Immune phenotypic linkage between colorectal cancer and liver metastasis

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Tumor regrowth between surgery and initiation of adjuvant therapy in patients with newly diagnosed glioblastoma

To assess incidence and degree of regrowth in glioblastoma between surgery and radiation therapy (RT) and to correlate regrowth with presurgical imaging and survival, we examined images of 32 patients with newly diagnosed glioblastoma who underwent MR spectroscopic imaging (MRSI), perfusion-weighted imaging (PWI), and diffusion-weighted imaging (DWI) prior to surgery, after surgery, and prior to RT/temozolomide. Contrast enhancement (CE) in the pre-RT MR image was compared with postsurgical DWI to differentiate tumor growth from postsurgical infarct. MRSI and PWI parameters were analyzed prior to surgery and pre-RT. Postsurgical MRI indicated that 18 patients had gross total and 14 subtotal resections. Twenty-one patients showed reduced diffusion, and 25 patients showed new or increased CE. In eight patients (25%), the new CE was confined to areas of postsurgical reduced diffusion. In the other 17 patients (53%), new CE was found to be indicative of tumor growth or a combination of tumor growth and surgical injury. Higher perfusion and creatine within nonenhancing tumor in the presurgery MR were associated with subsequent tumor growth. High levels of choline and reduced diffusion in pre-RT CE suggested active metabolism and tumor cell proliferation. Median survival was 14.6 months in patients with interim tumor growth and 24 months in patients with no growth. Increased volume or new onset of CE between surgery and RT was attributed to tumor growth in 53% of patients and was associated with shorter survival. This suggests that reducing the time between surgery and adjuvant therapy may be important. The acquisition of metabolic and physiologic imaging data prior to adjuvant therapy may also be valuable in assessing regions of new CE and nonenhancing tumor.     

DNA methylation based glioblastoma subclassification is related to tumoral T-cell infiltration and patient survival

BackgroundHistologically classified glioblastomas (GBM) can have different clinical behavior and response to therapy, for which molecular subclassifications have been proposed. We evaluated the relationship of epigenetic GBM subgroups with immune cell infiltrations, systemic immune changes during radiochemotherapy, and clinical outcome.Methods450K genome-wide DNA methylation was assessed on tumor tissue from 93 patients with newly diagnosed GBM, treated with standard radiochemotherapy and experimental immunotherapy. Tumor infiltration of T cells, myeloid cells, and Programmed cell death protein 1 (PD-1) expression were evaluated. Circulating immune cell populations and selected cytokines were assessed on blood samples taken before and after radiochemotherapy.ResultsForty-two tumors had a mesenchymal, 27 a receptor tyrosine kinase (RTK) II, 17 RTK I, and 7 an isocitrate dehydrogenase (IDH) DNA methylation pattern. Mesenchymal tumors had the highest amount of tumor-infiltrating CD3+ and CD8+ T cells and IDH tumors the lowest. There were no significant differences for CD68+ cells, FoxP3+ cells, and PD-1 expression between groups. Systemically, there was a relative increase of CD8+ T cells and CD8+ PD-1 expression and a relative decrease of CD4+ T cells after radiochemotherapy in all subgroups except IDH tumors. Overall survival was the longest in the IDH group (median 36 mo), intermediate in RTK II tumors (27 mo), and significantly lower in mesenchymal and RTK I groups (15.5 and 16 mo, respectively).ConclusionsMethylation based stratification of GBM is related to T-cell infiltration and survival, with IDH and mesenchymal tumors representing both ends of a spectrum. DNA methylation profiles could be useful in stratifying patients for immunotherapy trials.     

Cancer cell heterogeneity and plasticity: A paradigm shift in glioblastoma

Phenotypic plasticity has emerged as a major contributor to intra-tumoral heterogeneity and treatment resistance in cancer. Increasing evidence shows that glioblastoma (GBM) cells display prominent intrinsic plasticity and reversibly adapt to dynamic microenvironmental conditions. Limited genetic evolution at recurrence further suggests that resistance mechanisms also largely operate at the phenotypic level. Here we review recent literature underpinning the role of GBM plasticity in creating gradients of heterogeneous cells including those that carry cancer stem cell (CSC) properties. A historical perspective from the hierarchical to the nonhierarchical concept of CSCs towards the recent appreciation of GBM plasticity is provided. Cellular states interact dynamically with each other and with the surrounding brain to shape a flexible tumor ecosystem, which enables swift adaptation to external pressure including treatment. We present the key components regulating intra-tumoral phenotypic heterogeneity and the equilibrium of phenotypic states, including genetic, epigenetic, and microenvironmental factors. We further discuss plasticity in the context of intrinsic tumor resistance, where a variable balance between preexisting resistant cells and adaptive persisters leads to reversible adaptation upon treatment. Innovative efforts targeting regulators of plasticity and mechanisms of state transitions towards treatment-resistant states are needed to restrict the adaptive capacities of GBM.     

