Vasculogenic mimicry:a pivotal mechanism contributing to drug resistance in antiangiogenic therapy

The growth of solid tumors relies on establishing a robust blood supply,with angiogenesis playing a key role in this intricate process.Based on this understanding,therapeutic strategies targeting tumor angiogenesis have been developed.However,the clinical effectiveness of antiangiogenic therapy(AAT)in treating tumors has not lived up to expectations.In recent years,vasculogenic mimicry(VM)has attracted increasing attention from the academic community as a longstanding but often overlooked mechanism of nonangiogenic tumor vascularization.Within the tumor microenvironment,neoplastic cells can autonomously form vessel-like structures,creating a blood supply that does not rely on endothelial cells.This phenomenon,known as VM,is a critical marker of aggressive tumors and may play a significant role in conferring resistance to AAT.In this review,we thoroughly examine the evidence,clinical characteristics,and mechanisms of VM across various tumor types and explore its potential role and importance in resistance to AAT and the development of new antitumor therapies.

Vasculogenic Mimicry and Aberrant Expression of HIF-lα/E-cad Are Associated with Worse Prognosis of Esophageal Squamous Cell Carcinoma

Summary： This study aims to find good markers for predicting the prognosis of patients with eso- phageal squamous cell carcinoma （ESCC）. Vasculogenic mimicry （VM） and the expression of hy- poxia inducible factor-1α（HIF-1α）/E-cad protein in ESCC were investigated by immunostaining. The association between VM, HIF-1α/E-cad and clinicopathologic characteristics and 5-year-survival rate of patients with ESCC was analyzed. A total of 160 ESCC specimens were involved in this study and 28 specimens of normal esophageal mucosa served as controls. VM channels were identified in 78 （48.75%） of the 160 ESCC specimens and none of the normal esophageal mucosa was found to have VM. The rates of high-expression of HIF-1αand E-cad in ESCC were 43.75% and 38.75%, while the rates in control were 17.86% and 71.43%, respectively （P〈0.05 for all）. VM and the expression levels of HIF-1α and E-cad were significantly related to lymph node metastasis, serosa infiltration, PTNM staging and 5-year-survival rates of patients with ESCC （P〈0.05 for all）. VM was positively corre- lated with HIF-1α but negatively with E-cad, and HIF-let was negatively correlated with E-cad （P〈0.001 for all）. The 5-year-survival rate of patients with ESCC was 6.41% （5/78） in VM group and 65% （52/82） in non-VM group, 7.14% （5/70） in high HIF-1α expression group and 57.78% （52/90） in low HIF-1α expression group. Oppositely, the 5-year-survival rate in high E-cad expression group was 80.65% （50/62） and that in low E-cad expression group was 7.37% （7/98） （P〈0.05 for all）. Cox multifactor regression analysis indicated that lymph node metastasis, PTNM stage, VM and expres- sion levels of HIF-1α and E-cad were independent risk factors of patients with ESCC （P〈0.05 for all）. Combined detection ofVM, HIF-1α and E-cad plays an important role in predicting the invasion, me- tastasis and prognosis of patients with ESCC.

Notch4 Inhibition Suppresses Invasion and Vasculogenic Mimicry Formation of Hepatocellular Carcinoma Cells

Vasculogenic mimicry（VM） is a process by which aggressive tumor cells generate non-endothelial cell-lined channels in malignant tumors including hepatocellular carcinoma（HCC）. It has provided new insights into tumor behavior and has surfaced as a potential target for drug therapy. The molecular events underlying the process of VM formation are still poorly understood. In this study, we attempted to elucidate the relationship between Notch4 and VM formation in HCC. An effective si RNA lentiviral vector targeting Notch4 was constructed and transfected into Bel7402, a HCC cell line. VM networks were observed with a microscope in a 3 dimensional cell culture system. Cell migration and invasion were evaluated using wound healing and transwell assays. Matrix metalloproteinases（MMPs） activity was detected by gelatin zymography. Furthermore, the role of Notch4 inhibition in Bel7402 cells in vivo was examined in subcutaneous xenograft tumor model of mice. The results showed that downregulation of Notch4 destroyed VM network formation and inhibited migration and invasion of tumor cells in vitro（P〈0.05）. In vivo, tumor growth was also inhibited in subcutaneous xenograft model（P〈0.05）. The potential mechanisms might be related with down-regulation of MT1-MMP, MMP-2, MMP-9 expression and inhibition of the activation of MMP2 and MMP9. These results indicated that Notch4 may play an important role in VM formation and tumor invasion in HCC. Related molecular pathways may be used as novel therapeutic targets for HCC antiangiogenesis therapy.

