Publications by authors named "Anna Darabi"

37 Publications

Modeling SHH-driven medulloblastoma with patient iPS cell-derived neural stem cells.

Proc Natl Acad Sci U S A 2020 08 3;117(33):20127-20138. Epub 2020 Aug 3.

Department of Microbiology, Tumor and Cell biology (MTC), Karolinska Institutet, 171 65 Stockholm, Sweden;

Medulloblastoma is the most common malignant brain tumor in children. Here we describe a medulloblastoma model using Induced pluripotent stem (iPS) cell-derived human neuroepithelial stem (NES) cells generated from a Gorlin syndrome patient carrying a germline mutation in the sonic hedgehog (SHH) receptor We found that Gorlin NES cells formed tumors in mouse cerebellum mimicking human medulloblastoma. Retransplantation of tumor-isolated NES (tNES) cells resulted in accelerated tumor formation, cells with reduced growth factor dependency, enhanced neurosphere formation in vitro, and increased sensitivity to Vismodegib. Using our model, we identified to be a GLI target gene that is up-regulated in both Gorlin tNES cells and SHH-subgroup of medulloblastoma patients. Taken together, we demonstrate that NES cells derived from Gorlin patients can be used as a resource to model medulloblastoma initiation and progression and to identify putative targets.
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http://dx.doi.org/10.1073/pnas.1920521117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443968PMC
August 2020

Convection-enhanced delivery of temozolomide and whole cell tumor immunizations in GL261 and KR158 experimental mouse gliomas.

BMC Cancer 2020 Jan 3;20(1). Epub 2020 Jan 3.

Glioma Immunotherapy Group, Division of Neurosurgery, Department of Clinical Sciences, Lund University, Barngatan 4, 221-85, Lund, Sweden.

Background: Glioblastomas (GBM) are therapy-resistant tumors with a profoundly immunosuppressive tumor microenvironment. Chemotherapy has shown limited efficacy against GBM. Systemic delivery of chemotherapeutic drugs is hampered by the difficulty of achieving intratumoral levels as systemic toxicity is a dose-limiting factor. Although some of its effects might be mediated by immune reactivity, systemic chemotherapy can also inhibit induced or spontaneous antitumor immune reactivity. Convection-enhanced delivery of temozolomide (CED-TMZ) can tentatively increase intratumoral drug concentration while reducing systemic side effects. The objective of this study was to evaluate the therapeutic effect of intratumorally delivered temozolomide in combination with immunotherapy and whether such therapy can generate a cellular antitumor immune response.

Methods: Single bolus intratumoral injection and 3-day mini-osmotic pumps (Alzet®) were used to deliver intratumoral TMZ in C57BL6 mice bearing orthotopic gliomas. Immunotherapy consisted of subcutaneous injections of irradiated GL261 or KR158 glioma cells. Tumor size and intratumoral immune cell populations were analyzed by immunohistochemistry.

Results: Combined CED-TMZ and immunotherapy had a synergistic antitumor effect in the GL261 model, compared to CED-TMZ or immunotherapy as monotherapies. In the KR158 model, immunization cured a small proportion of the mice whereas addition of CED-TMZ did not have a synergistic effect. However, CED-TMZ as monotherapy prolonged the median survival. Moreover, TMZ bolus injection in the GL261 model induced neurotoxicity and lower cure rate than its equivalent dose delivered by CED. In addition, we found that T-cells were the predominant cells responsible for the TMZ antitumor effect in the GL261 model. Finally, CED-TMZ combined with immunotherapy significantly reduced tumor volume and increased the intratumoral influx of T-cells in both models.

Conclusions: We show that immunotherapy synergized with CED-TMZ in the GL261 model and cured animals in the KR158 model. Single bolus administration of TMZ was effective with a narrower therapeutic window than CED-TMZ. Combined CED-TMZ and immunotherapy led to an increase in the intratumoral influx of T-cells. These results form part of the basis for the translation of the therapy to patients with GBM but the dosing and timing of delivery will have to be explored in depth both experimentally and clinically.
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http://dx.doi.org/10.1186/s12885-019-6502-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6942363PMC
January 2020

The molecular landscape of ETMR at diagnosis and relapse.

Nature 2019 12 4;576(7786):274-280. Epub 2019 Dec 4.

Department of Pathology, St Jude Children's Research Hospital, Memphis, TN, USA.

Embryonal tumours with multilayered rosettes (ETMRs) are aggressive paediatric embryonal brain tumours with a universally poor prognosis. Here we collected 193 primary ETMRs and 23 matched relapse samples to investigate the genomic landscape of this distinct tumour type. We found that patients with tumours in which the proposed driver C19MC was not amplified frequently had germline mutations in DICER1 or other microRNA-related aberrations such as somatic amplification of miR-17-92 (also known as MIR17HG). Whole-genome sequencing revealed that tumours had an overall low recurrence of single-nucleotide variants (SNVs), but showed prevalent genomic instability caused by widespread occurrence of R-loop structures. We show that R-loop-associated chromosomal instability can be induced by the loss of DICER1 function. Comparison of primary tumours and matched relapse samples showed a strong conservation of structural variants, but low conservation of SNVs. Moreover, many newly acquired SNVs are associated with a mutational signature related to cisplatin treatment. Finally, we show that targeting R-loops with topoisomerase and PARP inhibitors might be an effective treatment strategy for this deadly disease.
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http://dx.doi.org/10.1038/s41586-019-1815-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908757PMC
December 2019

Extracellular lipid loading augments hypoxic paracrine signaling and promotes glioma angiogenesis and macrophage infiltration.

