The engineered agonistic anti-CD40 antibody potentiates the antitumor effects of ß-glucan by resetting TAMs.

Anti-CD40 antibodies (Abs) have been shown to induce antitumor T-cell responses. We reported that the engineered agonistic anti-CD40 Ab (5C11, IgG4 isotype) recognized human CD40 antigen expressed on a human B lymphoblastoid cell line as well as on splenic cells isolated from humanized CD40 mice. Of note, a single high dosage of 5C11 was able to prohibit tumor growth in parallel with an increase in the population of infiltrated CD8+ T cells. Furthermore, the antitumor effects of 5C11 were enhanced in the presence of ß-glucan along with an increase in the population of infiltrated CD8+ T cells. In addition, the numbers of CD86+ TAMs and neutrophils were elevated in the combination of 5C11 and ß-glucan compared with either 5C11 or ß-glucan alone. Furthermore, the abundance of Faecalibaculum, one of the probiotics critical for tumor suppression, was obviously increased in the combination of 5C11 and ß-glucan-treated mice. These data reveal a novel mechanism of tumor suppression upon the combination treatment of 5C11 and ß-glucan and propose that the combination treatment of agonistic anti-human CD40 antibody 5C11 and ß-glucan could be a promising therapeutic strategy for cancer patients.

Deficiency of N-linked glycosylation impairs immune function of B7-H6.

B7-H6 is a novel immune checkpoint molecule that triggers NK cell cytotoxicity, but the role of N-glycosylation in B7-H6 is poorly understood. We here identified the existence of N-glycosylation of B7-H6 in different cell lines and exogenous expression cells by PNGase F digestion and tunicamycin blockage. Subsequently, we demonstrated that B7-H6 contains 6 functional N-linked glycosylation sites by single site mutation and electrophoresis. Phylogenetical and structural analysis revealed that N43 and N208 glycan are conserved in jawed vertebrates and may thus contribute more to the biological functions. We further demonstrated that N43 and N208 glycosylation are essential for B7-H6 to trigger NK cell activation. Mechanistically, we found that N43 and N208 glycan contributed to the stability and membrane expression of B7-H6 protein. Lack of N208 glycosylation led to membrane B7-H6 shedding, while N43 mutation resulted in impaired B7-H6/NKp30 binding affinity. Together, our findings highlight the significance of N-linked glycosylation in B7-H6 biological functions and suggest potential targets for modulating NK cell-mediated immunity.

Radioimmunotherapy Targeting B7-H3 in situ glioma models enhanced antitumor efficacy by Reconstructing the tumor microenvironment.

Radionuclide drug conjugates (RDCs) with antibodies serve as a novel approach for the treatment of malignant tumors including glioblastoma. However, RDCs require optimal antibodies to work efficiently. Hu4G4, a novel B7-H3-targeting humanized monoclonal IgG1 antibody, is highly specific for the human B7-H3 protein (a marker of tumor cells, including glioblastoma cells). Herein, we established 131I-labeled hu4G4 (131I-hu4G4) and showed that it specifically bound to B7-H3 with high affinity (Kd = 0.99 ± 0.07 nM) and inhibited the growth of U87 cells in vitro. 131I-hu4G4 displayed potent in situ antitumor activity in a mouse model of glioma based on GL261 Red-Fluc-B7-H3 cells. More importantly, 131I-hu4G4 remodeled the tumor microenvironment and promoted the transformation of glioma from "cold" to "hot" tumors by promoting CD4+ and CD8+ T cell infiltration and the polarization of M2 to M1. Therefore, the antitumor activity observed with 131I-hu4G4, together with its ability to enhance antitumor immune responses, makes it a novel candidate for radioimmunotherapy of glioblastoma.

Development of a MMAE-based antibody-drug conjugate targeting B7-H3 for glioblastoma.

B7-H3 (immunoregulatory protein B7-homologue 3) is overexpressed in many cancer cells with limited expression in normal tissues, considered to be a promising target for tumor therapeutics. Clinical trials of antibody-drug conjugates (ADCs) against different targets for glioblastoma have been investigated and showed potent efficacies. In this study, we developed a homogeneous ADC 401-4 with a drug-to-antibody ratio (DAR) of 4, which was prepared by conjugation of Monomethyl auristatin E (MMAE) to a humanized anti-B7-H3 mAb 401, through a divinylsulfonamide-mediated disulfide re-bridging approach. In vitro studies, 401-4 displayed specific killing against B7-H3-expressing tumors and was more effective in cells with higher levels of B7-H3 for different glioblastoma cells. 401-4 was furthered labeled with Cy5.5 to yield a fluorescent conjugate 401-4-Cy5.5. The in vivo imaging studies showed that the conjugate accumulated in tumor regions and exhibited the ability to target-specific delivery. In addition, significant antitumor activities for 401-4 was observed against U87-derived tumor xenografts in a dose dependent manner.

