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Contains fulltext : 205270.pdf (publisher's version ) (Open Access)

Radboud University, 15 mei 2019, Promotores : Vries, I.J.M. de, Massuger, L.F.A.G. Co-promotores : Torensma, R., Altena, A.M. van, Contains fulltext : 202628.pdf (publisher's version ) (Open Access)

Contains fulltext : 247101.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 190940.pdf (publisher's version ) (Open Access), Lipid metabolism is altered in several cancer settings leading to different ratios of intermediates. Ovarian cancer is the most lethal gynecological malignancy. Cancer cells disperse in the abdominal space and ascites occurs. T cells obtained from ascites are unable to proliferate after an antigenic stimulus. The proliferation of ascites-derived T cells can be restored after culturing the cells for ten days in normal culture medium. No pathway aberrancies were detected. The acellular fraction of ascites can inhibit the proliferation of autologous as well as allogeneic peripheral blood lymphocytes, indicating the presence of soluble factors that interfere with T cell functionality. Therefore, we analyzed 109 lipid mediators and found differentially regulated lipids in suppressive ascitic fluid compared to normal abdominal fluid. Our study indicates the presence of lipid intermediates in ascites of ovarian cancer patients, which coincidences with T cell dysfunctionality. Since the immune system in the abdominal cavity is compromised, this may explain the high seeding efficiency of disseminated tumor cells. Further research is needed to fully understand the correlation between the various lipids and T cell proliferation, which could lead to new treatment options.

Contains fulltext : 196226.pdf (publisher's version ) (Open Access), Cancer stem cells (CSCs) have been identified as the source of tumor growth and disease recurrence. Eradication of CSCs is thus essential to achieve durable responses, but CSCs are resistant to current anti-tumor therapies. Novel therapeutic approaches that specifically target CSCs will, therefore, be crucial to improve patient outcome. Immunotherapies, which boost the body's own immune system to eliminate cancerous cells, could be an alternative approach to target CSCs. Vaccines of dendritic cells (DCs) loaded with tumor antigens can evoke highly specific anti-tumor T cell responses. Importantly, DC vaccination also promotes immunological memory formation, paving the way for long-term cancer control. Here, we propose a DC vaccination that specifically targets CSCs. DCs loaded with NANOG peptides, a protein required for maintaining stem cell properties, could evoke a potent anti-tumor immune response against CSCs. We hypothesize that the resulting immunological memory will also control newly formed CSCs, thereby preventing disease recurrence.

Item does not contain fulltext, AIM: Dendritic cells rapidly capture nanoparticles and induce a potent cellular immune response. It is yet unknown whether the immunological response induced by slow release of encapsulated versus soluble antigen and adjuvant is superior. MATERIALS & METHODS: The kinetics of poly(lactic-co-glycolic acid) PLGA nanoparticles antigen release was studied by the DQ-bovine serum albumin (BSA) self-quenching antigen model. The immunological response induced was evaluated by means of dendritic cell activation/maturation markers, cytokine production and their ability to drive antigen-specific T-cell proliferation. RESULTS & CONCLUSION: PLGA-encapsulated antigen and adjuvant showed an enhanced T-cell response when compared with soluble vaccine components by increasing antigenicity and adjuvanticity. Although the kinetic profile followed the same pattern, encapsulation increased strength and duration of the response.

Contains fulltext : 178035.pdf (publisher's version ) (Open Access)

Contains fulltext : 171662.pdf (Publisher’s version ) (Open Access), Antibodies are widely exploited as research/diagnostic tools and therapeutics. Despite providing exciting research opportunities, the multitude of available antibodies also offers a bewildering array of choice. Importantly, not all companies comply with the highest standards, and thus many reagents fail basic validation tests. The responsibility for antibodies being fit for purpose rests, surprisingly, with their user. This paper condenses the extensive experience of the European Monoclonal Antibody Network to help researchers identify antibodies specific for their target antigen. A stepwise strategy is provided for prioritising antibodies and making informed decisions regarding further essential validation requirements. Web-based antibody validation guides provide practical approaches for testing antibody activity and specificity. We aim to enable researchers with little or no prior experience of antibody characterization to understand how to determine the suitability of their antibody for its intended purpose, enabling both time and cost effective generation of high quality antibody-based data fit for publication.

Item does not contain fulltext, The tumor microenvironment is characterized by regulatory T cells, type II macrophages, myeloid-derived suppressor cells, and other immunosuppressive cells that promote malignant progression. Here we report the identification of a novel BDCA1(+)CD14(+) population of immunosuppressive myeloid cells that are expanded in melanoma patients and are present in dendritic cell-based vaccines, where they suppress CD4(+) T cells in an antigen-specific manner. Mechanistic investigations showed that BDCA1(+)CD14(+) cells expressed high levels of the immune checkpoint molecule PD-L1 to hinder T-cell proliferation. While this BDCA1(+)CD14(+) cell population expressed markers of both BDCA1(+) dendritic cells and monocytes, analyses of function, transcriptome, and proteome established their unique nature as exploited by tumors for immune escape. We propose that targeting these cells may improve the efficacy of cancer immunotherapy. Cancer Res; 76(15); 4332-46. (c)2016 AACR.

