B7-H4 is preferentially expressed in non-dividing brain tumor cells and in a subset of brain tumor stem-like cells

B7-H4, a newly discovered member of B7 family that negatively regulates T cell-mediated immunity, may facilitate tumor progression by undermining host immunity. Recent studies show that brain tumor stem-like cells (TSCs) contribute to tumorigenesis. However, the relationship between B7-H4 and the clinical behavior of brain TSCs remains unclear. In this study, we found that B7-H4 was expressed in cultured tumor cells from human gliomas (n = 5) and medulloblastomas (n = 3). Double immunostaining indicated that B7-H4 was primarily restricted to non-dividing (Ki67(-)) cultured tumor cells. Tumor cells cultured under medium conditions favoring the growth of neural stem cells were able to form primary and secondary spheres, along with expression of neural stem/progenitor cell markers. These cells differentiated into different neural lineages when cultured in differentiation medium, indicating that these cells have TSCs characteristics. Double immunostaining showed that TSCs consisted of proliferative (Ki67(+)) and quiescent (Ki67(-)) cells. We also found that B7-H4 was expressed in a small population of CD133(+) cells sorted by flow cytometry. Interestingly, both CD133(+) and CD133(-) cells were tumorigenic in SCID mice in vivo. However, CD133(+) cells-initiated glioblastomas showed a higher proliferation index, compared to CD133(-) cells-induced glioblastomas in vivo. Secondary glioma cells derived from CD133(+) or CD133(-) cell xenografts expressed B7-H4 as well. Our data suggest B7-H4 is preferentially expressed in non-dividing brain tumor cells and in a subpopulation of brain TSCs, and CD133(-) tumor cells also have the capacity to initiate brain formation in vivo.     

B7-H4 expression is elevated in human U251 glioma stem-like cells and is inducible in monocytes cultured with U251 stem-like cell conditioned medium

Previous studies indicated that B7-H4, the youngest B7 family, negatively regulates T cell-mediated immunity and is significantly overexpressed in many human tumors. Tumor stem cells are purported to play a role in tumor renewal and resistance to radiation and chemotherapy. However, the link between B7-H4 and tumor stem cells is unclear. In this study, we investigated B7-H4 expression in the medium of human glioma U251 cell cultures. Immunofluorescence results showed that U251 cells cultured in serum-free medium （supplemented with 2% B27, 20 ng/mL epidermal growth factor, 20 ng/mL basic fibroblast growth factor） maintained stem-like cell characteristics, including expression of stem cell marker CD133 and the neural progenitor cell markers nestin and SOX2. In contrast, U251 cells cultured in serum-containing medium highly expressed differentiation marker glial fibrillary acidic protein. Flow cytometry analysis showed serum-free medium-cultured U251 cells expressed higher intracellular B7-H4 than serum- containing medium-cultured U251 cells （24%-35% vs. 8%-11%, P 〈 0.001）. Immunofluorescence in purified monocytes from normal human peripheral blood mononuclear cells revealed moderate expression of BT-H4 after stimulation with conditioned medium from U251 cells cultured in serum-containing medium. Moreover, conditioned medium from U251 stem-like cells had a significant stimulation effect on B7-H4 expression compared with serum-containing conditioned medium （P 〈 0.01）. Negative costimulatory molecule B7-H4 was preferentially expressed in U251 stem-like cells, and conditioned medium from these cells more effectively induced monocytes to express BT-H4 than conditioned medium from U251 cells cultured in the presence of serum. Our results show that U251 stem-like cells may play a more crucial role in tumor immunoloregulation with high expression of B7-H4.     

CD133 is not present on neurogenic astrocytes in the adult subventricular zone, but on embryonic neural stem cells, ependymal cells, and glioblastoma cells