J Exp Clin Cancer Res 2019 Jun 7;38(1):241. Epub 2019 Jun 7.

Department of Clinical Sciences Lund, Section of Oncology and Pathology, Lund University, Barngatan 4, SE-221 85, Lund, Sweden.

Background: Primary brain tumors, in particular glioblastoma (GBM), remain among the most challenging cancers. Like most malignant tumors, GBM is characterized by hypoxic stress that triggers paracrine, adaptive responses, such as angiogenesis and macrophage recruitment, rescuing cancer cells from metabolic catastrophe and conventional oncological treatments. The unmet need of strategies to efficiently target tumor "stressness" represents a strong clinical motivation to better understand the underlying mechanisms of stress adaptation. Here, we have investigated how lipid loading may be involved in the paracrine crosstalk between cancer cells and the stromal compartment of the hypoxic tumor microenvironment.

Methods: Regions from patient GBM tumors with or without the lipid loaded phenotype were isolated by laser capture microdissection and subjected to comparative gene expression analysis in parallel with cultured GBM cells with or without lipid loading. The potential involvement of extracellular lipids in the paracrine crosstalk with stromal cells was studied by immunoprofiling of the secretome and functional studies in vitro as well as in various orthotopic GBM mouse models, including hyperlipidemic ApoE-/- mice. Statistical analyses of quantitative experimental methodologies were performed using unpaired Student's T test. For survival analyses of mouse experiments, log-rank test was used, whereas Kaplan-Meier was performed to analyze patient survival.

Results: We show that the lipid loaded niche of GBM patient tumors exhibits an amplified hypoxic response and that the acquisition of extracellular lipids by GBM cells can reinforce paracrine activation of stromal cells and immune cells. At the functional level, we show that lipid loading augments the secretion of e.g. VEGF and HGF, and may potentiate the cross-activation of endothelial cells and macrophages. In line with these data, in vivo studies suggest that combined local tumor lipid loading and systemic hyperlipidemia of ApoE-/- mice receiving a high fat diet induces tumor vascularization and macrophage recruitment, and was shown to significantly decrease animal survival.

Conclusions: Together, these data identify extracellular lipid loading as a potentially targetable modulator of the paracrine adaptive response in the hypoxic tumor niche and suggest the contribution of the distinct lipid loaded phenotype in shaping the glioma microenvironment.
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http://dx.doi.org/10.1186/s13046-019-1228-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6556032PMC
June 2019

The effect of locally delivered cisplatin is dependent on an intact immune function in an experimental glioma model.

Sci Rep 2019 04 4;9(1):5632. Epub 2019 Apr 4.

Glioma Immunotherapy Group, Division of Neurosurgery, Department of Clinical Sciences, Lund University, Lund, Sweden.

Several chemotherapeutic drugs are now considered to exert anti-tumour effects, by inducing an immune-promoting inflammatory response. Cisplatin is a potent chemotherapeutic agent used in standard medulloblastoma but not glioblastoma protocols. There is no clear explanation for the differences in clinical efficacy of cisplatin between medulloblastomas and glioblastomas, despite the fact that cisplatin is effective in vitro against the latter. Systemic toxicity is often dose limiting but could tentatively be reduced by intratumoral administration. We found that intratumoral cisplatin can cure GL261 glioma-bearing C57BL/6 mice and this effect was abolished in GL261-bearing NOD-scid IL2rγ (NSG) mice. Contrary to previous results with intratumoral temozolomide cisplatin had no additive or synergistic effect with whole cell either GL261 wild-type or GM-CSF-transfected GL261 cells whole cell vaccine-based immunotherapy. While whole tumour cell immunizations increased CD8 T-cells and decreased F4/80 macrophages intratumorally, cisplatin had no effect on these cell populations. Taken together, our results demonstrate that intratumoral cisplatin treatment was effective with a narrow therapeutic window and may be an efficient approach for glioma or other brain tumour treatment.
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http://dx.doi.org/10.1038/s41598-019-42001-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6449367PMC
April 2019

Establishment and characterization of an orthotopic patient-derived Group 3 medulloblastoma model for preclinical drug evaluation.

Sci Rep 2017 04 18;7:46366. Epub 2017 Apr 18.

Lund University, Faculty of Medicine, Department of Clinical Sciences Lund, Neurosurgery, Lund, Sweden.

Medulloblastomas comprise a heterogeneous group of tumours and can be subdivided into four molecular subgroups (WNT, SHH, Group 3 and Group 4) with distinct prognosis, biological behaviour and implications for targeted therapies. Few experimental models exist of the aggressive and poorly characterized Group 3 tumours. In order to establish a reproducible transplantable Group 3 medulloblastoma model for preclinical therapeutic studies, we acquired a patient-derived tumour sphere culture and inoculated low-passage spheres into the cerebellums of NOD-scid mice. Mice developed symptoms of brain tumours with a latency of 17-18 weeks. Neurosphere cultures were re-established and serially transplanted for 3 generations, with a negative correlation between tumour latency and numbers of injected cells. Xenografts replicated the phenotype of the primary tumour, including high degree of clustering in DNA methylation analysis, high proliferation, expression of tumour markers, MYC amplification and elevated MYC expression, and sensitivity to the MYC inhibitor JQ1. Xenografts maintained maintained expression of tumour-derived VEGFA and stromal-derived COX-2. VEGFA, COX-2 and c-Myc are highly expressed in Group 3 compared to other medulloblastoma subgroups, suggesting that these molecules are relevant therapeutic targets in Group 3 medulloblastoma.
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http://dx.doi.org/10.1038/srep46366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5394470PMC
April 2017

Preoperative systemic levels of VEGFA, IL-7, IL-17A, and TNF-β delineate two distinct groups of children with brain tumors.