Combined expressional analysis,bioinformatics and targeted proteomics identify new potential therapeutic targets in glioblastoma stem cells

Glioblastoma (GBM) is both the most common and the most lethal primary brain tumor. It is thought that GBM stem cells (GSCs) are critically important in resistance to therapy. Therefore, there is a strong rationale to target these cells in order to develop new molecular therapies.To identify molecular targets in GSCs, we compared gene expression in GSCs to that in neural stem cells (NSCs) from the adult human brain, using microarrays. Bioinformatic filtering identified 20 genes (PBK/TOPK, CENPA, KIF15, DEPDC1, CDC6, DLG7/DLGAP5/HURP, KIF18A, EZH2, HMMR/RHAMM/CD168, NOL4, MPP6, MDM1, RAPGEF4, RHBDD1, FNDC3B, FILIP1L, MCC, ATXN7L4/ATXN7L1, P2RY5/LPAR6 and FAM118A) that were consistently expressed in GSC cultures and consistently not expressed in NSC cultures. The expression of these genes was confirmed in clinical samples (TCGA and REMBRANDT). The first nine genes were highly co-expressed in all GBM subtypes and were part of the same protein-protein interaction network. Furthermore, their combined up-regulation correlated negatively with patient survival in the mesenchymal GBM subtype. Using targeted proteomics and the COGNOSCENTE database we linked these genes to GBM signalling pathways.Nine genes: PBK, CENPA, KIF15, DEPDC1, CDC6, DLG7, KIF18A, EZH2 and HMMR should be further explored as targets for treatment of GBM.     

EXPRESSION OF IMMUNE－RELATED MOLECULES IN GLIOBLASTOMA MULTIFORM CELLS

Objective: To investigate the expression of immune-related molecules in glioblastoma multiform(GBM) cells.Methods: The expression of major histocompatibility complex (MHC), β2-microglobulin, Fas, CD80 and CD86 molecules on the surface of GBM cells were evaluated by flow cytometry. The expression of TAP-l, TAP-2 and Tapasin in the GBM cells were evaluated by RT-PCR method. Results: MHC class I, 62 microglobulin, TAP-l,TAP-2 and tapasin were expressed in most GBM cell lines. Except U87, there was no MHC class II molecule expression on any of the other GBM cell lines. Fas was expressed on all the GBM cell lines examined.Conclusion: The mechanism by which GBM escapes immune surveillance may involve down regulation of expression of MHC class I molecules and MHC class Ⅱ molecules. MHC class I positive GBM may be the suitable target of immunotherapy.     

Nestin+/CD31+ cells in the hypoxic perivascular niche regulate glioblastoma chemoresistance by upregulating JAG1 and DLL4

BackgroundFailure of glioblastoma (GBM) therapy is often ascribed to different types of glioblastoma stem-like cell (GSLC) niche; in particular, a hypoxic perivascular niche (HPVN) is involved in GBM progression. However, the cells responsible for HPVNs remain unclear.MethodsImmunostaining was performed to determine the cells involved in HPVNs. A hypoxic chamber and 3-dimensional (3D) microfluidic chips were designed to simulate a HPVN based on the pathological features of GBM. The phenotype of GSLCs was evaluated by fluorescence scanning in real time and proliferation and apoptotic assays. The expression of JAG1, DLL4, and Hes1 was determined by immunostaining, ELISA, Western blotting, and quantitative PCR. Their clinical prognostic significance in GBM HPVNs and total tumor tissues were verified by clinical data and The Cancer Genome Atlas databases.ResultsNestin⁺/CD31⁺ cells and pericytes constitute the major part of microvessels in the HPVN, and the high ratio of nestin⁺/CD31⁺ cells rather than pericytes are responsible for the poor prognosis of GBM. A more real HPVN was simulated by a hypoxic coculture system in vitro, which consisted of 3D microfluidic chips and a hypoxic chamber. Nestin⁺/CD31⁺ cells in the HPVN were derived from GSLC transdifferentiation and promoted GSLC chemoresistance by providing more JAG1 and DLL4 to induce downstream Hes1 overexpression. Poor GBM prognosis correlated with Hes1 expression of tumor cells in the GBM HPVN, and not with total Hes1 expression in GBM tissues.ConclusionsThese results highlight the critical role of nestin⁺/CD31⁺ cells in HPVNs that acts in GBM chemoresistance and reveal the distinctive prognostic value of these molecular markers in HPVNs.     