Human brain tumor stem cells have been enriched using antibodies against the surface protein CD133. An antibody recognizing CD133 also served to isolate normal neural stem cells from fetal human brain, suggesting a possible lineage relationship between normal neural and brain tumor stem cells. Whether CD133-positive brain tumor stem cells can be derived from CD133-positive neural stem or progenitor cells still requires direct experimental evidence, and an important step toward such investigations is the identification and characterization of normal CD133-presenting cells in neurogenic regions of the embryonic and adult brain. Here, we present evidence that CD133 is a marker for embryonic neural stem cells, an intermediate radial glial/ependymal cell type in the early postnatal stage, and for ependymal cells in the adult brain, but not for neurogenic astrocytes in the adult subventricular zone. Our findings suggest two principal possibilities for the origin of brain tumor stem cells: a derivation from CD133-expressing cells, which are normally not present in the adult brain (embryonic neural stem cells and an early postnatal intermediate radial glial/ependymal cell type), or from CD133-positive ependymal cells in the adult brain, which are, however, generally regarded as postmitotic. Alternatively, brain tumor stem cells could be derived from proliferative but CD133-negative neurogenic astrocytes in the adult brain. In the latter case, brain tumor development would involve the production of CD133.     

胶质瘤干细胞、肿瘤相关巨噬细胞和B7-H1在脑胶质瘤组织中的表达及其意义

目的:探讨胶质瘤干细胞（glioma stem cells,GSCs）、肿瘤相关巨噬细胞（tumor-associated macrophages,TAMs）和负性共刺激分子B7-H1(B7 homolog 1)在不同病理级别脑胶质瘤组织中的表达及其相关性。方法:对30例低级别脑胶质瘤（WHOⅠ级、Ⅱ级）和30例高级别脑胶质瘤（WHOⅢ级、Ⅳ级）标本做连续石蜡包埋切片,用免疫组化SP法检测各组标本中GSCs、TAMs和B7-H1的表达情况,采用CD133作为GSCs的标志物,以CD68作为TAMs的标志物。用Image-Pro Plus 6.0软件分析每个视野阳性染色的积分光密度（IOD）,每个组织切片在400倍镜下分析20个视野。结果:CD133、CD68和B7-H1在高级别脑胶质瘤组织中的表达水平明显高于低级别脑胶质瘤组织（P＜0.01、P＜0.01、P＜0.01）;脑胶质瘤组织中B7-H1的空间表达位置在CD68+TAMs的分布区域较强;CD133与CD68的表达呈正相关（P＜0.001）,CD133与B7-H1的表达呈正相关（P＜0.001）。结论:随着病理级别的增高,脑胶质瘤组织中GSCs、TAMs和B7-H1的表达均增高,并且CD133与CD68和B7-H1的表达呈显著正相关。     

Expression of MHC I and NK ligands on human CD133<Superscript>+</Superscript> glioma cells: possible targets of immunotherapy

Mounting evidence suggests that gliomas are comprised of differentiated tumor cells and brain tumor stem cells (BTSCs). BTSCs account for a fraction of total tumor cells, yet are apparently the sole cells capable of tumor initiation and tumor renewal. BTSCs have been identified as the CD133-positive fraction of human glioma, whereas their CD133-negative daughter cells have limited proliferative ability and are not tumorogenic. It is well established that the bulk tumor mass escapes immune surveillance by multiple mechanisms, yet little is known about the immunogenicity of the CD133-positive fraction of the tumor mass. We investigated the immunogenicity of CD133-positive cells in two human astrocytoma and two glioblastoma multiforme samples. Flow cytometry analyses revealed that the majority of CD133-positive cells do not express detectable MHC I or natural killer (NK) cell activating ligands, which may render them resistant to adaptive and innate immune surveillance. Incubating CD133-positive cells in interferon gamma (INF-γ) significantly increased the percentage of CD133-positive cells that expressed MHC I and NK cell ligands. Furthermore, pretreatment of CD133-positive cells with INF-γ rendered them sensitive to NK cell-mediated lysis in vitro. There were no consistent differences in immunogenicity between the CD133-positive and CD133-negative cells in these experiments. We conclude that CD133-posistive and CD133-negative glioma cells may be similarly resistant to immune surveillance, but that INF-γ may partially restore their immunogenicity and potentiate their lysis by NK cells. Walter C. Low, and John R. Ohlfest contributed equally as senior authors. An erratum to this article can be found at     

PD-1 is expressed by tumor-infiltrating immune cells and is associated with poor outcome for patients with renal cell carcinoma.

