An Experimental Study Of Dendritic Cells Transfected With Cancer Stem Like Cells RNA Against 9L Brain Tumors

The most common malignant brain tumors are gliomas, representing 35.26～60.96%(average 44.69%) of all primary, malignant central nervous system tumors. Glioma is diffusely invasive, and highly infiltrative tumors spread into surrounding normal brain tissue. Because of this presentation, complete surgical resection of gliomas is not feasible. Despite advances in surgical techniques and adjuvant radiation and chemocherapies, the prognosis for patients with gliomas remains poor, with a median survival of 14.6 months. The traditional view of cancer believes that almost every cancer cell has the capacity to proliferate infinitely. In recent years, the concept that only a subpopulation of the cells in a cancer is endowed with the ability for self-renewal, extensive proliferation, multilineage differentiation and tumor initiation; meanwhile, the majority of cancer cells could not proliferate, or it can proliferate for a very short time. Such rare cancer cells were called "cancer stem cells". Up to now, cancer stem cells have been identified from many cancers including hematopietic malignancies, breast cancer and brain cancer et al.. Many studies suggest that stem-like cells in primary malignant brain tumors are called "brain tumor stem cells (BTSC)". Glioma is an organ composed of a heterogeneous combination of tumor cell types, every tumor cell in a glioma are different in their ability for proliferation, differentiation and sensitivity to anti-tumor drugs. Because most cancer stem cells reside in the GO phase of the cell cycle, additionally, cancer stem cells express higher levels of genes related to drug-resistance and anti-apoptosis, which make them resistant to traditional radiation and chemotherapy. Meanwhile, cancer stem cells have the ability for self-renewal, proliferation and differentiation, therefore, most of the tumor cells could be eradicated by traditional therapies, but these treatments may spare cancer stem cells, if cancer stem cells could not be eradicated, the tumor would recur sooner or later. Therefore brain tumor stem cells represented a critical target for cancer therapy, cancer may be cured by eradicating cancer stem cells, it is very necessary to develop novel therapies to eliminate cancer and cancer stem cells.For a long time, the central nervous system (CNS) has traditionally been regarded as an immunologically privileged site, as the blood-brain barried (BBB) had been thought to prevent components of the immune system from entering. However, many researches have demonstrated that lymphocytes could cross blood brain barried (BBB) and induce immunological reactions in the CNS under pathological conditions. Therefore, immune responses could be involved in the central nervous system diseases, which provide a theoretical basis for anti-tumor immunotherapy in the CNS.Dendtritic cells (DCs) are professional antigen presenting cells in vivo, which strong ingest and process antigen, and highly express major histocompatibility complex (MHC-Ⅰand MHC-Ⅱ) and costimulating molecule (CD80 and CD86). They can effectively present antigen to T cells and stimulate first immune response. There are several antigens to load DCs, such as dead tumor cells, cell lysates, apoptotic cells, purified protein, peptide, plasmid cDNA and nucleic acid (RNA and DNA) et al.. Because the presence of glioma heterogeneity, it is very difficult to get specific glioma antigens. The most appropriate host anti-tumor T cell response requires a series of epitopes, rather than confined to a particular epitope, therefore, it is a simple and effective method to prepare DC vaccination by loading DCs with the whole tumor cells or total RNA.In recent years, it has been reported that DC vaccination loaded with RNA encoding tumor-associated antigens or all tumor RNA could induce tumor-specific CTL against multiple epitopes. Up to now, DC vaccination loaded with tumor RNA has been successfully applied in many tumors including colon, rectum, prostate cancer, renal cell carcinoma, breast cancer, lung cancer and brain tumors. And it has been demonstrated that vaccination with DCs transfected with total RNA is more effective than other antigen-pulsed DCs.Compared with traditional vaccines, nucleic acid vaccine can induce the body's whole immune responses, the expression of peptide is more close to its natural conformation, and it can be modified