrients and oxygen to tumors. Unlike angiogenesis, VM is characterized by the development of new patterned three-dimensional vascular-like structures independent of endothelial cells. This phenomenon has been observed in many types of highly aggressive solid tumors. The presence of VM has also been associated with increased resistance to chemotherapy, low survival, and poor prognosis. MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are non-coding RNAs that regulate gene expression at the post-transcriptional level through different pathways. In recent years, these tiny RNAs have been shown to be expressed aberrantly in different human malignancies, thus contributing to the hallmarks of cancer. In this context, miRNAs and lncRNAs can be excellent biomarkers for diagnosis, prognosis, and the prediction of response to therapy. In this review, we discuss the role that the tumor microenvironment and the epithelial-mesenchymal transition have in VM. We include an overview of the mechanisms of VM with examples of diverse types of tumors. Finally, we describe the regulation networks of lncRNAs-miRNAs and their clinical impact with the VM. Knowing the key genes that regulate and promote the development of VM in tumors with invasive, aggressive, and therapy-resistant phenotypes will facilitate the discovery of novel biomarker therapeutics against cancer as well as tools in the diagnosis and prognosis of patients.

Investigation of the Prognostic Significance of Vasculogenic Mimicry and Its Inhibition by Sorafenib in Canine Mammary Gland Tumors.

Canine mammary gland tumor (CMT) is one of the most important tumors in intact female dogs, and due its similarity to human breast cancer (BC), it is considered a model in comparative oncology. A subset of mammary gland tumors can show aggressive behavior, and a recurrent histological finding is the presence of vasculogenic mimicry (VM). VM is a process in which highly aggressive cancer cells fuse, forming fluid-conducting channels without endothelial cells. Although, VM has been described in canine inflammatory carcinoma, no previous studies have investigated the prognostic and predictive significance of VM in CMT. Thus, this research aimed to investigate the prognostic significance of VM in vivo and the capacity of sorafenib to inhibit VM in vitro. VM was identified in situ in formalin-fixed paraffin-embedded CMT samples (n = 248) using CD31/PAS double staining. VM was identified in 33% of tumors (82/248). The presence of VM was more strongly related to tumor grade than to histological subtype. Patients with positive VM experienced shorter survival times than dogs without VM (P < 0.0001). Due to the importance of the VEGF-A/VEGFR-2 autocrine feed-forward loop in epithelial tumors, we investigated the association between VEGF-A and VEGFR-2 expression by neoplastic tumor cells and the associations of VEGF-A or VEGFR-2 expression with VM. Among the VM-positive samples, all (n = 82) showed high scores (3 or 4) for VEGF-A and VEGFR-2, indicating that VM was a common finding in tumors overexpressing VEGF-A and VEGFR-2. Thus, we cultured two CMT primary cell lines with VM abilities (CM9 and CM60) in vitro and evaluated the anti-tumoral effect of sorafenib. The CM9 cell line showed a half maximal inhibitory concentration (IC50) of 2.61 μM, and the CM60 cell line showed an IC50 of 1.34 μM. We performed a VM assay in vitro and treated each cell line with an IC50 dose of sorafenib, which was able to inhibit VM in vitro. Overall, our results indicated that VM was a prognostic factor for dogs bearing CMT and that sorafenib had an inhibitory effect on VM in CMT cancer cells in vitro.

ALDH1 Expression and Vasculogenic Mimicry Are Positively Associated with Poor Prognosis in Patients with Breast Cancer

Background/Aims: This study aimed to explore the prognostic value of aldehyde dehydrogenase 1 (ALDH1) expression and vasculogenic mimicry (VM) in patients with breast cancer. Methods: ALDH1 expression and the presence of VM were examined by immunohistochemistry and CD31/PAS double staining, respectively, using formalin-fixed paraffin-embedded tissues from 202 breast cancer patients. The mean follow-up period ranged from 15 to 115 months. The Kaplan-Meier method was used to plot survival curves. Prognostic values were assessed by multivariate analysis using the Cox regression model. Results: ALDH1 expression was strongly associated with VM (P = 0.005). ALDH1 expression was positively correlated with histological grade (P = 0.011). Both ALDH1 expression and VM were negatively related to the status of the estrogen receptor and progesterone receptor and were statistically increased in triple-negative breast cancer. Patients with ALDH1 expression or VM displayed poorer disease-free survival (DFS) and overall survival (OS) than ALDH1-negative or VM-negative patients, with the worst OS and DFS observed in ALDH1/VM-double-positive patients. ALDH1-positive and VM-positive were independent survival risk factors for DFS and OS. Conclusion: ALDH1 expression and VM are correlated with the survival rate of patients with breast cancer. ALDH1 and VM, either alone or together, are prognostic factors in patients with breast cancer.