Expression of Maspin in Non-small Cell Lung Cancer and Its Relationship to Vasculogenic Mimicry

Maspin belongs to the serine protease inhibitor （serpin） family and has been proven to be a suppressor of tumor growth and metastasis in many types of tumors. The purpose of this study was to investigate the expression of maspin in non-small cell lung cancer （NSCLC） and its relationship to vasculogenic mimicry （VM）. A total of 160 specimens of NSCLC were involved in this study and 20 specimens of normal lung tissue served as controls. VM, microvessel density （MVD） and the expression of maspin were detected by using immunohistochemical staining. The results showed that the positive rates of maspin and VM in the NSCLC group were 48.1% （77/160） and 36.9% （59/160）, respectively, which were significantly different from those in the control group with the positive rates of maspin and VM being 100% and 0% respectively （P〈0.05）. VM, MVD and the expression level of maspin were significantly related to tumor differentiation, lymph node metastasis, clinical stages and postoperative survival time （all P〈0.05）. The maspin expression in patients with squamous cell carcinoma was significantly higher than that in those with adenocarcinoma （P〈0.05）. The maspin expression was negatively correlated with VM and MVD, and there was a positive correlation between VM and MVD. Maspin-negative expression, VM and high MVD score were negatively related to the 5-year-survival rate. PTNM stages, VM, MVD and maspin expression were independent prognostic factors for NSCLC （P〈0.05）. It was suggested that the loss of expression of maspin may participate in the invasion and metastasis of NSCLC and it has a positive relationship to VM in NSCLC. Combined detection of maspin, VM and MVD may help predict the progression and prognosis of NSCLC.

Downregulation of Astrocyte Elevated Gene-1 Expression Combined with All-Trans Retinoic Acid Inhibits Development of Vasculogenic Mimicry and Angiogenesis in Glioma

Objective This study aimed to investigate the effects of downregulating astrocyte elevated gene-1(AEG-1)expression combined with all-trans retinoic acid(ATRA)on vasculogenic mimicry(VM)formation and angiogenesis in glioma.Methods U87 glioma cells were transfected with AEG-1 shRNA lentiviral vectors(U87-siAEG-1)and incubated in a medium containing 20µmol/L ATRA.Matrigel-based tube formation assay was performed to evaluate VM formation,and the cell counting kit-8(CCK-8)assay was used to analyze the proliferation of glioma cells in vitro.Reverse transcription-quantitative polymerase chain reaction and Western blot analysis were used to investigate the mRNA and protein expression of related genes,respectively.Glioma xenograft models were generated via subcutaneous implantation of glioma cells in nude mice.Tumor-bearing mice received an intraperitoneal injection of ATRA(10 mg/kg per day).Immunohistochemistry was used to evaluate the expression of related genes and the microvessel density(MVD)in glioma xenograft models.CD34/periodic acid-Schiff double staining was performed to detect VM channels in vivo.The volume and weight of tumors were measured,and a tumor growth curve was drawn to evaluate tumor growth.Results A combination of ATRA intervention and downregulation of AEG-1 expression significantly inhibited the proliferation of glioma cells in vitro and glioma VM formation in vitro and in vivo.It also significantly decreased MVD and inhibited tumor growth.Further,the expression levels of matrix metalloproteinase(MMP)-2,MMP-9,vascular endothelial-cadherin(VE-cadherin),and vascular endothelial growth factor(VEGF)in glioma significantly decreased in vivo and in vivo.Conclusion Hence,a combinatorial approach might be effective in treating glioma through regulating MMP-2,MMP-9,VEGF,and VE-cadherin expression.

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.

Effects of Vasculogenic Mimicry on Postoperative Recurrence and Progression of Non-Small Cell Lung Cancer

Vasculogenic mimicry(VM)in lung cancer shortens overall survival(OS)but its'associations with postoperative recurrence and progression of early non-small cell lung cancer(NSCLC)remain unclear.The purpose of this study was to analyze the association of VM with postoperative recurrence and progression of NSCLC as well as the effect of VM on postoperative recurrence-free survival(RFS).This study included NSCLC patients and detected VM in surgical specimens.The associations ofVM with the recurrence and progression were analyzed to assess the effect ofVM on postoperative RFS in NSCLC.A total of 80 NSCLC cases were followed up for 3 years.During follow-up,35 cases showed recurrence and progression where 5(6.25%)cases had simple local recurrence and the other 30(37.5%)cases had distant metastasis.The recurrence and progression rates in the first,second,and third years were 12.50%,23.75%,and 7.50%,respectively.The median RFS was 14.2 months.VM was detected in 30 out of 80 cases and was significantly correlated with tumor differentiation(r=0.365)and clinical stage(r=0.374)(both,P=0.001).Local recurrence of NSCLC was not correlated with VM,unlike distant metastasis(r=0.598,P<0.001).Average RFS was significantly longer in NSCLC patients without VM compared with the VM group 3 years post-operation(32 months versus 18 months,log-rank test P<0.001).Considering these,VM is significantly correlated with postoperative distant metastasis of NSCLC in which it is of a certain value for predicting poor prognosis in NSCLC.