PURPOSE: B7-H1 is expressed by clinically aggressive forms of renal cell carcinoma (RCC) and predicts adverse outcome. B7-H1 is known to impair host immunity via interaction with the Programmed Death-1 (PD-1) receptor, which is expressed by activated T cells. Levels of immune cells expressing PD-1 (PD-1(+)) in clinical RCC tumors have not been evaluated. Thus, we tested whether immune cell PD-1 expression is observed within aggressive RCC tumors. EXPERIMENTAL DESIGN: Between 2000 and 2003, 267 patients underwent nephrectomy at our institution for clear cell RCC and had fresh-frozen tissue available for review. These RCC specimens were immunostained using anti-PD-1 (clone MIH4) and outcome analyses were conducted. RESULTS: Mononuclear immune cell infiltration was observed in 136 (50.9%) specimens. PD-1(+) immune cells were present in 77 of these 136 (56.6%) tumors. In contrast, RCC tumor cells did not express PD-1. Patients with PD-1(+) immune cells were significantly more likely to harbor B7-H1(+) tumor cells (P < 0.001), larger tumors (P = 0.001), and tumors of higher nuclear grade (P = 0.001). Likewise, intratumoral PD-1(+) immune cells were associated with advanced tumor-node-metastasis stage (P = 0.005), coagulative tumor necrosis (P = 0.027), and sarcomatoid differentiation (P = 0.008). With a median follow-up of 2.9 years, 52 patients died from RCC. Univariately, patients with PD-1(+) immune cells were at significant risk of cancer-specific death compared with PD-1(-) patients (risk ratio, 2.24; P = 0.004). CONCLUSIONS: Levels of immune cells expressing PD-1 were increased in patients with high-risk RCC tumors. Interactions between immune cell PD-1 and B7-H1 may promote cancer progression by contributing to immune dysfunction in patients with RCC.     

B7-H1 blockade augments adoptive T-cell immunotherapy for squamous cell carcinoma

In this report, we demonstrate that B7-H1, a B7 family molecule implicated in tumor immune evasion, is constitutively expressed on 66% of freshly isolated squamous cell carcinomas of the head and neck (SCCHN). To define the potential impact of tumor-associated B7-H1 on immunotherapy, the B7-H1-negative mouse SCC line, SCCVII, was transfected to express B7-H1. Although all of the animals succumbed to B7-H1/SCCVII tumors even after adoptive T-cell immunotherapy, the infusion of B7-H1 blocking monoclonal antibody with activated T cells cured 60% of animals. These data support B7-H1 blockade as a new approach to enhance the efficacy of T-cell immunotherapy.     

Cancer stem cells in nervous system tumors

Most current research on human brain tumors is focused on the molecular and cellular analysis of the bulk tumor mass. However, evidence in leukemia and more recently in solid tumors such as breast cancer suggests that the tumor cell population is heterogeneous with respect to proliferation and differentiation. Recently, several groups have described the existence of a cancer stem cell population in human brain tumors of different phenotypes from both children and adults. The finding of brain tumor stem cells (BTSCs) has been made by applying the principles for cell culture and analysis of normal neural stem cells (NSCs) to brain tumor cell populations and by identification of cell surface markers that allow for isolation of distinct tumor cell populations that can then be studied in vitro and in vivo. A population of brain tumor cells can be enriched for BTSCs by cell sorting of dissociated suspensions of tumor cells for the NSC marker CD133. These CD133+ cells, which also expressed the NSC marker nestin, but not differentiated neural lineage markers, represent a minority fraction of the entire brain tumor cell population, and exclusively generate clonal tumor spheres in suspension culture and exhibit increased self-renewal capacity. BTSCs can be induced to differentiate in vitro into tumor cells that phenotypically resembled the tumor from the patient. Here, we discuss the evidence for and implications of the discovery of a cancer stem cell in human brain tumors. The identification of a BTSC provides a powerful tool to investigate the tumorigenic process in the central nervous system and to develop therapies targeted to the BTSC. Specific genetic and molecular analyses of the BTSC will further our understanding of the mechanisms of brain tumor growth, reinforcing parallels between normal neurogenesis and brain tumorigenesis.     