Establishment of a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay to Measure Soluble B7-H5 in Patients with Cancer.

B7-H5, an immune checkpoint molecule, is markedly upregulated in multiple cancers and plays an important role in tumor progression and immune escape. However, the expression and significance of soluble B7-H5 (sB7-H5) in cancer remain unclear. Herein, we generated two novel mouse anti-human B7-H5 monoclonal antibodies (mAbs) 2E5 and 7B10, which had different epitopes. Based on the two mAbs, a sandwich enzyme-linked immunosorbent assay (ELISA) system was developed. Using this ELISA, we found that compared with healthy controls (HCs), sB7-H5 levels were significantly increased in the serum of patients with gastric cancer (GC), colorectal cancer (CRC), and lung cancer (LC) and were associated with TNM stage and metastasis. Receiver operating characteristic (ROC) curve analysis showed that sB7-H5 has diagnostic value for GC, CRC, and LC. Collectively, our findings delineate that sB7-H5 may be used as a predictor for diagnosis of cancer and a potential therapeutic target for cancer treatment.

SIK2 promotes ovarian cancer cell motility and metastasis by phosphorylating MYLK.

Salt-inducible kinase 2 (SIK2; also known as serine/threonine-protein kinase SIK2) is overexpressed in several cancers and has been implicated in cancer progression. However, the mechanisms by which SIK2 regulates cancer cell motility, migration and metastasis in ovarian cancer have not been fully discovered. Here, we identify that SIK2 promotes ovarian cancer cell motility, migration and metastasis in vitro and in vivo. Mechanistically, SIK2 regulated cancer cell motility and migration by myosin light chain kinase, smooth muscle (MYLK)-meditated phosphorylation of myosin light chain 2 (MYL2). SIK2 directly phosphorylated MYLK at Ser343 and activated its downstream effector MYL2, promoting ovarian cancer cell motility and metastasis. In addition, we found that adipocytes induced SIK2 phosphorylation at Ser358 and MYLK phosphorylation at Ser343, enhancing ovarian cancer cell motility. Moreover, SIK2 protein expression was positively correlated with the expression of MYLK-pS343 in ovarian cancer cell lines and tissues. The co-expression of SIK2 and MYLK-pS343 was associated with reduced median overall survival in human ovarian cancer samples. Taken together, SIK2 positively regulates ovarian cancer motility, migration and metastasis, suggesting that SIK2 is a potential candidate for ovarian cancer treatment.

Interlocked feedback loops balance the adaptive immune response.

Adaptive immune responses can be activated by harmful stimuli. Upon activation, a cascade of biochemical events ensues the proliferation and the differentiation of T cells, which can remove the stimuli and undergo cell death to maintain immune cell homeostasis. However, normal immune processes can be disrupted by certain dysregulations, leading to pathological responses, such as cytokine storms and immune escape. In this paper, a qualitative mathematical model, composed of key feedback loops within the immune system, was developed to study the dynamics of various response behaviors. First, simulation results of the model well reproduce the results of several immune response processes, particularly pathological immune responses. Next, we demonstrated how the interaction of positive and negative feedback loops leads to irreversible bistable, reversible bistable and monostable, which characterize different immune response processes: cytokine storm, normal immune response, immune escape. The stability analyses suggest that the switch-like behavior is the basis of rapid activation of the immune system, and a balance between positive and negative regulation loops is necessary to prevent pathological responses. Furthermore, we have shown how the treatment moves the system back to a healthy state from the pathological immune response. The bistable mechanism that revealed in this work is helpful to understand the dynamics of different immune response processes.

Identification of Candidate Gene Signatures and Regulatory Networks in Endometriosis and its Related Infertility by Integrated Analysis.