DPY19L3 promotes vasculogenicmimicry by its C-mannosyltransferase activity

C-mannosylation is a post-translational modification that occurs intracellularly in the endoplasmic reticulum.In humans,biosynthesis of C-mannosylation in proteins containing thrombospondin type 1 repeat is catalyzed by the DPY19 family;nonetheless,biological functions of protein C-mannosylation are not yet fully understood,especially in tumor progression.Vasculogenic mimicry(VM)is the formation of fluid-conducting channels by highly invasive and genetically deregulated tumor cells,enabling the tumors to form matrix-embedded vasculogenic structures,containing plasma and blood cells to meet the metabolic demands of rapidly growing tumors.In this study,we focused on DPY19L3,a C-mannosyltransferase,and aimed to unravel its role in VM.Knockout of DPY19L3 inhibited the formation of VM in HT1080 human fibrosarcoma cells.Re-expression of wild-type DPY19L3 recovered VM formation;however,DPY19L3 isoform2,an enzymatic activity-defect mutant,did not restore it,suggesting that the C-mannosyltransferase activity of DPY19L3 is crucial to its function.Furthermore,the knockdown of DPY19L3 in MDA-MB-231 breast cancer cells hindered its network formation ability.Altogether,our findings suggest that DPY19L3 is required for VM formation and stipulate the relevance of C-mannosylation in oncogenesis.

Contribution of cancer stem cells to tumor vasculogenic mimicry

Vasculogenic mimicry(VM),a newly-defined pattern of tumor blood supply,provides a special passage without endothelial cells and is conspicuously different from angiogenesis and vasculogenesis.The biological features of the tumor cells that form VM remain unknown.Cancer stem cells(CSCs)are believed to be tumorinitiating cells,capable of self-renewal and multipotent differentiation,which resemble normal stem cells in phenotype and function.Recently CSCs have been shown to contribute to VM formation as well as angiogenesis.These findings challenge the previous understanding of the cellular basis of VM formation.In this review,we present evidence for participation of CSCs in VM formation.We also discuss the potential mechanisms and possible interaction of CSCs with various elements in tumor microenvironment niche.Based on the importance of VM in tumor progression,it constitutes a novel therapeutic target for cancer.

Vasculogenic mimicry is a key prognostic factor for laryngeal squamous cell carcinoma： a new pattern of blood supply

Background Recurrence and local lymph node metastasis affected the prognosis of patients with laryngeal cancer. The aim of this study was to analyze clinical and pathological significance of vasculogenic mimicry （VM） in laryngeal squamous cell carcinoma （LSCC） and evaluate its contribution to prognosis. Methods Data of 168 cases of LSCC were reviewed retrospectively to reveal clinical pathology and prognostic significance of VM. CD31 and periodic acid-Schiff double staining was used to identify VM. Results VM in LSCC contributed to lymph node metastasis （P=0.003） and clinical progression. VM correlated to histopathology grade （P=0.001） of LSCC. VM was an adverse prognostic factor for both disease-specific survival （P=0.039） and metastasis-free survival （P=0.042） by univariate survival analyses. And it was an independent prognostic factor for only disease-specific survival （P=0.003） by multivariate survival analyses. Conclusions VM existed in LSCC. LSCC with VM has more Dotential to invasion and metastasis.