3D培养在神经胶质瘤研究中的应用

传统的肿瘤研究依赖于肿瘤细胞系构建肿瘤模型,并在细胞基础上测定肿瘤细胞的耐药性等,但在临床实验中大多数肿瘤药物以失败告终,其中最主要的原因是细胞系不能模拟肿瘤细胞在人体内微环境的特性,3D培养技术在肿瘤研究中的应用愈加广泛。本文主要从3D培养在神经胶质瘤研究中的应用进行综述。     

Tumor microvasculature supports proliferation and expansion of glioma‐propagating cells

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. The identification of ‘cancer stem cells’ (CSC) has shed new light on the potential mechanism of therapy resistance of these tumors. Because these cells appear to be more resistant to conventional treatments, they are thought to drive tumor regrowth after therapy. Therefore, novel therapeutic approaches that target these cells are needed. Tumor cells interact with their microenvironment. It has been reported that close contact between CSCs and tumor microvascular endothelium in GBM is important for CSCs to preserve their undifferentiated state and self‐renewal ability. However, our understanding of this interaction is still rudimentary. This is in part due to a lack of suitable in vitro models that accurately represent the in vivo situation. Therefore, we set up a co‐culture system consisting of primary brain tumor microvascular endothelial cells (tMVECs) and glioma propagating cells (GPCs) derived from biopsies of GBM patients. We found that tMVECs support the growth of GPCs resulting in higher proliferation rates comparing to GPCs cultured alone. This effect was dependent on direct contact between the 2 cell types. In contrast to GPCs, the FCS‐cultured cell line U87 was stimulated by culturing on tMVEC‐derived ECM alone, suggesting that both cell types interact different with their microenvironment. Together, these results demonstrate the feasibility and utility of our system to model the interaction of GPCs with their microenvironment. Identification of molecules that mediate this interaction could provide novel targets for directed therapy for GBM. © 2009 UICC     

Molecular interactions between breast cancer cells and the bone microenvironment drive skeletal metastases

Breast cancer cells preferentially spread to bone. Bone metastases are currently incurable and therefore better treatments need to be developed. Metastasis is an inefficient, multi-step process. Specific aspects of both breast cancer cells and the bone microenvironment contribute to the development of bone metastases. Breast cancers express chemokine receptors, integrins, cadherins, and bone-resorbing and bone-forming factors that contribute to the successful and preferential spread of tumor to bone. Bone is rich in growth factors and cell types that make it a hospitable environment for breast cancer growth. Once breast cancer cells enter the bone, a highly complex vicious cycle develops, in which breast cancer cells secrete factors that act on bone cells and other cells within the bone (stem cells, T cells, platelets, adipocytes, fibroblasts, and endothelial cells), causing them to secrete factors that act on adjacent cancer cells. The steps in the metastatic cascade and the vicious cycle within bone offer unique targets for adjuvant treatments to treat and cure bone metastases.     

PD-1/PD-L1与胶质母细胞瘤关系的研究进展

胶质母细胞瘤（glioblastoma,GBM）是目前已知最具侵袭性和致命性的原发脑肿瘤,虽然进行了广泛的研究,预后依旧很差,中位生存期仅为15个月左右。而随着人们对血脑屏障的进一步认知以及中枢神经系统与颈深部淋巴系统密切联系的发现,使得GBM患者可能在免疫治疗这种新颖治疗方式中获得受益。PD-1/PD-L1为免疫调节轴上最主要的靶点,针对PD-1/PD-L1的治疗模式在多种类型肿瘤中取得了突破性进展,但在GBM方面仍处于初始阶段。本文就PD-1/PD-L1检查点与GBM的密切关系进行系统描述,并整理、总结近年来应用于GBM的PD-1/PD-L1免疫抑制剂治疗的临床进展,以期为临床治疗提供参考。     