by gene construction in order to increasing vaccine targets. The strategy of using RNA has several potential advantages. First, RNA can be isolated from a very small number of cells, and RNA can be effectively amplified byRT-PCR, therefore, unlike tumor-extract vaccines, a small amount of tumor tissue is sufficient to prepare the material for vaccination; Second, the identity of the tumor antigens and the human leukocyte antigen (HLA) of patient does not need to be known and the presence of multiple tumor antigens reduces the risk of antigen-negative escape mutants; Third, the half-life of RNA is approximately less than 24 hours, and RNA can only be expressed in cytoplasm; however, DNA must-enter the nucleus for transcription, non-integrated DNA can stay in cells for months, so unlike DNA-based vaccines, there is little danger of incorporation of RNA sequences into the host genome, which means DCs transfected with RNA has no risk of tumor. Finally, specific antigen of RNA could be amplified by PCR, which can induce antigen-specific CTL against tumor, meanwhile, it also reduce the risk of auto-immune diseases. It has been reported that no autoimmune responses have been detected in mice vaccinated with DCs transfected with total tumor RNA. Therefore, DCs transfected with total tumor RNA is a simple, feasible and safe method for tumor immunotherapy.Recently, with the development of research on cancer stem cells, several researchers began to prepare DC vaccination by loading antigens of cancer stem cells, they found that vaccination strategy with dendritic cells loaded with cancer stem cells is more effective than traditional vaccination. These findings suggested that DCs loaded with cancer stem cells may induce CTL that target cancer stem cells.Therefore, our strategy is based on the research background that DCs is the most powerful APCs in vivo and brain tumor stem cells play a key role in tumor initiation. Firstly,9L cancer stem-like cells were isolated from 9L gliosarcoma cell line by neurosphere assay, the basic characteristics of 9L cancer stem-like cells were investigated; Then, total tumor RNA from 9L tumorspheres (9LTS) and 9L monolayer cells were extracted, and DC vaccination with total RNA from 9LTS and 9L monolayer cells were prepared; Finally, we immunized 9L cells/F344 rats by DC vaccination, the effects of DC transfected with total tumor RNA of 9L cancer stem cells were observed.The study includes three chapters:ChapterⅠIsolation and characterization of cancer stem-like cells in rat 9L cell lineObjective:To establish a simplified culture system to isolate cancer stem-like cells from rat 9L cell line, observe their morphology, and investigate the expression marker of CD 133 and Nestin, their ability for differenitaion and tumorigenicity.Methods:Rat 9L gliomasarcoma cells were cultured in serum-containing medium (DMEM/F12+10%FBS) and serum-free medium (DMEM/F12+20ng/mL bFGF+ 20ng/mL EGF). Floating 9L tumor spheres were formed in serum-free medium. The morphology was observed by optic microscope, the immunophenotype and the ability for differentiation were tested by Immunocytochemistry. To investigate the tumorigenicity,9L tumor sphere cells and 9L monolayer cells were implanted into the right striatum of F344 rats, the survival were observed.Results:Rat 9L gliomasarcoma cells were cultured in serum-containing medium as adherent cells, they grow processes just like fibroblast, showing short spindle, long spindle-shape, star-shape and long fiber-shape.9L tumor spheres were formed in serum-free medium, these 9L tumor speres could be digested by 0.01% and passaged for at least 1 month. The majority of the 9L tumor sphere cells and 9L monolayer cells were positive for CD 133 and Nestin by immunocytochemistry and flow cytometry. When these 9L tumor spheres were transferred into serum-containing medium, they were positive for GFAP, NSE and Galc by immunocytochemistry.14 days after tumor cell inoculation, the tumor cell-implanted rat brains were removed. We found that the phenotype of implanted 9L monolayer cells were similar to that of 9L tumor spheres and most of the tumor cells derived from both 9L monolayer cells and 9L tumor spheres were positive for Nestin and CD 133 by immunohistochemistry. Tumors derived from 9L tumor spheres were much larger and showed diffuse infiltrating growth patterns, they were widely invaded the ipsilateral hemisphere. In contrast,9L monolayer cells gave rise to less invasive