The Expression and Functional Significance of Vascular Endothelial-Cadherin, CD44, and Vimentin in Oral Squamous Cell Carcinoma.

Objectives:Ninety percent of head and neck cancers are squamous cell carcinoma which develops in the oral cavity. Metastasis is the main causative factor for death in 90% of all cancer-related deaths and begins with the invasion of tumor cells through the walls of small blood vessels or lymph vessels. A growing body of evidence has shown that vasculogenic mimicry (VM) facilitates tumor growth and cancer metastasis. The current study aimed to present the role of vascular endothelial (VE)-cadherin, CD44, and vimentin in inducing VM and epithelial-mesenchymal transition (EMT) and to identify the cancer stem cell (CSC) niche in different grades of oral squamous cell carcinoma (OSCC).Materials and Methods:A total of 63 OSCC samples (21 samples each grade) were collected from the archive of Pathology Department of Besat educational hospital, Hamadan, Iran, from 2000 to 2015. VE-cadherin, CD44, and vimentin/periodic acid–Schiff (PAS) double-staining were used to validate VM. VM was identified by the detection of PAS-positive loops surrounded by tumor cells. Chi-square test was used to examine the differences between the variables. Significant level was set at 0.05. Pearson's correlation was used to assess the co-localization of the markers.Results:There were statistically significant differences between tumor grade and the expression levels of VE-cadherin, CD44, and vimentin (P = 0.000). In addition, significant differences were found between tumor grade and microvessel density (P = 0.000) and between tumor grade and VM (P = 0.000).Conclusion:Our results may disclose a definite relationship between VE-cadherin, CD44 and vimentin expression levels, VM formation, EMT, CSCs, and microvessel count in OSCC samples. For this reason, it is suggested that VE-cadherin, CD44, and vimentin are related to angiogenesis and VM formation in OSCC, therefore, in tumor progression and metastasis. Recently, antitumor angiogenic therapies have been challenged. The presence of VM may explain the failure of antiangiogenic treatments.

Vasculogenic mimicry persists during glioblastoma xenograft growth

Background: Vasculogenic mimicry (VM) describes the functional plasticity of aggressive tumor cells to form newly recognized microcirculation, lined by tumor cells rather than endothelial cells, in solid tumors. The presence of VM in glioma is significantly associated with high tumor grade and poor prognosis. However, whether VM is a regular feature or only a temporary phenomenon during glioma growth is unknown. This study was designed to observe VM during the growth of subcutaneous and orthotopic xenograft glioma in Balb/c nude mice. Methods: The human glioblastoma cell line (U87) was used as xenografts in Balb/c nude mice models. The xenografts were obtained at different stages of tumor growth, and evaluated for VM and endothelium-dependent vessels by dual staining for endothelial marker CD34 and periodic acid-Schiff (PAS). Results: It was found that the PAS-positive patterns which were identified as VM were persistent during tumor growth of both subcutaneous and orthotropic xenografts. Further analysis showed that the microvessel density (MVD) of endothelium-dependent vessels was positively correlated with the tumor size of subcutaneous xenograft (r = 0.406, P = 0.009), while no significant correlation was found between VM density (VMD) and the tumor size (r = 0.107, P = 0.512). Furthermore, VMD was negatively correlated with MVD (r = −0.404, P = 0.010). Conclusion: The study results revealed that both VM and endothelium-dependent vessels coexist persistently during glioblastoma xenograft growth, indicating that VM may complement microcirculation in gliomas.

AB052. ZEB1 promotes vasculogenic mimicry formation in prostate cancer is associated with epithelial-mesenchymal transition

BackgroundThis study investigated the role of ZEB1 in vasculogenic mimicry (VM) formation and the interplay between VM and epithelial-mesenchymal transition (EMT).MethodsNinety-two prostate cancer tissue specimens were stained by CD34 and periodic acid Schiff. Then, we stained ZEB1 protein in the consecutive sections. Moreover, prostate cancer cells were subjected to ZEB1 knockdown using ZEB1 siRNA and then to 3D culture assay. EMT related maker was also evaluated.ResultsThe data showed that the presence of VM and high ZEB1 expression were associated with higher Gleason score, TNM stage, and lymph node and distant metastases. ZEB1 knockdown reduced VM formation and the expression of EMT-related in prostate cancer cells.ConclusionsIn the current study was the first to reveal that ZEB1 played an important role in VM formation in prostate cancer ex vivo and in vitro. Mechanistically, this process may have a relationship with EMT.