Interactions between microenvironment and cancer cells in two animal models of bone metastasis

The preferential proliferation of cancer cells in the bone microenvironment is poorly characterised. Expression pattern of bone marrow and other organ microenvironment in contact with osteolytic (Walker W256) and osteoblastic (MatLyLu MLL) metastases were investigated. Fisher and Copenhagen rats received, respectively, W256 and MLL cells injection. Bone and soft tissues were analysed by immunochemistry for DKK1, cathepsin K, RANKL, MCSF or IL6 expression. Tartrate-resistant acid phosphatase (TRAcP)-positive cells were detected by a histoenzymatic technique. In bone, expressions of MCSF and DKK1 were shown in stromal cells of the bone marrow, in contact with metastatic foci of both tumours. Many stromal cells were found RANKL positive in the vicinity of the tumours. Cells expressing cathepsin K and multinucleated TRAcP+ cells were found in direct contact with trabeculae but also in bone marrow spaces near metastatic cells. In extraosseous tumours, cells in contact with malignant cells did not expressed DKK1, MCSF, cathepsin K and IL6. Some RANKL+ cells were found in the periphery of subcutaneous tumours but may represent Langerhans cells. Abnormal presence of TRAcP+ cells was never observed in the vicinity of malignant cells. Interaction between stromal and cancer cells induces the expression on the formers of characteristics leading to osteoclastogenesis only in the bone microenvironment.     

The interaction between cancer associated fibroblasts and tumor associated macrophages via the osteopontin pathway in the tumor microenvironment of hepatocellular carcinoma

Background: Cancer-tumor associated macrophage (TAM)-cancer associated fibroblast (CAF) interactions are an important factor in the tumor microenvironment of hepatocellular carcinoma.Materials and Methods: Hepatic stellate cells (HSCs) were cultured with cancer cell-conditioned medium (Ca.-CM), TAM-CM and CAF-CM, and the expression of CAF markers were evaluated by RT-PCR. Whether HSCs cultured with Ca.-CM, TAM-CM and CAF-CM contributed to the enhanced malignancy of cancer cells was examined using proliferation, invasion and migration assays. Furthermore, the differences between these three types of CM were evaluated using cytokine arrays.Results: HSCs cultured with Ca.-CM, TAM-CM and CAF-CM showed significantly increased mRNA expression of αSMA, FAP and IL-6. All HSCs cultured with each CM exhibited significantly increased proliferation, invasion and migration of cancer cells. The osteopontin concentration was significantly higher in HSCs cultured with TAM-CM than the other CAF-CMs. Osteopontin inhibition significantly reduced osteopontin secretion from HSCs cultured with TAM-CM and suppressed the proliferation and invasion of cancer cells enhanced by HSCs cultured with TAM-CM.Conclusions: We observed enhanced osteopontin secretion from TAMs, and this increased osteopontin further promoted osteopontin secretion from HSCs cultured with TAM-CM, leading to increased malignancy. For the first time, we demonstrated the importance of cancer-TAM-CAF interactions via osteopontin in hepatocellular carcinoma.     

To grab the stroma by the horns: From biology to cancer therapy with mesenchymal stem cells

Mesenchymal stem or stromal cells (MSCs) are precursor cells that play important roles in tumorigenesis. MSCs are recruited to tumors from local and distant sources to form part of the tumor microenvironment. MSCs influence tumor progression by interacting with cancer cells, endothelial cells, immune cells, and cancer stem cells, in a context-dependent network. This review aims to synthesize this emerging yet controversial field to identify key questions regarding the mechanisms of MSC mobilization and survival in blood; homing to tumors, metastases, and premetastatic sites; spatiotemporal organization and differentiation; and interaction with immune cells and cancer stem cells. Understanding the fundamental biology underlying mesenchymal stem cell and tumor interactions has the potential to inform our knowledge of cancer initiation and progression as well as lead to novel therapeutics for cancer. Furthermore, knowledge of endogenous mechanisms can be used to “program” exogenous MSCs for targeted chemotherapeutic delivery to tumors and metastases. Emerging studies will provide crucial insight into the mechanisms of tumor interactions with the whole organism including MSCs.     