Contains fulltext : 172739.pdf (Publisher’s version ) (Open Access)

Item does not contain fulltext, Ovarian cancer is a devastating disease with a high relapse rate. Due to a mostly asymptomatic early stage and lack of early diagnostic tools, the disease is usually diagnosed in a late stage. Surgery and chemotherapy with taxanes and platinum compounds are very effective in reducing tumor burden. However, relapses occur frequently and there is a lack of credible second-line options. Therefore, new treatment modalities are eagerly awaited. The presence and influx of immune cells in the ovarian cancer tumor microenvironment are correlated with survival. High numbers of infiltrating T cells correlate with improved progression free and overall survival, while the presence of regulatory T cells and expression of T cell inhibitory molecules is correlated with a poor prognosis. These data indicate that immunotherapy, especially cell-based immunotherapy could be a promising novel addition to the treatment of ovarian cancer. Here, we review the available data on the immune contexture surrounding ovarian cancer and discuss novel strategies and targets for immunotherapy in ovarian cancer. In the end the addition of immunotherapy to existing therapeutic options could lead to a great improvement in the outcome of ovarian cancer, especially when targeting cancer stem cells.

Contains fulltext : 153018.pdf (Publisher’s version ) (Open Access)

Item does not contain fulltext, Cancer cells evolve from normal cells throughout life and are usually recognized by our immune system and destroyed, a process called immunosurveillance. Unfortunately, in some instances cancer cells paralyze our immune system, resulting in outgrowth and spreading of the tumor. Understanding the complexity of immunomodulation by tumors is important for the development of therapeutical strategies. Nowadays, various approaches have been developed to enhance anti-tumor immune responses and abrogate the immune dampening effect of the tumor and its surrounding environment, including dendritic cell-based vaccines, therapies to counteract myeloid derived suppressor cell function within the tumor and antagonists of inhibitory signaling pathways to overcome 'immune checkpoints'. The challenge is now to find the right combination of immune based therapies to fully restore immune function and provide a more efficacious and enduring anti-tumor response.

Item does not contain fulltext, OBJECTIVE: Glycogen synthase kinase-3beta (Gsk-3beta)/beta-catenin signaling regulates development of the secondary palate. It has been unclear about the effects of Gsk-3beta/beta-catenin signaling on palatal fusion and osteogenic differentiation in palatal shelves. DESIGN: In this study, palatal shelves from mouse embryonic day 13 (E13) were cultured in vitro with or without lithium chloride (LiCl). Palatal fusion was evaluated by haematoxylin-eosin staining. The expression of osteogenic markers in palatal shelves was measured by quantitative PCR, and immunohistochemical staining. Cell proliferation and apoptosis were examined by Ki-67 immunohistochemical and TUNEL staining, respectively. Gsk-3beta expression was evaluated by quantitative PCR and Western blotting. beta-catenin protein expression was evaluated by Western blotting. RESULTS: After the treatment with 10 mM LiCl, palatal shelves failed to fuse, and the mRNA and protein levels of osteogenic markers were reduced compared with controls. The number of Ki67-positive cell in the palatal osteoid was significantly higher in the LiCl group than in the controls. The apoptotic cells in the midline epithelial seam were reduced by LiCl. Gsk-3beta mRNA and protein expression levels decreased and beta-catenin protein expression levels increased by treatment of LiCl. CONCLUSION: Our findings show that LiCl-mediated GSK3beta inhibition prevents palatal fusion and osteogenic differentiation in palatal shelves by increased beta-catenin signaling. It indicated that overactivation of canonical Wnt signaling might impair the fusion of the secondary palate.

Item does not contain fulltext, Immune suppression is a very stable property of multipotent stromal cells also known as mesenchymal stem cells (MSCs). All cell lines tested showed robust immune suppression not affected by a long culture history. Several mechanisms were described to account for this capability. Since several of the described mechanisms were not causing the immune suppression, the expression pattern of cord-blood-derived MSCs by microarray experiments was determined. Dendritic cells cocultured with cord blood MSCs were compared with cord blood MSCs. Putative immune suppressive candidates were tested to explain this inhibition. We find that cord blood MSCs themselves are hardly immunogenic as tested with allogeneic T-cells. Dendritic cells cocultured with second-party T-cells evoked abundant proliferation that was inhibited by third-party cord blood MSCs. Optimal inhibition was seen with one cord blood MSC for every dendritic cell. Blocking human leukocyte antigen G only saw partial recovery of proliferation. Several cytokines, gangliosides, enzymes like arginase, NO synthase, and indole amine 2,3-dioxygenase as well as the induction of Treg were not involved in the inhibition. The inhibiting moiety was identified as prostaglandin B2 by lipid metabolite analysis of the culture supernatant and confirmed with purified prostaglandin B2.