HIF-1\u03b1 promoted vasculogenic mimicry formation in hepatocellular carcinoma through LOXL2 up-regulation in hypoxic tumor microenvironment

BackgroundThe incidence and mortality rates of hepatocellular carcinoma (HCC) have steadily increased in recent years. A hypoxic microenvironment is one of the most important characteristics of solid tumors which has been shown to promote tumor metastasis, epithelial-mesenchymal transition and angiogenesis. Epithelial-mesenchymal transition and vasculogenic mimicry have been regarded as crucial contributing factors to cancer progression. HIF-1α functions as a master transcriptional regulator in the adaptive response to hypoxia. Lysyl oxidases like 2 (LOXL2) is a member of the lysyl oxidase family, which main function is to catalyze the covalent cross-linkages of collagen and elastin in the extracellular matrix. Recent work has demonstrated that HIF-1α promotes the expression of LOXL2, which is believed to amplify tumor aggressiveness. LOXL2 has shown to promote metastasis and is correlated with poor prognosis in hepatocellular carcinoma. The purpose of our study is to explore the role of HIF-1α in progression and metastasis of hepatocellular carcinoma by promoting the expression of LOXL2 as well as the potential regulatory mechanism.MethodsHIF-1α, LOXL2 expression and CD31/periodic acid-Schiff double staining in HCC patient samples were examined by immunohistochemical staining. shRNA plasmids against HIF-1α was used to determine whether LOXL2 been increased by HIF-1α. We monitored a series of rescue assays to demonstrate our hypothesis that LOXL2 is required and sufficient for HIF-1α induced EMT and VM formation, which mediates cellular transformation and takes effect in cellular invasion. Then we performed GeneChip® Human Transcriptome Array (HTA) 2.0 in HepG2 cells, HepG2 cells overexpressed LOXL2 and HepG2 cells treated with CoCl2.ResultsIn clinical HCC tissues, it confirmed a positive relationship between HIF-1α and LOXL2 protein. Importantly, HIF-1α and LOXL2 high expression and the presence of vasculogenic mimicry were correlated to poor prognosis. HIF-1α was found to induce EMT, HCC cell migration, invasion and VM formation by regulating LOXL2. The results of microarray assays were analyzed.ConclusionHIF-1α plays an important role in the development of HCC by promoting HCC metastasis, EMT and VM through up-regulating LOXL2. This study highlights the potential therapeutic value of targeting LOXL2 for suppression of HCC metastasis and progression.

Role of X‑linked inhibitor of apoptosis‑associated factor‑1 in vasculogenic mimicry in ovarian cancer.

X-linked inhibitor of apoptosis‑associated factor1 (XAF1) was identified as a novel X-linked inhibitor of apoptosis (XIAP) binding partner that may reverse the anti‑apoptotic effect of XIAP. Previous studies have revealed that XAF1 serves an important role in cancer angiogenesis. Vasculogenic mimicry (VM) describes the formation of fluid‑conducting channels by highly invasive and genetically dysregulated tumor cells. VM is critical for tumor blood supply and is associated with aggressive actions and metastasis. The aim of present study was to investigate the potential association between XAF1 expression with VM of ovarian cancer, and evaluate the role of XAF1 in tumor cell migration and invasion of SKOV3 cells. VM structure and XAF1 expression were detected in 94 tissue samples of advanced epithelial ovarian cancer (EOC). Invasion and migration of the SKOV3 human ovarian carcinoma cell line were identified by Transwell assay. It was revealed that the presence of VM was associated with high grade advanced ovarian cancer. Reduced XAF1 expression was significantly associated with presence of VM. Overexpression of XAF1 significantly reduced invasion and migration of SKOV3 cells, and inhibited vascular endothelial growth factor protein expression. Furthermore, vasculature was suppressed by overexpression of XAF1 invivo in xenograft models. In conclusion, XAF1 expression was associated with VM in ovarian cancer, suggesting a potential role of XAF1 in the formation of VM in EOC. These findings may facilitate the development of novel therapeutic agents for the treatment of ovarian cancer.