Evidence of brain tumor stem progenitor-like cells with low proliferative capacity in human benign pituitary adenoma

Tumor stem cells have been implicated as cancer-initiating cells in malignant brain tumors. However, whether benign brain tumors also contain tumor stem cells are largely unexplored. Here, we investigated whether tumor stem-like cells were present in pituitary adenoma similar to malignant brain tumors. By immunocytochemistry, we found that pituitary adenoma tissues expressed neural stem cell marker. These cells could form neurospheres in vitro, expressed neural stem/progenitor cell markers and generated daughter cells with the capacity to differentiate into three neural lineages. Importantly, compared with non-invasive pituitary adenomas, we found that CD133 expression was significantly increased in invasive pituitary adenomas, suggesting that the proliferative capacity was correlated with the malignance of pituitary adenomas. Finally, invasive pituitary adenomas cells displayed lower proliferative ability than glioblastoma. Our data indicate that a subpopulation of stem/progenitor-like cells are present in pituitary adenomas, and these cells may be responsible for benign tumor initiation and maintenance.     

Expression of the B7-related molecule B7-H1 by glioma cells: a potential mechanism of immune paralysis

Human glioblastoma is a highly lethal tumor that is known for its immune inhibitory capabilities. B7-homologue 1 (B7-H1), a recently identified homologue of B7.1/2 (CD80/86), has been described to exert costimulatory and immune regulatory functions. We investigated the expression and the functional activity of B7-H1 in human glioma cells in vitro and in vivo. Although lacking B7.1/2 (CD80/86), all 12 glioma cell lines constitutively expressed B7-H1 mRNA and protein. Exposure to IFN-gamma strongly enhanced B7-H1 expression. Immunohistochemical analysis of malignant glioma specimens revealed strong B7-H1 expression in all 10 samples examined, whereas no B7-H1 expression could be detected on normal brain tissues. To elucidate the functional significance of glioma cell-related B7-H1 expression, we performed coculture experiments of glioma cells with alloreactive CD4+ and CD8+ T cells. Glioma-related B7-H1 was identified as a strong inhibitor of CD4+ as well as CD8+ T-cell activation as assessed by increased cytokine production (IFN-gamma, interleukin-2, and interleukin-10) and expression levels of the T-cell activation marker (CD69) in the presence of a neutralizing antibody against B7-H1 (mAb 5H1). B7-H1 expression may thus significantly influence the outcome of T-cell tumor cell interactions and represents a novel mechanism by which glioma cells evade immune recognition and destruction.     

Cancer stem cells and brain tumors

Besides the role of normal stem cells in organogenesis, cancer stem cells are thought to be crucial for tumorigenesis. Most current research on human tumors is focused on molecular and cellular analysis of the bulk tumor mass. However, evidence in leukemia and, more recently, in solid tumors suggests that the tumor cell population is heterogeneous. In recent years, several groups have described the existence of a cancer stem cell population in different brain tumors. These neural cancer stem cells (NCSC) can be isolated by cell sorting of dissociated suspensions of tumor cells for the neural stem cell marker CD133. These CD133+ cells -which also express nestin, an intermediate filament that is another neural stem cell marker- represent a small fraction of the entire brain tumor population. The stem-like cancer cells appear to be solely responsible for propagating the disease in laboratory models. A promising new approach to treating glioblastoma proposes targeting cancer stem cells. Here, we summarize progress in delineating NCSC and the implications of the discovery of this cell population in human brain tumors.     

Identification of a cancer stem cell in human brain tumors

Most current research on human brain tumors is focused on the molecular and cellular analysis of the bulk tumor mass. However, there is overwhelming evidence in some malignancies that the tumor clone is heterogeneous with respect to proliferation and differentiation. In human leukemia, the tumor clone is organized as a hierarchy that originates from rare leukemic stem cells that possess extensive proliferative and self-renewal potential, and are responsible for maintaining the tumor clone. We report here the identification and purification of a cancer stem cell from human brain tumors of different phenotypes that possesses a marked capacity for proliferation, self-renewal, and differentiation. The increased self-renewal capacity of the brain tumor stem cell (BTSC) was highest from the most aggressive clinical samples of medulloblastoma compared with low-grade gliomas. The BTSC was exclusively isolated with the cell fraction expressing the neural stem cell surface marker CD133. These CD133+ cells could differentiate in culture into tumor cells that phenotypically resembled the tumor from the patient. The identification of a BTSC provides a powerful tool to investigate the tumorigenic process in the central nervous system and to develop therapies targeted to the BTSC.     