Pediatr Blood Cancer 2016 12 29;63(12):2112-2122. Epub 2016 Jul 29.

Glioma Immunotherapy Group, Faculty of Medicine, Department of Clinical Sciences Lund, Neurosurgery, Lund University, Lund, Sweden.

Background: Primary brain tumors are the most common solid tumors in children. Increasing evidence demonstrates diverse intratumoral immune signatures, which are tentatively reflected in peripheral blood.

Procedure: Twenty cytokines were analyzed in preoperative plasma samples from five healthy children and 45 children with brain tumors, using a multiplex platform (MesoScale Discovery V-PLEX ). Tumor types included medulloblastoma (MB), ependymoma, sarcoma, high-grade glioma, pilocytic astrocytoma, and other low-grade gliomas.

Results: A panel of four cytokines [VEGFA, interleukin (IL)-7, IL-17A, and tumor necrosis factor (TNF)-β] delineated two distinct patient groups, identified as VEGFA IL-7 IL-17A TNF-β (Group A) and VEGFA IL-7 IL-17A TNF-β (Group B). Healthy controls and the vast majority of patients with MB were found within Group A, whereas patients with other tumor types were equally distributed between the two groups. Unrelated to A/B affiliation, we detected trends toward increased IL-10 and decreased IL-12/23 and TNF-α in several tumor types. Finally, a small number of patients displayed evidence of enhanced systemic immune activation, including elevated levels of interferon-γ, granulocyte monocyte colony-stimulating factor, IL-6, IL-12/23, and TNF-α. Following tumor resection, cytokine levels in a MB patient approached the levels of healthy controls.

Conclusions: We identify common features and individual differences in the systemic immune profiles of children with brain tumors. Overall, patients with MB displayed a uniform cytokine profile, whereas other tumor diagnoses did not predict systemic immunological status in single patients. Future characterization and monitoring of systemic immune responses in children with brain tumors will have important implications for the development and implementation of immunotherapy.
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http://dx.doi.org/10.1002/pbc.26158DOI Listing
December 2016

Wnt/β-catenin pathway regulates MGMT gene expression in cancer and inhibition of Wnt signalling prevents chemoresistance.

Nat Commun 2015 Nov 25;6:8904. Epub 2015 Nov 25.

Department of Women's and Children's Health, Childhood Cancer Research Unit, Karolinska Institutet, Stockholm S-17176, Sweden.

The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is commonly overexpressed in cancers and is implicated in the development of chemoresistance. The use of drugs inhibiting MGMT has been hindered by their haematologic toxicity and inefficiency. As a different strategy to inhibit MGMT we investigated cellular regulators of MGMT expression in multiple cancers. Here we show a significant correlation between Wnt signalling and MGMT expression in cancers with different origin and confirm the findings by bioinformatic analysis and immunofluorescence. We demonstrate Wnt-dependent MGMT gene expression and cellular co-localization between active β-catenin and MGMT. Pharmacological or genetic inhibition of Wnt activity downregulates MGMT expression and restores chemosensitivity of DNA-alkylating drugs in mouse models. These findings have potential therapeutic implications for chemoresistant cancers, especially of brain tumours where the use of temozolomide is frequently used in treatment.
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http://dx.doi.org/10.1038/ncomms9904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4674781PMC
November 2015

A standardized and reproducible protocol for serum-free monolayer culturing of primary paediatric brain tumours to be utilized for therapeutic assays.

Sci Rep 2015 Jul 17;5:12218. Epub 2015 Jul 17.

Glioma Immunotherapy Group, Division of Neurosurgery, Department of Clinical Sciences Lund, Lund University, Lund, Sweden.

In vitro cultured brain tumour cells are indispensable tools for drug screening and therapeutic development. Serum-free culture conditions tentatively preserve the features of the original tumour, but commonly comprise neurosphere propagation, which is a technically challenging procedure. Here, we define a simple, non-expensive and reproducible serum-free cell culture protocol for establishment and propagation of primary paediatric brain tumour cultures as adherent monolayers. The success rates for establishment of primary cultures (including medulloblastomas, atypical rhabdoid tumour, ependymomas and astrocytomas) were 65% (11/17) and 78% (14/18) for sphere cultures and monolayers respectively. Monolayer culturing was particularly feasible for less aggressive tumour subsets, where neurosphere cultures could not be generated. We show by immunofluorescent labelling that monolayers display phenotypic similarities with corresponding sphere cultures and primary tumours, and secrete clinically relevant inflammatory factors, including PGE2, VEGF, IL-6, IL-8 and IL-15. Moreover, secretion of PGE2 was considerably reduced by treatment with the COX-2 inhibitor Valdecoxib, demonstrating the functional utility of our newly established monolayer for preclinical therapeutic assays. Our findings suggest that this culture method could increase the availability and comparability of clinically representative in vitro models of paediatric brain tumours, and encourages further molecular evaluation of serum-free monolayer cultures.
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http://dx.doi.org/10.1038/srep12218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505308PMC
July 2015

Al adjuvants can be tracked in viable cells by lumogallion staining.

