Triggering Of TLR2Expressed By Glioma Stem Cells Nhances Invasion And Migration

Gliomas are the most common central nervous system (CNS) malignant primarytumors which account for40-60%. The most significant biological characteristic of gliomasis their local invasiveness. Current therapy for gliomas mainly includes maximal saferesection of the tumor mass followed by radiotherapy and chemotherapy. But thesetreatments can hardly prevent tumor recurrences in the long run. The fundamental cause offailure of clinical treatments is incomplete resection of gliomas because of its localinvasiveness. Therefore, it is of great importance and necessary to explore the biologicalmechanisms of gliomas’ local invasivenessPrevious studies on Toll-like receptors (TLRs) mainly focused on their roles ofmediating innate immunity and acquired immunity. In recent years, it has been discoveredthat TLRs expression is not restricted to immune cells but also can be found in tumortissues and a variety of tumor cell lines and they play an important role in development andprogression of tumors. Tumor progression is a complex interaction process between tumorsand hosts,among which proliferation, apoptosis, mobility and relase of proteolytic enzymeof cancer cells are all important parts of research.TLRs which were initially found in Drosophila are conserved sequences preserved inevolution of species. TLRs are referred to as pathogen-associated molecular patterns(PAMPs). TLRs are type Ⅰtransmembrane proteins with ectodomains containingleucine-rich repeats and Toll/interleukin1(IL-1) receptor (TIR) domains. So far,10and12funtional TLRs have been discovered in human and mice. TLR1-TLR9are conserved inboth human and mice. Mouse TLR10is loss of its function due to retroviral insertion andTLR11, TLR12and TLR13have been lost from human genome. TLRs were discoveredmainly expressed by macrophages and dendritic cells (DCs). However, recent studies havedemonstrated that the expression of TLRs is not restricted to immune cells, but also can befound in many tumors which play a significant role in tumor progression.The hypothesis of cancer stem cells (CSCs) which was proposed in recent years supposes that a small population of cells within tumors with stem cell-like properties areresponsible for tumorigenesis and giving rise to a diversity of more differentiated cells.However, expression of TLRs in CSCs and its role in tumor progression have not beenreported.It has been reported that TLRs are expressed by gliomas, but the role of TLRsexpressed by gliomas and its mechanisms need to be further explored. In order to determineexpression of TLRs in gliomas and to explore whether TLRs play a role in glioma invasionand migration, we first detected the expression of TLRs in mouse glioma cell line GL261and then we determined TLR2may be a significant potential factor that can effect theinvasive and migration capacity of glioma cells. TLR2activation of glioma cells bysynthetic TLR2ligand Pam3CSK4modulated MMP2and MMP9expression at mRNAlevel. We also observed the role of TLR2on survival periods of glioma-bearing mice andthe invasiveness of cancer cells to the adjacent normal tissue. The main results andconclusions are as follows:1. Expression of TLRs in GL261was detected by quantity PCR analysis and gliomastem cells (GSCs) showed an enhancement of invasive and migration ability and TLR2expression was much higher in GSCs at mRNA and protein levels compared to GL261.Quantitative PCR analysis revealed that GL261expressed large quantity of TLR2, but littleor no TLR1,TLR5,TLR7or TLR11at mRNA levels. In order to investigate whether theexpression of TLR2is different between GSCs and GL261, we isolated GSCs throughneurosphere culture in serum-free media which was regarded as a classic approach to enrichGSCs. Then immunofluorescence analysis was conducted to identify their characteristics ofcancer stem cells including expressions of Nestin,Oligo2and Sox2which were generallyaccepted as GSCs markers. As expected, expressions of Nestin,Oligo2and Sox2by sphereswere positively. So we esteemed these spheres as GSCs and used them to perform thefollowing experiments. Compared to GL261, the expression of TLR2in GSCs was muchhigher at mRNA and protein levels confirmed by quantitative PCR and Western Blotting.Transwell invasion and migration assays exhibited that GSCs exhibited significantincreasement of invasive and migration activity by1.55and1.68fold (P <0.05).2. Stimulation TLR2with TLR2ligand Pam3CSK4enhanced invasive and migrationactivity of GSCs. Possible mechanism for this phenomenon was supposed that up-regulation expression of MMP2and MMP9. Transwell invasion and migration assaysrevealed that significant enhancements of invasive and migration activities by2.23and2.28fold (P <0.05) after48h1μg/ml Pam3CSK4treatment. After silencing TLR2efficientlyconfirmed by quantitative PCR analysis and Western Blotting, invasive and migrationcapacities of GSCs were not increased in spite of Pam3CSK4treatment. TLR2activationcould up-regulate expression of MMP2and MMP9.4hours and6hours later afterPam3CSK4stimulation, MMP2and MMP9expression of GSCs reached its peak detectedby quantitative PCR analysis. Similar effects were not appeared after silencing TLR2.3. The role of TLR2expression played in survivals of tumor-bearing mice and theinvasiveness. Survivals of glioma-bearing mice were markedly prolonged after silencingTLR2of GSCs. However, survival periods of glioma-bearing mice were not notablyextended after silencing TLR2of GL261. The invasiveness was decreased after TLR2silencing of GSCs confirmed by hematoxylin and eosin (H&E) staining of the brainsections. However, local invasiveness was similar after blocking TLR2.