B7-H3, a potential therapeutic target, is expressed in diffuse intrinsic pontine glioma

Diffuse intrinsic pontine glioma (DIPG) is a brain cancer with a median survival of only 1 year. Lack of molecular characterization of this tumor impedes the development of novel therapies. Membrane protein B7-H3, aka CD276, involved in interactions with host defenses in certain cancers, has been shown to be over-expressed in the majority of malignant neuroectodermal tumors including adult high-grade glioma. Targeting B7-H3 with a monoclonal antibody has demonstrated safety and efficacy in the salvage treatment of stage IV childhood neuroblastoma, another neuroectodermal tumor. It thus stands to reason that B7-H3 might serve as a therapeutic target in DIPG. B7-H3 immunoreactivity was determined in DIPG and non-diffuse brainstem glioma specimens with immunohistochemistry. In addition, B7-H3 mRNA expression was evaluated with microarrays in another set of specimens. All of the nine (100 %) DIPG specimens were shown to be B7-H3 immunoreactive. In the non-diffuse brainstem glioma group, none of the eight WHO grade I specimens showed B7-H3 immunoreactivity and nine of the 24 WHO grade II specimens (37.5 %) showed B7-H3 immunoreactivity. The association between histological grade and B7-H3 immunoreactivity was statistically highly significant. B7-H3 mRNA expression was also significantly higher in DIPG samples than in normal brain and juvenile pilocytic astrocytoma (WHO grade I) specimens. In summary, B7-H3 is over-expressed in DIPG. Given the need for novel treatment in this disease, antibody-based immunotherapy against B7-H3 in DIPG warrants further investigation.     

The Brain Microenvironment Preferentially Enhances the Radioresistance of CD133+ Glioblastoma Stem-like Cells

Brain tumor xenografts initiated from glioblastoma (GBM) CD133⁺ tumor stem-like cells (TSCs) are composed of TSC and non-TSC subpopulations, simulating the phenotypic heterogeneity of GBMs in situ. Given that the discrepancies between the radiosensitivity of GBM cells in vitro and the treatment response of patients suggest a role for the microenvironment in GBM radioresistance, we compared the response of TSCs and non-TSCs irradiated under in vitro and orthotopic conditions. As a measure of radioresponse determined at the individual cell level, γH2AX and 53BP1 foci were quantified in CD133⁺ cells and their differentiated (CD133^-) progeny. Under in vitro conditions, no difference was detected between CD133⁺ and CD133^- cells in foci induction or dispersal after irradiation. However, irradiation of orthotopic xenografts initiated from TSCs resulted in the induction of fewer γH2AX and 53BP1 foci in CD133⁺ cells compared to their CD133^- counterparts within the same tumor. Xenograft irradiation resulted in a tumor growth delay of approximately 7 days with a corresponding increase in the percentage of CD133⁺ cells at 7 days after radiation, which persisted to the onset of neurologic symptoms. These results suggest that, although the radioresponse of TSCs and non-TSCs does not differ under in vitro growth conditions, CD133⁺ cells are relatively radioresistant under intracerebral growth conditions. Whereas these findings are consistent with the suspected role for TSCs as a determinant of GBM radioresistance, these data also illustrate the dependence of the cellular radioresistance on the brain microenvironment.     

Tumor B7-H1 is associated with poor prognosis in renal cell carcinoma patients with long-term follow-up

B7-H1 participates in T-cell costimulation functioning as a negative regulator of immunity. Recent observations suggest that B7-H1 is expressed by renal cell carcinoma (RCC) tumor cells and is associated with poor prognosis. However, outcome analyses have been restricted to patients with fresh-frozen tissue and limited follow-up. We report the clinical effect of B7-H1 in RCC patients with a median of 10 years of follow-up. Between 1990 and 1994, 306 patients underwent nephrectomy for clear cell RCC and had paraffin tissue available for review. We did immunohistochemistry with anti-B7-H1 and conducted outcome analyses. Among the 306 patients, 73 (23.9%) harbored tumors with B7-H1 expression. Patients with tumor B7-H1 were at a significantly increased risk of both death from RCC [risk ratio (RR), 3.92; P < 0.001] and overall mortality (RR, 2.37; P < 0.001). The 5-year cancer-specific survival rates were 41.9% and 82.9% for patients with and without tumor B7-H1, respectively. In a multivariate model, tumor B7-H1 remained associated with cancer-specific death even after adjusting for tumor-node-metastasis stage, grade, and performance status (RR, 2.00; P = 0.003). In the subset of 268 patients with localized RCC, tumor B7-H1 was significantly associated with metastatic cancer progression (RR, 3.46; P < 0.001) and death from RCC (RR, 4.13; P < 0.001) even after adjusting for stage, grade, and performance status (RR, 1.78, P = 0.036). RCC patients with tumor B7-H1 are at significant risk of rapid cancer progression and accelerated rates of mortality. B7-H1 may function as a key determinant in RCC, abrogating immune responses directed against this immunogenic tumor.     