J Immunol Methods 2015 Jul 17;422:87-94. Epub 2015 Apr 17.

Department of Biomedical Science, Faculty of Health and Society, Malmö University, Malmö SE-205 06, Sweden. Electronic address:

The mechanism behind the adjuvant effect of aluminum salts is poorly understood notwithstanding that aluminum salts have been used for decades in clinical vaccines. In an aqueous environment and at a nearly neutral pH, the aluminum salts form particulate aggregates, and one plausible explanation of the lack of information regarding the mechanisms could be the absence of an efficient method of tracking phagocytosed aluminum adjuvants and thereby the intracellular location of the adjuvant. In this paper, we want to report upon the use of lumogallion staining enabling the detection of phagocytosed aluminum adjuvants inside viable cells. Including micromolar concentrations of lumogallion in the culture medium resulted in a strong fluorescence signal from cells that had phagocytosed the aluminum adjuvant. The fluorescence appeared as spots in the cytoplasm and by confocal microscopy and co-staining with probes presenting fluorescence in the far-red region of the spectrum, aluminum adjuvants could to a certain extent be identified as localized in acidic vesicles, i.e., lysosomes. Staining and detection of intracellular aluminum adjuvants was achieved not only by diffusion of lumogallion into the cytoplasm, thereby highlighting the presence of the adjuvant, but also by pre-staining the aluminum adjuvant prior to incubation with cells. Pre-staining of aluminum adjuvants resulted in bright fluorescent particulate aggregates that remained fluorescent for weeks and with only a minor reduction of fluorescence upon extensive washing or incubation with cells. Both aluminum oxyhydroxide and aluminum hydroxyphosphate, two of the most commonly used aluminum adjuvants in clinical vaccines, could be pre-stained with lumogallion and were easily tracked intracellularly after incubation with phagocytosing cells. Staining of viable cells using lumogallion will be a useful method in investigations of the mechanisms behind aluminum adjuvants' differentiation of antigen-presenting cells into inflammatory cells. Information will be gained regarding the phagosomal pathways and the events inside the phagosomes, and thereby the ultimate fate of phagocytosed aluminum adjuvants could be resolved.
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http://dx.doi.org/10.1016/j.jim.2015.04.008DOI Listing
July 2015

Aberrant immunostaining pattern of the CD24 glycoprotein in clinical samples and experimental models of pediatric medulloblastomas.

J Neurooncol 2015 May 29;123(1):1-13. Epub 2015 Mar 29.

Glioma Immunotherapy Group, Division of Neurosurgery, Department of Clinical Sciences Lund, Lund University, Barngatan 2B, 221 85, Lund, Sweden,

The CD24 glycoprotein is a mediator of neuronal proliferation, differentiation and immune suppression in the normal CNS, and a proposed cancer biomarker in multiple peripheral tumor types. We performed a comparative analysis of CD24 gene expression in a large cohort of pediatric and adult brain tumors (n = 813), and further characterized protein expression in tissue sections (n = 39), primary brain tumor cultures (n = 12) and a novel orthotopic group 3 medulloblastoma xenograft model. Increased CD24 gene expression was demonstrated in ependymomas, medulloblastomas, anaplastic astrocytomas and glioblastomas, although medulloblastomas displayed higher expression than all other tumor entities. Preferential expression of CD24 in medulloblastomas was confirmed at protein level by immunostaining and computerized image analysis of cryosections. Morphologies and immunophenotyping of CD24(+) cells in tissue sections tentatively suggested disparate functions in different tumor subsets. Notably, protein staining of medulloblastoma cells was associated with prominent cytoplasmic and membranous granules, enabling rapid and robust identification of medulloblastoma cells in clinical tissue samples, as well as in experimental model systems. In conclusion, our results implicate CD24 as a clinically and experimentally useful medulloblastoma immunomarker. Although our results encourage further functional studies of CD24 as a potential molecular target in subsets of brain tumors, the promiscuous expression of CD24 in vivo highlights the importance of specificity in the future design of such targeted treatment.
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http://dx.doi.org/10.1007/s11060-015-1758-5DOI Listing
May 2015

Unequivocal identification of intracellular aluminium adjuvant in a monocytic THP-1 cell line.

Sci Rep 2014 Sep 5;4:6287. Epub 2014 Sep 5.

The Birchall Centre, Lennard-Jones Laboratories, Keele University, Keele, Staffordshire, ST5 5BG, UK.

Aluminium-based adjuvants (ABA) are the predominant adjuvants used in human vaccinations. While a consensus is yet to be reached on the aetiology of the biological activities of ABA several studies have identified shape, crystallinity and size as critical factors affecting their adjuvanticity. In spite of recent advances, the fate of ABA following their administration remains unclear. Few if any studies have demonstrated the unequivocal presence of intracellular ABA. Herein we demonstrate for the first time the unequivocal identification of ABA within a monocytic T helper 1 (THP-1) cell line, using lumogallion as a fluorescent molecular probe for aluminium. Use of these new methods revealed that particulate ABA was only found in the cell cytoplasm. Transmission electron microscopy revealed that ABA were contained within vesicle-like structures of approximately 0.5-1 μm in diameter.
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http://dx.doi.org/10.1038/srep06287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4155332PMC
September 2014

HLA-I antigen presentation and tapasin influence immune responses against malignant brain tumors - considerations for successful immunotherapy.