Expression von EphA2 in metastasierten und nicht metastasierten primären uvealen Melanomen

Little is known about how the expression of Ephrin type-A receptor 2 (EphA2) influences cell-cell adhesion, migration, angiogenesis, and the formation of vasculogenic mimicry (VM) channels in uveal melanomas or how this may be related to the rate of metastasis. Paraffin embedded sections of 50 histopathologically well characterised primary uveal melanomas (mean largest tumour diameter: 16.3 mm) were evaluated with respect to the expression of EphA2. Systemic metastasis was detected in 29 patients. The remaining 21 patients were followed for a mean of 10 years. Tumour angiogensis was analysed by endoglin expression (CD105), the activity of the mature vascular system (von Willebrand factor) and the presence of VM (CD31/PAS staining). All uveal melanomas expressed EphA2, with a mean of 95.93 % positive cells ± SD: 6.3 %. There was no significant association between EphA2 and the rate of metastases (p = 0.196), endoglin expression (p = 0.652), VM (p = 0.267) or with any other clinical or histopathological factors (p < 0.05). However, there was significant up-regulation of EphA2 in the nucleus of the metastatic uveal melanoma subgroup, while cytoplasmatic localisation in the subgroup was associated with better prognosis (p = 0.006). There were low levels of EphA2 expression in the specific retinal layers, the ciliary and corneal epithelium, and the choroidal and corneal endothelium. Nuclear expression of EphA2 in this series of large tumours was significantly associated with an increased rate of metastasis. On the other hand, cytoplasmic localisation was associated with a better prognosis. As there was no correlation between EphA2 expression and angiogenesis, the mature vasculature or VM, EphA2 appears to become less important in the advanced stages of the disease.

Vasculogenic Mimicry in Prostate Cancer: The Roles of EphA2 and PI3K.

BACKGROUND. Aggressive tumor cells can form perfusable networks that mimic normal vasculature and enhance tumor growth and metastasis. A number of molecular players have been implicated in such vasculogenic mimicry, among them the receptor tyrosine kinase EphA2, which is aberrantly expressed in aggressive tumors. Here we study the role and regulation of EphA2 in vasculogenic mimicry in prostate cancer where this phenomenon is still poorly understood.METHODS. Vasculogenic mimicry was characterized by tubules whose cellular lining was negative for the endothelial cell marker CD34 but positive for periodic acid-Schiff staining, and/or contained red blood cells. Vasculogenic mimicry was assessed in 92 clinical samples of prostate cancer and analyzed in more detail in three prostate cancer cell lines kept in three-dimensional culture. Tissue samples and cell lines were also assessed for total and phosphorylated levels of EphA2 and its potential regulator, Phosphoinositide 3-Kinase (PI3K). In addition, the role of EphA2 in vasculogenic mimicry and in cell migration and invasion were investigated by manipulating the levels of EphA2 through specific siRNAs. Furthermore, the role of PI3K in vasculogenic mimicry and in regulating EphA2 was tested by application of an inhibitor, LY294002.RESULTS. Immunohistochemistry of prostate cancers showed a significant correlation between vasculogenic mimicry and high expression levels of EphA2, high Gleason scores, advanced TNM stage, and the presence of lymph node and distant metastases. Likewise, two prostate cancer cell lines (PC3 and DU-145) formed vasculogenic networks on Matrigel and expressed high EphA2 levels, while one line (LNCaP) showed no vasculogenic networks and lower EphA2 levels. Specific silencing of EphA2 in PC3 and DU-145 cells decreased vasculogenic mimicry as well as cell migration and invasion. Furthermore, high expression levels of PI3K and EphA2 phosphorylation at Ser897 significantly correlated with the presence of vasculogenic mimicry and in vitro inhibition of PI3K by LY294002 disrupted vasculogenic mimicry, potentially through a reduction of EphA2 phosphorylation at Ser897.CONCLUSIONS. The expression levels of PI3K and EphA2 are positively correlated with vasculogenic mimicry both in vivo and in vitro. Moreover, phosphorylation levels of EphA2 regulated by PI3K are also significantly associated with vasculogenic mimicry in vivo. Based on its functional implication in vasculogenic mimicry in vitro, EphA2 signaling may be a potential therapeutic target in advanced prostate cancer.