Contains fulltext : 125214.pdf (publisher's version ) (Open Access), Ovarian cancer accounts for only 3% of all cancers in women, but it causes more deaths than any other gynecologic cancer. Treatment with chemotherapy and cytoreductive surgery shows a good response to the therapy. However, in a large proportion of the patients the tumor grows back within a few years. Cancer stem cells, that are less responsive to these treatments, are blamed for this recurrence of disease. Immune therapy either cellular or humoral is a novel concept to treat cancer. It is based on the notice that immune cells invade the tumor. However, the tumor invest heavily to escape from immune elimination by recruiting several immune suppressive mechanisms. These processes are normally in place to limit excessive immune activation and prevent autoimmune phenomena. Here, we discuss current knowledge about the immune (suppressive) status in ovarian cancer. Moreover, we discuss the immunological targets of ovarian cancer stem cells.

Item does not contain fulltext, The identification of growth and differentiation pathways that are responsible for the proliferation and survival of cancer stem cells (CSCs) has opened avenues for the discovery of novel therapeutic targets. In the initial phase of an anticancer immune response, T cells specific for tumor-associated antigens develop in patients and, at least under selected circumstances, are able to eliminate malignant cells. However, it remains unknown whether CSC-specific T cells are also operational. We found naturally occurring multifunctional CD4+ and CD8+ T cells specific for the stem cell marker OCT4 among the peripheral blood mononuclear cells (PBMCs) of both healthy individuals and ovarian cancer patients. Moreover, lymphocytes isolated from the ascites of patients affected by ovarian malignancies also contained OCT4-specific T cells. OCT4-reactive CD4+ T cells did not produce interferon gamma (IFNgamma) and IFNgamma-inducible protein 10 (IP-10) but were capable of proliferation upon stimulation with dendritic cells (DCs) loaded with an OCT4-derived peptide or OCT4 mRNA. OCT4-reactive CD8+ cells did not proliferate in response to a similar challenge, yet produced IP-10 as well as sufficient amounts of IFNgamma to induce IP-10 . Furthermore, CD8+ cytotoxic T cells were able to release their lysosomal components, as indicated by the mobilization of CD107a. These results demonstrate the existence of anti-CSC specific T cells in ovarian cancer patients.

Radboud Universiteit Nijmegen, 01 oktober 2013, Promotor : Figdor, C.G. Co-promotores : Torensma, R., Spriel, A.B. van, Contains fulltext : 118381.pdf (Publisher’s version ) (Open Access)

Item does not contain fulltext, Bone marrow (BM) stromal cells (MSCs), also known as mesenchymal stem cells, display a high degree of heterogeneity. To shed light on the causes of this heterogeneity, MSCs were collected from either human BM (n=5) or adipose tissue (AT) (n=5), and expanded using 2 different culture methods: one based on fetal calf serum, and one based on human platelet lysate. After initial expansion, MSCs were frozen, and the vials were transported to 3 different laboratories and grown for 1 passage using the same brand of culture plastic, medium, and supplements. Subsequently, the cells were harvested and assayed for their gene expression profile using the Affymetrix exon microarray platform. Based on gene expression profiles, the most discriminative feature was the anatomical harvesting site, followed by culture methodology. Remarkably, genes in the WNT pathway were expressed at higher levels in BM-derived MSCs than in AT-derived MSCs. Although differences were found between laboratories, cell culture location only slightly affects heterogeneity. Furthermore, individual donors contributed marginally to the observed differences in transcriptomes. Finally, BM-derived MSCs displayed the highest level of similarity, irrespective their culture conditions, when compared to AT-derived cells.