Anticancer Agents Med Chem 2014 ;14(8):1094-100

Lund University, BMC D14 Immunology, Tornavägen 10, 221 84 Lund, Sweden.

Human leukocyte antigen class I (HLA-I) presents antigenic peptides to cytotoxic CD8+ T cells (CTLs). This is a pivotal step in the generation of CTL responses. Both the quantity and quality of peptide-HLA-I (pHLA-I) complexes are crucial for CTL responses, but the level of HLA-I expression per se is also directly involved in dictating NK-cell responses. Antigen processing machinery (APM) proteins are involved in the maturation of HLA-I and in the selection of which peptides are - or are not - presented. Thus, these proteins are key players in shaping the immune response to cells in health and disease. In this review, we recap the most important features of APM components and their synergistic work to assure proper pHLA-I cell surface expression. We pay special attention to the HLA-I dedicated multifunctional protein, tapasin, and in relation to the different tapasin-dependency of HLA-I allomorphs we also discuss allomorph specific traits in maturation, structure and linkage to malignant diseases and brain tumors in particular. We next discuss the possibilities of restoring or manipulating the immune responses against brain tumors. In this context we discuss IFNγ therapy, cytostatics and irradiation. Finally, we integrate current views and knowledge to set the direction for future emphasis in the area of immunotherapy against brain tumors.
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http://dx.doi.org/10.2174/1871520614666140825110001DOI Listing
May 2015

Intratumorally implanted mesenchymal stromal cells potentiate peripheral immunotherapy against malignant rat gliomas.

J Neuroimmunol 2014 Sep 23;274(1-2):240-3. Epub 2014 Jul 23.

Bengzon Group, Lund Stem Cell Center, Division of Neurosurgery, Department of Clinical Sciences, Lund University, BMC B10, SE-221 84 Lund, Sweden.

Bone marrow-derived mesenchymal stromal cells (MSCs) target glioma extensions and micro-satellites efficiently when implanted intratumorally. Here, we report that intratumoral implantation of MSCs and peripheral immunotherapy with interferon-gamma (IFNγ) producing tumor cells improve the survival of glioma-bearing rats (54% cure rate) compared to MSC alone (0% cure rate) or immunotherapy alone (21% cure rate) by enforcing an intratumoral CD8(+) T cell response. Further analysis revealed that the MSCs up-regulate MHC classes I and II in response to IFNγ treatment in vitro and secrete low amounts of immunosuppressive molecules prostaglandin E2 and interleukin-10.
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http://dx.doi.org/10.1016/j.jneuroim.2014.07.014DOI Listing
September 2014

Enhancer hijacking activates GFI1 family oncogenes in medulloblastoma.

Nature 2014 Jul 22;511(7510):428-34. Epub 2014 Jun 22.

Data Management Facility, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, Heidelberg 69120, Germany.

Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery, radiation and chemotherapy, posing a considerable burden of toxicity to the developing child. Genomics has illuminated the extensive intertumoral heterogeneity of medulloblastoma, identifying four distinct molecular subgroups. Group 3 and group 4 subgroup medulloblastomas account for most paediatric cases; yet, oncogenic drivers for these subtypes remain largely unidentified. Here we describe a series of prevalent, highly disparate genomic structural variants, restricted to groups 3 and 4, resulting in specific and mutually exclusive activation of the growth factor independent 1 family proto-oncogenes, GFI1 and GFI1B. Somatic structural variants juxtapose GFI1 or GFI1B coding sequences proximal to active enhancer elements, including super-enhancers, instigating oncogenic activity. Our results, supported by evidence from mouse models, identify GFI1 and GFI1B as prominent medulloblastoma oncogenes and implicate 'enhancer hijacking' as an efficient mechanism driving oncogene activation in a childhood cancer.
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http://dx.doi.org/10.1038/nature13379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4201514PMC
July 2014

Immunizations with unmodified tumor cells and simultaneous COX-2 inhibition eradicate malignant rat brain tumors and induce a long-lasting CD8(+) T cell memory.

J Neuroimmunol 2014 Sep 28;274(1-2):161-7. Epub 2014 Jun 28.

Glioma Immunotherapy Group, Division of Neurosurgery, Department of Clinical Sciences, Lund University, Barngatan 2B, SE-221 85 Lund, Sweden.

Malignant brain tumors induce pronounced immunosuppression, which diminishes immune responses generated by immunotherapy. Here we report that peripheral immunotherapy, using irradiated unmodified whole tumor cells, and systemic cyclooxygenase-2 inhibition induce cure in glioma-bearing rats (60% cure rate), whereas neither monotherapy was sufficient to cure any animal. Moreover, the combined therapy protected against secondary tumor challenges (89% cure rate) and the secondary immune response was correlated with increased plasma interferon-gamma levels and CD8(+) T cells systemically and intratumorally. In conclusion, we demonstrate that cyclooxygenase-2 inhibition is sufficient to render unmodified tumor cells immunogenic in immunotherapy of experimental brain tumors.
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http://dx.doi.org/10.1016/j.jneuroim.2014.06.019DOI Listing
September 2014

Intratumoral COX-2 inhibition enhances GM-CSF immunotherapy against established mouse GL261 brain tumors.