tumor masses. Kaplan-Meier analysis for survival was shown in Figure 4 and confirmed that 9L tumor spheres are significantly more aggressive than monolayer cells. The median survival of rats injected with 9L tumor spheres was 15 days, whereas that of rats injected with 9L monolayer cells was 21 days (P<0.001)Conclusion:9L tumor sphere cells possessed the ability of stem cells for proliferation, multilineage differentiation and tumor initiation,9L cancer stem like cells were enriched in tumor spheres by neurosphere assay. CD133 and Nestin could not be specific markers for cancer stem cells in 9L cells.9L cancer stem cells was just a part of CD 133+ cells.ChapterⅡIsolation and Culture of F344 rat's dendritic cellsObjective:To establish a method of isolating and culturing dendritic cells (DCs) from the bone marrow of F344 rats.Methods:Dendritic cells were obtained from the bone marrow of F344 rat's limbs by RPMI 1640 lavation were cultured in the RPMI 1640 in vitro under the condition of 40ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 40ng/ml interleukin-4 (IL-4). Tumor necrosis factor-alpha (TNF-α, 100ng/ml) was added into the medium in day 7 to stimulate its maturation. Flow cytometry was used to detect the expression of surface molecules (0X62, CD80 and CD86) on DCs on day 7 and day 10.Results:DCs could be isolated from inbed F344 bone marrow by GM-CSF and IL-4. DCs expressed surface antigens, including 0X62 63.12%, CD80 50.71%, CD86 47.36% on the 7th day; 0X62 96.45%, CD80 90.49%, CD86 92.55% on the 10th day. About 5×106 dendritic cells could be obtained from each F344 rats (male, weight 200g).Conclusion:The culture method of DCs from bone marrow of F344 rats has been successfully established, which provide cell source for DC immunotherapy against glioma. ChapterⅢDendritic cells transfected with cancer stem like cells RNA against 9L brain tumorsObjective:To investigate the anti-tumor efficacy of DCs vaccination transfected with total RNA of cancer stem cells and to discuss the mechanism of immune response, to provide experimental basis for clinical application.Methods:Dendritic cells were isolated from F344 bone marrow cells, then these dendritic cells were transfected with total RNA of 9L cancer stem cells or 9L monolayer cells. F344 rats bearing with 9L brain tumors were treated by subcutaneous injection of either PBS, unpulsed DCs, DCs transfected with 9L monolayer cells RNA (DC-9LTS) or DCs transfected with 9L tumor spheres RNA (DC-9L) on day 3,10,17.21 days after tumor implantation, brains and serum were obtained from different groups, lymphocytes infiltration was detected by immunohistochemistry, the concentration of IFN-γwas tested by ELISA. Survival time was observed and determined using the method of Kaplan Meier analysis.Results:survival time analysis:Rats vaccinated with DCs transfected with 9L tumor spheres RNA (DC-9LTS) and monolayer cells RNA (DC-9L) expired with median survival dates of 36 and 31 days, respectively. Animinals bearing intracranial 9L gliosarcoma were vaccinated with un-pulsed DCs vaccine all expired with a median survival of 21 days. Kaplan-Meier survival curve showed that rats treated with DC-9LTS had longer survival than other groups (P<0.01). there is significant difference among DC-9L group and DC group, PBS group (P<0.01). There is no significant difference between DC group and PBS group (χ2=0.071, P=0.789).Results of the concentration of IFN-γ:21 days after tumor implantation, serum were harvested for IFN-gamma assay. The concentration of IFN-gamma was 157.08±7.25pg/ml (DC-9LTS group),132.64±1.77pg/ml (DC-9L group), 96.43±4.06pg/ml (DC group),95.15±6.68pg/ml (PBS group) and 99.00±7.74pg/ml (control group). And the concentration of IFN-gamma of DC-9LTS group was much higher than other groups (P<0.05).Results of the expression of CD4 and CD8 in brain tumors:DC-9LTS can effectively enhance T-cell infiltration, a large number of CD8+ cells were detected in and around the tumor in DC-9LTS group, compared with DC-9L group, DC and PBS group (P<0.001). Expression of CD8+ cell was not detected in DC group and PBS group. However no expression of CD4+ cells was observed in all groupsConclusion:Immunotherapy using DCs transfected with 9L CSCs total RNA is more effective for the treatment of 9L brain gliomas, the strategy prolonged the survival of 9L glioma-bearing rats significantly, which provide a scientific foundation for further investigation of this approach to eradicate gliomas.