Endometriosis is a common gynecological disease associated with infertility, and it represents an economic burden worldwide. However, the molecular mechanisms underlying endometriosis development have not yet been fully elucidated. Here, we aimed to identify reliable key genes and the related regulatory network that may be involved in endometriosis. Differentially expressed genes (DEGs) were identified through integrated analysis of four expression datasets of endometriosis from Gene Expression Omnibus. Gene functional analysis and protein-protein interaction network construction were performed to reveal the potential function of DEGs. Subsequently, candidate hub genes were defined and validated in GSE105764 dataset, and the associated regulatory networks were constructed. Additionally, GSE120103 dataset was applied to identify the differential expression between the infertile and fertile groups of patients with stage IV endometriosis. Finally, real-time quantitative polymerase chain reaction analysis was performed to identify the differential expression of hub genes in the collected clinical specimens. Robust rank aggregation integrated analysis determined 158 DEGs. Epithelial cell differentiation was the most significantly enriched biological process, and leukocyte transendothelial migration was the most significantly enriched pathway. Eight hub genes including CLDN3, CLDN5, CLDN7, CLDN11, HOXC8, HOXC6, HOXB6, and HOXB7 were identified, and most of these were validated as abnormally expressed genes in both the infertile group and patients with endometriosis. Transcriptional factors and microRNAs related to these genes were identified. Altogether, our integrated analysis identified critical gene signatures, involved pathways, and regulatory networks, which could provide clinically significant insights into the molecular mechanisms underlying endometriosis and its related infertility.

MTHFD2 promotes ovarian cancer growth and metastasis via activation of the STAT3 signaling pathway.

Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is a bifunctional enzyme located in the mitochondria. MTHFD2 has been reported to be overexpressed in several malignant tumors and is implicated in cancer development. This study aimed to investigate the effect of MTHFD2 on ovarian cancer progression. The expression of MTHFD2 was detected by bioinformatic analysis, immunohistochemistry, RT-qPCR (real-time quantitative PCR analysis), and western blot analysis. The effects of MTHFD2 depletion on cell proliferation, migration, and invasion were determined through in vitro experiments. Cell cycle progression and apoptosis were accessed by flow cytometry. The related signaling pathway protein expression was determined by western blot analysis. We found that MTHFD2 is highly expressed in both ovarian cancer tissues and cell lines. MTHFD2 deletion suppressed cell proliferation and metastasis. Knockdown of MTHFD2 induces cell apoptosis and G2/M arrest, whereas the number of cells in S phase increased with MTHFD2 overexpression. Mechanically, our results indicate that an inhibitory effect of MTHFD2 knockdown may be mediated by the downregulation of cyclin B1/Cdc2 complex and the inhibitory effect on its activity. Additionally, MTHFD2 could regulate cell growth and aggressiveness via activation of STAT3 and the STAT3-induced epithelial-mesenchymal transition signaling pathway. These findings indicate that MTHFD2 is overexpressed in ovarian cancer and regulates cell proliferation and metastasis, presenting an attractive therapeutic target.

Inhibition of long non-coding RNA XIST upregulates microRNA-149-3p to repress ovarian cancer cell progression.

Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) play critical roles in human diseases. We aimed to clarify the role of lncRNA X-inactive specific transcript (XIST)/miR-149-3p/forkhead box P3 (FOXP3) axis in ovarian cancer (OC) cell growth. XIST, miR-149-3p and FOXP3 expression in OC tissues and cell lines was assessed, and the predictive role of XIST in prognosis of OC patients was analyzed. The OC cell lines were screened and accordingly treated with silenced/overexpressed XIST plasmid or miR-149-3p mimic/inhibitor, and then the proliferation, invasion, migration, colony formation ability, apoptosis, and cell cycle distribution of OC cells were measured. Effect of altered XIST and miR-149-3p on tumor growth in vivo was observed. Online website prediction and dual luciferase reporter gene were implemented to detect the targeting relationship of lncRNA XIST, miR-149-3p, and FOXP3. XIST and FOXP3 were upregulated, whereas miR-149-3p was downregulated in OC tissues and cells. High XIST expression indicated a poor prognosis of OC. Inhibition of XIST or elevation of miR-149-3p repressed proliferation, invasion, migration, and colony formation ability, and promoted apoptosis and cell cycle arrest of HO-8910 cells. In SKOV3 cells upon treatment of overexpressed XIST or reduction of miR-149-3p, there exhibited an opposite tendency. Based on online website prediction, dual luciferase reporter gene, and RNA pull-down assays, we found that there was a negative relationship between XIST and miR-149-3p, and miR-149-3p downregulated FOXP3 expression. This study highlights that knockdown of XIST elevates miR-149-3p expression to suppress malignant behaviors of OC cells, thereby inhibiting OC development.

Erratum: B7-H3 promotes the cell cycle-mediated chemoresistance of colorectal cancer cells by regulating CDC25A: Erratum.