Int J Cancer 2014 Jun 25;134(11):2748-53. Epub 2013 Nov 25.

Glioma Immunotherapy Group, Division of Neurosurgery, Department of Clinical Sciences, Lund University, Lund, Sweden.

Immunotherapy has shown effectiveness against experimental malignant brain tumors, but the clinical results have been less convincing most likely due to immunosuppression. Prostaglandin E2 (PGE2 ) is the key immunosuppressive product of cyclooxygenase-2 (COX-2) and increased levels of PGE2 and COX-2 have been shown in several tumor types, including brain tumors. In the current study, we report enhanced cure rate of mice with established mouse GL261 brain tumors when immunized with granulocyte macrophage-colony stimulating factor (GM-CSF) secreting tumor cells and simultaneously treated with the selective COX-2 inhibitors parecoxib systemically (5 mg/kg/day; 69% cure rate) or valdecoxib intratumorally (5.3 µg/kg/day; 63% cure rate). Both combined therapies induced a systemic antitumor response of proliferating CD4(+) and CD8(+) T cells, and further analysis revealed T helper 1 (Th 1) cell supremacy. The GL261 tumor cell line produced low levels of PGE2 in vitro, and co-staining at the tumor site demonstrated that a large fraction of the COX-2(+) cells were derived from CD45(+) immune cells and more specifically macrophages (F4/80(+)), indicating that tumor-infiltrating immune cells constitute the primary source of COX-2 and PGE2 in this model. We conclude that intratumoral COX-2 inhibition potentiates GM-CSF immunotherapy against established brain tumors at substantially lower doses than systemic administration. These findings underscore the central role of targeting COX-2 during immunotherapy and implicate intratumoral COX-2 as the primary target.
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http://dx.doi.org/10.1002/ijc.28607DOI Listing
June 2014

Aluminium based adjuvants and their effects on mitochondria and lysosomes of phagocytosing cells.

J Inorg Biochem 2013 Nov 9;128:229-36. Epub 2013 Aug 9.

Department of Biomedical Laboratory Science, Faculty of Health and Society, Malmö University, SE-205 06 Malmö, Sweden.

Aluminium oxyhydroxide, Al(OH)3 is one of few compounds approved as an adjuvant in human vaccines. However, the mechanism behind its immune stimulating properties is still poorly understood. In vitro co-culture of an aluminium adjuvant and the human monocytic cell line THP-1 resulted in reduced cell proliferation. Inhibition occurred at concentrations of adjuvant several times lower than would be found at the injection site using a vaccine formulation containing an aluminium adjuvant. Based on evaluation of the mitochondrial membrane potential, THP-1 cells showed no mitochondrial rupture after co-culture with the aluminium adjuvant, instead an increase in mitochondrial activity was seen. The THP-1 cells are phagocytosing cells and after co-culture with the aluminium adjuvant the phagosomal pathway was obstructed. Primary or early phagosomes mature into phagolysosomes with an internal pH of 4.5 - 5 and carry a wide variety of hydrolysing enzymes. Co-culture with the aluminium adjuvant yielded a reduced level of acidic vesicles and cathepsin L activity, a proteolytic enzyme of the phagolysosomes, was almost completely inhibited. THP-1 cells are an appropriate in vitro model in order to investigate the mechanism behind the induction of a phagocytosing antigen presenting cell into an inflammatory cell by aluminium adjuvants. Much information will be gained by investigating the phagosomal pathway and what occurs inside the phagosomes and to elucidate the ultimate fate of phagocytosed aluminium particles.
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http://dx.doi.org/10.1016/j.jinorgbio.2013.08.003DOI Listing
November 2013

PD-L1 expression by neurons nearby tumors indicates better prognosis in glioblastoma patients.

J Neurosci 2013 Aug;33(35):14231-45

Neuroinflammation Unit, Biotech Research and Innovation Centre, University of Copenhagen, DK-2200 Copenhagen, Denmark.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor. In general, tumor growth requires disruption of the tissue microenvironment, yet how this affects glioma progression is unknown. We studied program death-ligand (PD-L)1 in neurons and gliomas in tumors from GBM patients and associated the findings with clinical outcome. Remarkably, we found that upregulation of PD-L1 by neurons in tumor-adjacent brain tissue (TABT) associated positively with GBM patient survival, whereas lack of neuronal PD-L1 expression was associated with high PD-L1 in tumors and unfavorable prognosis. To understand the molecular mechanism of PD-L1 signaling in neurons, we investigated PD-L1 function in cerebellar and cortical neurons and its impact on gliomas. We discovered that neuronal PD-L1-induced caspase-dependent apoptosis of glioma cells. Because interferon (IFN)-β induces PD-L1 expression, we studied the functional consequences of neuronal Ifnb gene deletion on PD-L1 signaling and function. Ifnb-/- neurons lacked PD-L1 and were defective in inducing glioma cell death; this effect was reversed on PD-L1 gene transfection. Ifnb-/- mice with intracerebral isografts survived poorly. Similar to the observations in GBM patients, better survival in wild-type mice was associated with high neuronal PD-L1 in TABT and downregulation of PD-L1 in tumors, which was defective in Ifnb-/- mice. Our data indicated that neuronal PD-L1 signaling in brain cells was important for GBM patient survival. Reciprocal PD-L1 regulation in TABT and tumor tissue could be a prognostic biomarker for GBM. Understanding the complex interactions between tumor and adjacent stromal tissue is important in designing targeted GBM therapies.
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http://dx.doi.org/10.1523/JNEUROSCI.5812-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6618508PMC
August 2013

Intratumoral temozolomide synergizes with immunotherapy in a T cell-dependent fashion.