Effect of TLR4 and B7-H1 on Immune Escape of Urothelial Bladder Cancer and its Clinical Significance

Background/Aim: Toll-like receptor 4 (TLR4) and B7-H1, both normally expressed restricted to immune cells,are found to be aberrantly expressed in a majority of human tumors and may play important roles in regulationof tumor immunity. It has been shown that urothelial bladder cancer (UBC) patients can manifest tumoralimmune escape which may be a potential critical factor in tumor pathogenesis and progression. However, so far,the mechanisms of UBC-related immune escape have not been clarified. The aim of this study was to investigatethe effect of TLR4 and B7-H1 on immune escape of UBC. Methods: Bladder cancer T24 cells were pre-incubatedwith LPS and co-cultured with tumor specific CTLs. CTL cytotoxicity and apoptosis rates were measured by MTTassay and flow cytometry, respectively. The effects of an ERK inhibitor on B7-H1 expression and CTL cytotoxicityagainst T24 cells were also evaluated. In addition, TLR4, B7-H1 and PD-1 protein expression was analyzed byimmunohistochemistry in 60 UBC specimens and 10 normal urothelia. Results: TLR4 activation protected T24cells from CTL killing via B7-H1 overexpression. However PD98059, an inhibitor of ERK, enhanced CTL killingof T24 cells by reducing B7-H1 expression. TLR4 expression was generally decreased in UBC specimens, whileB7-H1 and PD-1 were greatly overexpressed. Moreover, expression of both B7-H1 and PD-1 was significantlyassociated with UICC stage and WHO grade classification. Conclusions: TLR4 and B7-H1 may contribute toimmune escape of UBC. Targeting B7-H1 or the ERK pathway may offer new immunotherapy strategies forbladder cancer.     

人脑胶质瘤移植瘤中CD133+细胞表型与微生态环境的关系

目的 研究CD133+细胞在肿瘤组织中的分布特征,探讨其与微生态环境的关系.方法 用CD133+肿瘤球或转红色荧光蛋白(RFP)基因的胶质瘤干细胞SU-3行裸小鼠原位、右腋皮下和腹腔等异位接种,致瘤后取荷瘤鼠全脑及整个皮下移植瘤组织行HE染色以及CD133免疫组织化学和免疫荧光染色,分别在光镜、荧光显微镜和激光共聚焦显微镜下观察移植瘤的组织结构和CD133+肿瘤细胞的分布特征.结果 在光镜下,原位接种的CD133+肿瘤细胞的分布有一定规律性,从形态上可分为成簇、成对和单个分布;从部位上可分为肿瘤血管旁、血管内皮、正常组织中和脑室内.皮下接种的CD133+肿瘤细胞成簇或散在分布于肌肉和脂肪组织中,还可见CD133免疫复合物分布于血管壁及其基质.在荧光显微镜和激光扫描共聚焦显微镜下可见,有些肿瘤血管内皮、血管旁的细胞共表达CD133和RFP.结论 在荷瘤鼠体内成簇生长的CD133+细胞实际上是类似体外生长的肿瘤干细胞(CSCs)球,而成对和单个生存的CD133+细胞分别代表CSCs与微生态环境相关的对称和不对称分裂;沿血管分布的CD133+细胞是依赖微生态环境生存的细胞,而远离血管或远离肿瘤组织、孤立于正常组织中的CD133+细胞是播散的CSCs或神经干细胞,属不受微生态环境控制的细胞.