Establishment Of A Stem-like Cancer Cell Line From Glioblastoma And The Role Of CD40 Signaling On Its Biological Characteristics

Glioma, especially high-grade glioblastoma mutiforme (GBM), is the most common and highly aggressive primary brain tumors. Despite intensive multimodal treatment combining surgery, chemotherapy and radiation, the outcome of patients remains remain dismal. Recent studies have demonstrated the existence of a small subpopulation of cells with stem-like features called cancer stem cells (CSC) that are self renewal, highly tumorigenic and chemo-radio resistant and therefore the concept of CSC may provide explanations for the failure to induce long-term remission since traditional therapies act only on differentiated tumor cells but not CSCs. However, the biological features of CSC and its interaction with tumor microenvironment are rarely understood. So, there is an urgent need for new and clinically relevant in vitro model for studying CSC biology and the crosstalk with tumor microenvironment to conduct preclinical screening of drugs for malignant brain tumors. In our previous study, it was shown that CD40 expression has a major impact on tumor growth, invasion and glioma angiogenesis indicating that over-expression of CD40 plays a significant role in tumorigensis. To investigate the relations between CD40 signaling and CSC, we isolated stem-like cancer cells from a GBM patient and explore tumor microenvironment especially CD40 and its downstream signals on the biofunctions of CSC.1. Establishment of a stem-like cancer cell line from glioblastomaWe have isolated glioma stem cells from surgical specimen of a high-grade glioma patient and established a stable stem-like cancer cell line which was named G1335. Then, we further identified its stemness by growth and proliferation features, cancer stem cell markers, chromosomes, invasiveness, chemoresistance and tumorigenicity. It was demonstrated that the cell line contain a subpopulation of cells that can form neurospheres in defined stem cell medium with growth factors. The cells share many characteristics of stem cells, including expression neural stem cell marker nestin, glioma stem cell marker CD133 and differentiated glioma cell marker GFAP, showing self-renewal and multipotent differentiation ability. Compared with previously-established cell lines, G1335 has obviously highly chemoresistance and great tumorigenicity. It is a typical stem-like cancer cell line that could be a potential tool for glioma stem cell research.2. The role of tumor microenvironment factors on enhancement of CD40 expression of G1335The tumor microenvironment is being increasingly recognized as an important determinant of tumor progression as well as of therapeutic response. In this section, we investigated the role of some typical tumor microenvironment factors such as hypoxia, cytokines such as IFN-γ、TNF-α、IL-6 on the expression of CD40 on G1335. The results showed that hypoxia could effectively upregulate IL-6 receptor gp130 expression on the membrane of G1335. IFN-γ、TNF-α、IL-6 could upregulate the expression of CD40 and hypoxia and IL-6 showed synergic effects in upexpression of CD40. Therefore, it was speculated that tumor microenvironment plays an very important role in tumorigensis. Hypoxia environment and cytokines stimulation could upregulate CD40 expression, forming CD40-IL-6/gp130 positive feedback loop which eventually give tumor cell constant and stronger CD40 signaling for tumor angiogenesis and invasive growth.3. The role of CD40 signaling on the biological characteristics of G1335 and the underlying mechanismsIn this section, the relationship between CD40 and VEGF, IL-6, CXCR4 as well as their contributions to neovascularization and glioma stem cell invasive growth under tumor microenvironment were investigated.By FCM, ELISA and Transwell analysis, it was proved CD40 signaling could stimulate VEGF and IL-6 secretion which are important for tumor angiogenesis and growth. IL-6 is not only able to promote neurosphere formation and proliferation, but also elevate CXCR4 expression on the membrane of G1335. CXCR4 is essential for chemotaxis of cancer stem cell metastatic spreading to areas where SDF-1αis highly concentrated. SDF-1α/CXCR4 axis is known to favor cancer stem cell survival and growth. Besides, some inhibitory components such as co-stimulatory molecules TGF-β、PD-L1 could be upexpressed with the ligation of sCD40L which may contribute to immunoescape of cancer stem cells. Through comparison, we found considered difference on cell growth between groups with or without sCD40L stimulation. And by in vivo study, cells pretreated with sCD40L could evidently promote tumor growth and vascularization when subcutaneously injected into nude mice. Immunohistochemical staining showed that the vessel density of the tumor stimulated by sCD40L was much higher than that of control groups, suggesting over expression of CD40 might be involved in tumorigenesis.Taken together, under tumor microenvironment, CD40 and VEGF, IL-6, CXCR4 have close relation with angiogenesis and invasion of cancer stem cells, providing new target molecules and strategies for immunotherapy of cancer stem cells.