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Radboud Universiteit Nijmegen, 23 januari 2013, Promotores : Vries, I.J.M. de, Figdor, C.G. Co-promotores : Torensma, R., Schreibelt, G., Contains fulltext : 101057.pdf (publisher's version ) (Open Access)

Contains fulltext : 118796.pdf (publisher's version ) (Open Access), Bone marrow-derived mesenchymal stem cells (MSCs) have the capacity to regenerate renal tubule epithelia and repair renal function without fusing with resident tubular cells. The goal of the present project was to investigate the role of MSCs secreted cytokines on tubule cell viability and regeneration after a toxic insult, using a conditionally immortalized human proximal tubule epithelial cell (ciPTEC) line. Gentamicin was used to induce nephrotoxicity, and cell viability and migration were studied in absence and presence of human MSC-conditioned medium (hMSC-CM) i.e. medium containing soluble factors produced and secreted by MSCs. Exposure of ciPTEC to 0-3000mug/ml gentamicin for 24h caused a significant dose-dependent increase in cell death. We further demonstrated that the nephrotoxic effect of 2000mug/ml gentamicin was recovered partially by exposing cells to hMSC-CM. Moreover, exposure of ciPTEC to gentamicin (1500-3000mug/ml) for 7 days completely attenuated the migratory capacity of the cells. In addition, following scrape-wounding, cell migration of both untreated and gentamicin-exposed cells was increased in the presence of hMSC-CM, as compared to exposures to normal medium, indicating improved cell recovery. Our data suggest that cytokines secreted by MSCs stimulate renal tubule cell regeneration after nephrotoxicity.

Contains fulltext : 127334.pdf (publisher's version ) (Closed access), PURPOSE: The aim of this study was to examine the expression of established stem cell markers in ascites and tumor tissue obtained from ovarian cancer patients. METHODS: Mononuclear cells present in ascites were collected by density gradient centrifugation. Intracellular flowcytometry was used to assess the putative presence of stem cell markers. RT-PCR was used to detect full length Oct4A, a splice variant Oct4B, implicated in glioma and breast cancer, Oct4 pseudogenes and c-Myc. Genes were cloned and sequenced to determine putative mutations. Confocal laser scanning microscopy was performed to localize the markers in ascites cells as well as in tumor tissue. Material from carcinomas other than epithelial ovarian carcinoma served as control. RESULTS: A small quantity of cells in ascites and in tumor tissue of ovarian cancer patients was detected that expresses c-Myc, Oct4A and Nanog. Besides Oct4A, present in the nucleus, also the cytoplasmic resident Oct4B splice variant was detected. Remarkably, c-Myc was found partially in the cytoplasm. Since no mutations in c-Myc were found that could explain the cytoplasmic localization, we hypothesize that this is due an IL-6 induced c-Myc shuttle factor. CONCLUSIONS: The expression of stem cell genes was detected in a small proportion of tumor cells present in ascites as well as in tumor tissue. IL-6 plays an important role in the induction of c-Myc.

Contains fulltext : 117752.pdf (publisher's version ) (Open Access), Many processes regulating immune responses are initiated by G-protein coupled receptors (GPCRs) and report biochemical changes in the microenvironment. Dendritic cells (DCs) are the most potent antigen-presenting cells and crucial for the regulation of innate and adaptive immune responses. The lipid mediator Prostaglandin E2 (PGE2) via four GPCR subtypes (EP1-4) critically regulates DC generation, maturation and migration. The role of PGE2 signaling in DC biology was unraveled by the characterization of EP receptor subtype expression in DC progenitor cells and DCs, the identification of the signaling pathways initiated by these GPCR subtypes and the classification of DC responses to PGE2 at different stages of differentiation. Here, we review the advances in PGE2 signaling in DCs and describe the efforts still to be made to understand the spatio-temporal fine-tuning of PGE2 responses by DCs.

Radboud Universiteit Nijmegen, 29 maart 2012, Promotores : Kuijpers-Jagtman, A.M., Katsaros, C. Co-promotores : Hoff, J.W. Von den, Torensma, R., Contains fulltext : 92739.pdf (publisher's version ) (Open Access)

1 september 2012, Item does not contain fulltext, Human dendritic cells (DCs) infiltrate solid tumors, but this infiltration occurs in favorable and unfavorable disease prognoses. The statistical inference is that tumor-infiltrating DCs (TIDCs) play no conclusive role in predicting disease progression. This is remarkable because DCs are highly specialized antigen-presenting cells linking innate and adaptive immunity. DCs either boost the immune system (enhancing immunity) or dampen it (leading to tolerance). This dual effect explains the dual outcomes of cancer progression. The reverse functional characteristics of DCs depend on their maturation status. This review elaborates on the markers used to detect DCs in tumors. In many cases, the identification of DCs in human cancers relies on staining for S-100 and CD1a. These two markers are mainly expressed by Langerhans cells, which are one of several functionally different DC subsets. The activation status of DCs is based on the expression of CD83, DC-SIGN, and DC-LAMP, which are nonspecific markers of DC maturation. The detection of TIDCs has not kept pace with the increased knowledge about the identification of DC subsets and their maturation status. Therefore, it is difficult to draw a conclusion about the performance of DCs in tumors. We suggest a novel selection of markers to distinguish human DC subsets and maturation states. The use of these biomarkers will be of pivotal importance to scrutinize the prognostic significance of TIDCs.