[This corrects the article DOI: 10.7150/jca.37255.].

CTL Attenuation Regulated by PS1 in Cancer-Associated Fibroblast.

Objective: Cancer-associated fibroblasts (CAFs) were associated with tumor progression in the tumor microenvironment (TME). However, their immunosuppressive roles in protecting cancer cells from the attack by cytotoxic T lymphocytes (CTLs) are not fully clear. In this study, we investigated whether and how CAFs regulate tumor-infiltrating lymphocytes as well as their role in tumor immunosuppression. Methods: Eighty-three cases of ovarian cancer and 10 controls were analyzed for CAFs and CD8+ tumor-infiltrating lymphocytes by gene array and immunohistochemistry. We evaluated presenilin 1 (PS1) expression in CAFs, CTL penetration, tumor burden, dendritic cell function, and migration of tumor-infiltrating lymphocytes and their function in vivo and in vitro after silencing PS1. In addition, the pathway via which PS1 affects the TME was also evaluated. Results: PS1 was highly expressed in CAFs, and its silencing significantly promoted CD8+ CTL proliferation and penetration in multiple ovarian models (p < 0.05), resulting in tumor regression and growth inhibition. Interleukin (IL)-1ß was identified as a major immune inhibitor in the TME, and it was significantly decreased after PS1 silencing (p < 0.05), which was regulated by the WNT/ß-catenin pathway. It was also showed that high expression of IL-1ß in CAFs inhibits CTL penetration significantly (p < 0.05). Conclusion: Highly expressed PS1 in CAFs plays a crucial role in regulating tumor-infiltrating lymphocyte populations in the TME via the WNT/ß-catenin pathway. Targeting PS1 may retrieve functional CTLs in the TME and improve the efficacy of current immunotherapies.

Preferential Expression of B7-H6 in Glioma Stem-Like Cells Enhances Tumor Cell Proliferation via the c-Myc/RNMT Axis.

B7 homologue 6 (B7-H6), a newly identified member of the B7 costimulatory molecule family, is not only a crucial regulator of NK cell-mediated immune responses through binding to NKp30 but also has clinical implications due to its abnormal expression in human cancers. Here, we show that B7-H6 expression is abnormally upregulated in glioma tissue and that B7-H6 is coexpressed with stem cell marker Sox2. Intriguingly, B7-H6 was rarely detected on the surface of glioma cell lines but was abundantly expressed in glioma stem-like cells (GSLCs) that were derived from the glioma cell lines in vitro. Surprisingly, B7-H6 was the only one that was preferentially expressed in the GSLCs among the B7 family members. Functionally, knockdown of B7-H6 in GSLCs by siRNAs led to the inhibition of cell proliferation, with decrease in the expression of the oncogene Myc as well as inactivation of PI3K/Akt and ERK/MAPK signaling pathways. Moreover, we determined that three genes CBL (Casitas B-Lineage Lymphoma Proto-Oncogene), CCNT1 (Cyclin T1), and RNMT (RNA guanine-7 methyltransferase) were coexpressed with B7-H6 and c-myc in glioma tissue samples from the TCGA database and found, however that only RNMT expression was inhibited by the knockdown of B7-H6 expression in the GSLCs, suggesting the involvement of RNMT in the B7-H6/c-myc axis. Extending this to 293T cells, we observed that knocking out of B7-H6 with CRISPR-Cas9 system also suppressed cell proliferation. Thus, our findings suggest B7-H6 as a potential molecule for glioma stem cell targeted immunotherapy.

B7-H3 promotes the cell cycle-mediated chemoresistance of colorectal cancer cells by regulating CDC25A.

Colorectal cancer (CRC) is one of the most common malignancies, and chemoresistance is one of the key obstacles in the clinical outcome. Here, we studied the function of B7-H3 in regulating cell cycle-mediated chemoresistance in CRC. The ability of B7-H3 in regulating chemoresistance was investigated via cell viability, clonogenicity, apoptosis and cycle analysis in vitro. Moreover, the role of B7-H3/CDC25A axis in regulating chemoresistance in vivo in the xenograft tumor models by intraperitoneal injection of oxaliplatin (L-OHP) and CDC25A inhibitors. The correlation between B7-H3 and CDC25A was examined in the CRC patients by immunohistochemistry (IHC) and pathological analyses. We found that B7-H3 could effectively enhance the resistance to a chemotherapeutic drug (oxaliplatin or 5-fluorouracil) via CDC25A. B7-H3 regulated the expression of CDC25A by the STAT3 signaling pathway in CRC cells. Furthermore, overexpression of B7-H3 enhanced chemoresistance by reducing the G2/M phase arrest in a CDC25A-dependent manner. Silencing CDC25A or treatment with CDC25A inhibitor could reverse the B7-H3-induced chemoresistance of cancer cells. Moreover, both B7-H3 and CDC25A were significantly upregulated in CRC samples compared with normal adjacent tissues and that the levels correlated with tumor stage. CDC25A was positively correlated with B7-H3 expression in this cohort. Taken together, our findings provide an alternative mechanism by which CRC cells can acquire chemoresistance via the B7-H3/CDC25A axis.