Inhibition of telomerase in the endothelial cells disrupts tumor angiogenesis in glioblastoma xenografts

Tumor angiogenesis is a complex process that involves a series of interactions between tumor cells and endothelial cells (ECs). In vitro, glioblastoma multiforme (GBM) cells are known to induce an increase in proliferation, migration and tube formation by the ECs. We have previously shown that in human GBM specimens the proliferating ECs of the tumor vasculature express the catalytic component of telomerase, hTERT, and that telomerase can be upregulated in human ECs by exposing these cells to GBM in vitro. Here, we developed a controlled in vivo assay of tumor angiogenesis in which primary human umbilical vascular endothelial cells (HUVECs) were subcutaneously grafted with or without human GBM cells in immunocompromised mice as Matrigel implants. We found that primary HUVECs did not survive in Matrigel implants, and that telomerase upregulation had little effect on HUVEC survival. In the presence of GBM cells, however, the grafted HUVECs not only survived in Matrigel implants but developed tubule structures that integrated with murine microvessels. Telomerase upregulation in HUVECs enhanced such effect. More importantly, inhibition of telomerase in HUVECs completely abolished tubule formation and greatly reduced survival of these cells in the tumor xenografts. Our data demonstrate that telomerase upregulation by the ECs is a key requisite for GBM tumor angiogenesis.     

MGMT modulates glioblastoma angiogenesis and response to the tyrosine kinase inhibitor sunitinib

Angiogenesis inhibitors, such as sunitinib, represent a promising strategy to improve glioblastoma (GBM) tumor response. In this study, we used the O⁶-methylguanine methyltransferase (MGMT)-negative GBM cell line U87MG stably transfected with MGMT (U87/MGMT) to assess whether MGMT expression affects the response to sunitinib. We showed that the addition of sunitinib to standard therapy (temozolomide [TMZ] and radiation therapy [RT]) significantly improved the response of MGMT-positive but not of MGMT-negative cells. Gene expression profiling revealed alterations in the angiogenic profile, as well as differential expression of several receptor tyrosine kinases targeted by sunitinib. MGMT-positive cells displayed higher levels of vascular endothelial growth factor receptor 1 (VEGFR-1) compared with U87/EV cells, whereas they displayed decreased levels of VEGFR-2. Depleting MGMT using O⁶-benzylguanine suggested that the expression of these receptors was directly related to the MGMT status. Also, we showed that MGMT expression was associated with a dramatic increase in the soluble VEGFR-1/VEGFA ratio, thereby suggesting a decrease in bioactive VEGFA and a shift towards an antiangiogenic profile. The reduced angiogenic potential of MGMT-positive cells is supported by: (i) the decreased ability of their secreted factors to induce endothelial tube formation in vitro and (ii) their low tumorigenicity in vivo compared with the MGMT-negative cells. Our study is the first to show a direct link between MGMT expression and decreased angiogenicity and tumorigenicity of GBM cells and suggests the combination of sunitinib and standard therapy as an alternative strategy for GBM patients with MGMT-positive tumors.     

Connective tissue growth factor associated with oncogenic activities and drug resistance in glioblastoma multiforme

Connective tissue growth factor (CTGF or CCN2) is a secreted protein that belongs to the CCN [cysteine‐rich CYR61/CTGF/nephroblastoma‐overexpressed gene] family. These proteins have been implicated in various biological processes, including stimulation of cell proliferation, migration, angiogenesis and tumorigenesis. In a previous study, we found that CTGF mRNA was elevated in primary gliomas, and a significant correlation existed between CTGF mRNA levels versus tumor grade, histology and patient survival. In this study, the role of CTGF in glioma tumorigenesis was explored. Forced expression of CTGF in glioblastoma multiforme (GBM) cells accelerated their growth in liquid culture and soft agar, stimulated cells migration in Boyden chamber assays and significantly increased their ability to form large, vascularized tumors in nude mice. CTGF induced the expression of the antiapoptotic proteins, Bcl‐xl, Survivin and Flip. Overexpression of CTGF caused the U343 GBM cells to survive for longer than 40 days in serum‐free medium and resist antitumor drugs including tumor necrosis factor (TNF), TNF‐related apoptosis‐inducing ligand, VELCADE (bortezomib, proteasome inhibitor) and temozolomide. Our data suggest that CTGF plays an important role in glioma progression, by supporting tumor cells survival and drug resistance.