Angioarchitecture and CD133^+ tumor stem cell distribution in intracranial hemangiopericytoma A comparative study with meningioma

Angioarchitecture plays an important role in the malignant development of intracranial hemangiopericytoma. It remains poorly understood whether high frequency of hemorrhage during clinical surgery for intracranial hemangiopericytoma is associated with angioarchitecture. The present study utilized hematoxylin-eosin staining, and immunohistochemical staining with epithelial membrane antigen, vimentin, CD34, von Willebrand factor （vWF） and CD133 to observe characteristics of angioarchitecture. In addition, silver stains were used to demonstrate changes in reticular fibers in the wall of vessel channels in intracranial hemangiopericytoma and meningioma. Five patterns of angioarchitecture were identified in intracranial hemangiopericytoma, namely tumor cell islands, vasculogenic mimicry, mosaic blood vessels, sprouting angiogenesis, and intussusceptive angiogenesis. Several CD133＋ tumor cells were found to form tumor cell islands. A connection between vWF ^＋ and vWF channels was detected in the pattern of intussusceptive angiogenesis, and some vimentin^＋ tumor cells were embedded in the periodic acid-Schiff positive channel wall. Incomplete threads of reticular fibers formed the walls of larger pseudo-vascular channels and some tumor clumps or scattered tumor cells were detected ＂floating＂ in them. The angioarchitecture, specific markers and reticular fibers of intracranial hemangiopericytoma were significantly different from meningioma. Angioarchitecture provides a functional vascular network for vascular evolution in intracranial hemangiopericytoma and contributes to significant intra-operative bleeding.

Exosomes derived from pulmonary metastatic sites enhance osteosarcoma lung metastasis by transferring the miR-194/215 cluster targeting MARCKS

Osteosarcoma,a prevalent primary malignant bone tumor,often presents with lung metastases,severely impacting patient survival rates.Extracellular vesicles,particularly exosomes,play a pivotal role in the formation and progression of osteosarcoma-related pulmonary lesions.However,the communication between primary osteosarcoma and exosome-mediated pulmonary lesions remains obscure,with the potential impact of pulmonary metastatic foci on osteosarcoma progression largely unknown.This study unveils an innovative mechanism by which exosomes originating from osteosarcoma pulmonary metastatic sites transport the miR-194/215 cluster to the primary tumor site.This transportation enhances lung metastatic capability by downregulating myristoylated alanine-rich C-kinase substrate(MARCKS)expression.Addressing this phenomenon,in this study we employ cationic bovine serum albumin(CBSA)to form nanoparticles(CBSA-anta-194/215)via electrostatic interaction with antagomir-miR-194/215.These nanoparticles are loaded into nucleic acid-depleted exosomal membrane vesicles(anta-194/215@Exo)targeting osteosarcoma lung metastatic sites.Intervention with bioengineered exosome mimetics(anta-194/215@Exo)not only impedes osteosarcoma progression but also significantly prolongs the lifespan of tumor-bearing mice.These findings suggest that pulmonary metastatic foci-derived exosomes initiate primary osteosarcoma lung metastasis by transferring the miR-194/215 cluster targeting MARCKS,making the miR-194/215 cluster a promising therapeutic target for inhibiting the progression of patients with osteosarcoma lung metastases.

Association between glioblastoma cell-derived vessels and poor prognosis of the patients

Background:Vessels with different microcirculation patterns are required for glioblastoma(GBM)growth.However,details of the microcirculation patterns in GBM remain unclear.Here,we examined the microcirculation patterns of GBM and analyzed their roles in patient prognosis together with two well-known GMB prognosis factors(O^(6)-methylguanine DNA methyltransferase[MGMT]promoter methylation status and isocitrate dehydrogenase[IDH]mutations).Methods:Eighty GBM clinical specimens were collected from patients diagnosed between January 2000 and December 2012.The microcirculation patterns,including endothelium-dependent vessels(EDVs),extracellular matrix-dependent vessels(ECMDVs),GBM cell-derived vessels(GDVs),and mosaic vessels(MVs),were evaluated by immunohistochemistry(IHC)and immunofluorescence(IF)staining in both GBM clinical specimens and xenograft tissues.Vascular density assessments and three-dimensional reconstruction were performed.MGMT promoter methylation status was determined by methylation-specific PCR,and IDH1/2 mutations were detected by Sanger sequencing.The relationship between the microcirculation patterns and patient prognosis was analyzed by Kaplan-Meier method.Results:All 4 microcirculation patterns were observed in both GBM clinical specimens and xenograft tissues.EDVs were detected in all tissue samples,while the other three patterns were observed in a small number of tissue samples(ECMDVs in 27.5%,GDVs in 43.8%,and MVs in 52.5%tissue samples).GDV-positive patients had a median survival of 9.56 months versus 13.60 months for GDV-negative patients(P=0.015).In MGMT promoter-methylated cohort,GDV-positive patients had a median survival of 6.76 months versus 14.23 months for GDV-negative patients(P=0.022).Conclusion:GDVs might be a negative predictor for the survival of GBM patients,even in those with MGMT promoter methylation.