MicroRNA-708 Suppresses Cell Proliferation and Enhances Chemosensitivity of Cervical Cancer Cells to cDDP by Negatively Targeting Timeless.

PURPOSE: Cervical cancer is the fourth most common cause of cancer-associated mortality in women worldwide. Previous studies have reported that microRNAs (miRNAs) are involved in multiple biological aspects of cancer progression by regulating gene expression. Here, we investigated the role of microRNA-708 (miR-708) in cervical cancer. METHODS: The expression levels of miR-708 in cervical cancer tissues and paired-normal cervical tissues were tested by quantitative polymerase chain reaction (qPCR). The interaction between miR-708 and Timeless was identified by bioinformatics method, dual-luciferase reporter assay, and Western blotting. The effects of over-expression of miR-708 on cell proliferation and cisplatin sensitivity were determined by Cell Counting Kit-8 (CCK-8) and colony formation assay. Cell cycle and apoptosis were analyzed by flow cytometry. DNA damage induced by over-expression of miR-708 was determined by comet assay. Expression levels of the genes involved in repair of DNA damage were analyzed by Western blotting. RESULTS: MiR-708 was down-regulated in cervical cancer tissues compared with paired-normal cervical tissues. By bioinformatics method, Western blotting, and dual-luciferase reporter assay, we found that Timeless was a direct target of miR-708. Furthermore, miR-708 suppressed cellular viability, colony formation, promoted apoptosis, and induced DNA damage levels. MiR-708 also enhanced chemosensitivity of cervical cancer cells to cDDP via impairing the ATR/CHK1 signaling pathway. CONCLUSION: We conclude that miR-708 suppresses cell proliferation, facilitates cisplatin efficacy, and impairs DNA repair pathway in cervical cancer cells. These results demonstrate that miR-708 might be a candidate therapeutic target for future cervical cancer therapy.

B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2.

Accumulating evidence suggests that aerobic glycolysis is important for colorectal cancer (CRC) development. However, the underlying mechanisms have yet to be elucidated. B7-H3, an immunoregulatory protein, is broadly overexpressed by multiple tumor types and plays a vital role in tumor progression. In this study, we found that overexpression of B7-H3 effectively increased the rate of glucose consumption and lactate production, whereas knockdown of B7-H3 had the opposite effect. Furthermore, we showed that B7-H3 increased glucose consumption and lactate production by promoting hexokinase 2 (HK2) expression in CRC cells, and we also found that HK2 was a key mediator of B7-H3-induced CRC chemoresistance. Depletion of HK2 expression or treating cells with HK2 inhibitors could reverse the B7-H3-induced increase in aerobic glycolysis and B7-H3-endowed chemoresistance of cancer cells. Moreover, we verified a positive correlation between the expression of B7-H3 and HK2 in tumor tissues of CRC patients. Collectively, our findings suggest that B7-H3 may be a novel regulator of glucose metabolism and chemoresistance via controlling HK2 expression in CRC cells, a result that could help develop B7-H3 as a promising therapeutic target for CRC treatment.

Paeoniflorin exerts antitumor effects by inactivating S phase kinase-associated protein 2 in glioma cells.

Paeoniflorin (PF), a natural compound isolated from Paeoniae radix, has been shown to exert antitumor effects in various types of human cancers including glioma. However, the mechanism of action is not well understood. S-phase kinase-associated protein (Skp)2 functions as an oncogene in many cancers. In the present study, we investigated whether Skp2 mediates the anti-glioma activity of PF. We found that PF inhibited glioma cell proliferation, migration and invasion, and induced G2/M arrest and apoptosis. Skp2 expression was downregulated in glioma cells treated with PF. PF-induced antitumor effects in glioma cells were abolished by Skp2 overexpression but were enhanced by RNA interference of Skp2. Moreover, PF treatment inhibited U87 cell-derived tumor growth in a xenograft mouse model. These results demonstrate that PF exerts its antitumor effects in part by inhibiting Skp2 expression in glioma cells and could be a promising therapeutic agent for glioma therapy.