Cancer Immunol Immunother 2013 Sep 18;62(9):1463-74. Epub 2013 Jun 18.

Glioma Immunotherapy Group, Division of Neurosurgery, Department of Clinical Sciences in Lund, BMC D14, Lund University, 221 84, Lund, Sweden.

Despite temozolomide (TMZ) treatment, the prognosis for patients with glioblastoma multiforme is still dismal. As dose escalation of TMZ is limited by systemic toxicity, intratumoral delivery emerges as an attractive treatment modality, which may sustain cytotoxic drug concentrations intratumorally and induce immunogenic cell death. Both clinical and experimental gliomas have responded to immunotherapy, but the benefit of simultaneous chemo- and immunotherapy is inadequately studied. Here, we monitored survival of GL261-bearing C57BL/6 mice following a 3-day treatment with either intratumoral TMZ (micro-osmotic pump, 4.2 mg/kg/day) or systemic TMZ (i.p. injections, 50 mg/kg/day) alone, or combined with immunization using GM-CSF secreting GL261 cells. Peripheral and intratumoral leukocytes were analyzed by flow cytometry and immunohistochemistry. Intratumoral TMZ induced higher survival rate than systemic TMZ (45 vs. 8%). When T cells were depleted following intratumoral TMZ, the therapeutic effect was completely abrogated (0 % survival). Intratumoral TMZ synergistically increased survival rate of immunized mice (from 25 to 83%), while systemic TMZ failed (0%). While systemic TMZ induced a transient leukopenia, intratumoral TMZ and immunotherapy sustained the proliferation of CD8+ T cells and decreased the number of intratumoral immunosuppressive cells. In conclusion, intratumoral TMZ alone or in combination with immunotherapy could cure glioma-bearing mice, due to attenuation of local immunosuppression and increase in potential effector immune cells.
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http://dx.doi.org/10.1007/s00262-013-1449-zDOI Listing
September 2013

Absence of Epstein-Barr and cytomegalovirus infection in neuroblastoma cells by standard detection methodologies.

Pediatr Blood Cancer 2013 Sep 9;60(9):E91-3. Epub 2013 Apr 9.

Division of Clinical Genetics, Department of Laboratory Medicine, Lund University, Lund, Sweden.

Indications exist in the scientific literature that infection with human herpes family viruses may contribute to the pathogenesis of neuroblastoma (NB). However, systematic investigations regarding viral presence in NB cells have been scarcely reported. Here, the presence of DNA from Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) was assessed by PCR in 12 NBs, supplemented with RNA in situ hybridization, immunohistochemical detection, and high-throughput DNA sequencing. These standard methods did not detect infection by EBV or HCMV in NB cells in any tumor, while occasional immune cells were positive for EBV RNA or HCMV protein in four cases.
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http://dx.doi.org/10.1002/pbc.24535DOI Listing
September 2013

IFNγ in combination with IL-7 enhances immunotherapy in two rat glioma models.

J Neuroimmunol 2013 May 23;258(1-2):91-5. Epub 2013 Mar 23.

Division of Neurosurgery, Department of Clinical Sciences, Lund University, Lund, Sweden.

Peripheral immunization, using a combination of interferon-gamma (IFNγ)- and interleukin-7 (IL-7)-producing tumor cells, eradicated 75% of pre-established intracerebral N32 rat glioma tumors, and prolonged survival in the more aggressive RG2 model. Rats immunized with IFNγ- and IL7-transduced N32 cells displayed increases in IFNγ plasma levels and proliferating circulating T cells when compared with rats immunized with N32-wild type cells. Following irradiation, the expression of MHC I and II was high on N32-IFNγ cells, but low on RG2-IFNγ cells. In conclusion, IFNγ and IL-7 immunizations prolong survival in two rat glioma models.
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http://dx.doi.org/10.1016/j.jneuroim.2013.02.017DOI Listing
May 2013

Inhibition of cyclooxygenase-2 enhances immunotherapy against experimental brain tumors.

Cancer Immunol Immunother 2012 Aug 3;61(8):1191-9. Epub 2012 Jan 3.

Glioma Immunotherapy Group, The Rausing Laboratory, BMC D14, Divison of Neurosurgery, Department of Clinical Sciences, Lund University, Lund, Sweden.