[Identification and characterization of monoclonal antibody Y4F11 against human B7-H3].

Objective To generate mouse anti-human monoclonal antibodies against the molecules expressed on immune cells from malignant pleural effusions, and identify the antigen recognized by the monoclonal antibody named Y4F11. Methods The cells separated from pleural effusions of lung cancer patients were used to immunize BALB/c mice. Hybridoma technology was used for cell fusion and screening; the monoclonal antibody named Y4F11 was chosen as the object for the following experiment. Flow cytometry was performed to analyze the molecules expressed on naive and activated T, B, NK cells and monocytes; Co-immunoprecipitation was conducted to get the antigen pulled down by Y4F11. Mass spectrometry sequencing and BLASTP were used to identify the antigen molecules. Western blotting, dot-blotting and flow cytometry were utilized to identify Y4F11. Results The study obtained 78 hybridoma clones secreting antibodies stably which could recognize surface molecules on cells from pleural effusions. The hybridoma named Y4F11 was chosen as the following experiment object for further identification. The molecule which Y4F11 recognized was about 50 Kd, and further was confirmed as B7-H3 molecule. Furthermore, Y4F11 could recognize B7-H3 expressed on B7-H3 gene-transferred cells as well as B7-H3 fusion protein. Besides, the expression pattern of B7-H3 on immune cells was confirmed by Y4F11 antibody. All these results indicated that the antigen molecule recognized by Y4F11 antibody was B7-H3. Conclusion A hybridoma cell line with stable expression of monoclonal antibody B7-H3 has been successfully obtained and could be used for biological function study on B7-H3.

TGF-ß1 promotes colorectal cancer immune escape by elevating B7-H3 and B7-H4 via the miR-155/miR-143 axis.

Transforming growth factor-beta 1 (TGF-ß1) suppresses T cell function, promoting tumor immune escape. Yet, whether the depression of TGF-ß1 on T cell function is mediated by co-inhibitory molecules B7-H3 and B7-H4 remains largely unclear. Here, we demonstrated that TGF-ß1 elevated the expression of miR-155 in colorectal cancer cells through SMAD3 and SMAD4. The upregulated miR-155 attenuated miR-143 by inhibiting its direct target, the transcription factor CEBPB. Consequently, the direct target genes of miR-143, B7-H3 and B7-H4, were augmented in the cytoplasm and membrane of tumor cells. Over-expression of B7-H3 and B7-H4 in HCT-116 cells induced T cells to secrete TGF-ß1 and the immunosuppressive cytokines IL-2, IL-6, and IL-17. Restoration of miR-143 inhibited the growth of HCT-116 xenograft tumors in mice, and also repressed the expression of B7-H3 and B7-H4 in the tumors. Thus, this study reveals the mechanism by which TGF-ß1 leads to T cell-mediated tumor evasion through an increase in B7-H3 and B7-H4 expression.

[Elevated expression of B7-H6 in U87 cells-derived glioma stem like cells is associated with biological characteristics].

Objective To investigate the expression and biological significance of costimulatory molecule B7-H6, a member of B7 family, in glioma stem like cells (GSLCs). Methods In virtue of the ability of forming neurospheres in vitro , GSLCs were isolated from U87 cells by cell sub-cloning. Real-time quantitative PCR and flow cytometry were performed to detect the expressions of stem cell related markers (c-myc, Sox2, CD133, nestin, and CXCR4), as well as the expressions of B7 family molecules. The different doses of adriamycin, carboplatin, cisplatin, were used to treat GSLCs for testing their chemotherapy-resistance. After the expression of B7-H6 in GSLCs was knockdown by siRNA, CCK-8 method was used to detect cell proliferation. Results GSLCs were successfully isolated from U87 cells, which formed neurospheres in vitro . The expressions of multiple stem cell markers were up-regulated and the GSLCs showed enhanced chemo therapy-resistance. B7 family members, B7-H1, B7-H3, B7-H4 and B7-H6 were expressed in GSLCs. Compared with primary U87 cells, GSLCs presented with a remarkably increased expression of B7-H6 on cell membrane. When B7-H6 was silenced by siRNA, cell proliferation was inhibited along with the decrease of c-myc expression. Conclusion The expression of B7-H6 is up-regulated in U87-derived GSLCs, which is associated with the biological characteristics of GSLCs.