Glioblastoma multiforme is the most common and aggressive malignant brain tumor in humans, and the prognosis is very poor despite conventional therapy. Immunotherapy represents a novel treatment approach, but the effect is often weakened by release of immune-suppressive molecules such as prostaglandins. In the current study, we investigated the effect of immunotherapy with irradiated interferon-γ (IFN-γ)-secreting tumor cells and administration of the selective cyclooxygenase-2 (COX-2) inhibitor parecoxib as treatment of established rat brain tumors. COX-2 inhibition and immunotherapy significantly enhanced the long-term cure rate (81% survival) compared with immunotherapy alone (19% survival), and there was a significant increase in plasma IFN-γ levels in animals treated with the combined therapy, suggesting a systemic T helper 1 immune response. COX-2 inhibition alone, however, did neither induce cure nor prolonged survival. The tumor cells were identified as the major source of COX-2 both in vivo and in vitro, and unmodified tumor cells produced prostaglandin E(2) in vitro, while the IFN-γ expressing tumor cells secreted significantly lower levels. In conclusion, we show that immunotherapy of experimental brain tumors is greatly potentiated when combined with COX-2 inhibition. Based on our results, the clinically available drug parecoxib may be added to immunotherapy against human brain tumors. Furthermore, the discovery that IFN-γ plasma levels can be used to determine the ongoing in vivo immune response has translational potential.
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http://dx.doi.org/10.1007/s00262-011-1196-yDOI Listing
August 2012

Detection of human cytomegalovirus in medulloblastomas reveals a potential therapeutic target.

J Clin Invest 2011 Oct 26;121(10):4043-55. Epub 2011 Sep 26.

Karolinska Institutet, Department of Women's and Children's Health, Childhood Cancer Research Unit, Stockholm, Sweden.

Medulloblastomas are the most common malignant brain tumors in children. They express high levels of COX-2 and produce PGE2, which stimulates tumor cell proliferation. Human cytomegalovirus (HCMV) is prevalent in the human population and encodes proteins that provide immune evasion strategies and promote oncogenic transformation and oncomodulation. In particular, HCMV induces COX-2 expression; STAT3 phosphorylation; production of PGE2, vascular endothelial growth factor, and IL-6; and tumor formation in vivo. Here, we show that a large proportion of primary medulloblastomas and medulloblastoma cell lines are infected with HCMV and that COX-2 expression, along with PGE2 levels, in tumors is directly modulated by the virus. Our analysis indicated that both HCMV immediate-early proteins and late proteins are expressed in the majority of primary medulloblastomas. Remarkably, all of the human medulloblastoma cell lines that we analyzed contained HCMV DNA and RNA and expressed HCMV proteins at various levels in vitro. When engrafted into immunocompromised mice, human medulloblastoma cells induced expression of HCMV proteins. HCMV and COX-2 expression correlated in primary tumors, cell lines, and medulloblastoma xenografts. The antiviral drug valganciclovir and the specific COX-2 inhibitor celecoxib prevented HCMV replication in vitro and inhibited PGE2 production and reduced medulloblastoma tumor cell growth both in vitro and in vivo. Ganciclovir did not affect the growth of HCMV-negative tumor cell lines. These findings imply an important role for HCMV in medulloblastoma and suggest HCMV as a novel therapeutic target for this tumor.
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http://dx.doi.org/10.1172/JCI57147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3195466PMC
October 2011

Intratumoral IL-7 delivery by mesenchymal stromal cells potentiates IFNgamma-transduced tumor cell immunotherapy of experimental glioma.

J Neuroimmunol 2010 Jan 14;218(1-2):140-4. Epub 2009 Nov 14.

The Rausing Laboratory, BMC D14, Division of Neurosurgery, Department of Clinical Sciences, Lund University, SE-221 84 Lund, Sweden.

The present study reports regression of pre-established experimental rat gliomas as a result of combining peripheral immunization using interferon gamma (IFNgamma) transduced autologous tumor cells with local intratumoral delivery of interleukin 7 (IL-7) by mesenchymal stromal cells. IL-7 alone significantly decreased the tumor area and this effect was enhanced with IFNgamma immunization. A higher density of intratumoral T-cells was observed in animals receiving combined therapies compared to rats receiving either cytokine alone suggesting that the therapeutic effect is dependent on a T-cell response.
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http://dx.doi.org/10.1016/j.jneuroim.2009.10.017DOI Listing
January 2010

The outermost N-terminal region of tapasin facilitates folding of major histocompatibility complex class I.

Eur J Immunol 2009 Oct;39(10):2682-94

Department of Experimental Immunology, Institute of International Health, Immunology and Microbiology, University of Copenhagen, Panum Institute, DK-2200 Copenhagen, Denmark.

Tapasin (Tpn) is an ER chaperone that is uniquely dedicated to MHC-I biosynthesis. It binds MHC-I molecules, integrates them into peptide-loading complexes, and exerts quality control of the bound peptides; only when an "optimal peptide" is bound will the MHC-I be released and exported to the cell surface for presentation to T cells. The exact mechanisms of Tpn quality control and the criteria for being an optimal peptide are still unknown. Here, we have generated a recombinant fragment of human Tpn, Tpn(1-87) (representing the 87 N-terminal and ER-luminal amino acids of the mature Tpn protein). Using a biochemical peptide-MHC-I-binding assay, recombinant Tpn(1-87) was found to specifically facilitate peptide-dependent folding of HLA-A*0201. Furthermore, we used Tpn(1-87) to generate a monoclonal antibody, alphaTpn(1-87)/80, specific for natural human Tpn and capable of cellular staining of ER localized Tpn. Using overlapping peptides, the epitope of alphaTpn(1-87)/80 was located to Tpn(40-44), which maps to a surface-exposed loop on the Tpn structure. Together, these results demonstrate that the N-terminal region of Tpn can be recombinantly expressed and adopt a structure, which at least partially resembles that of WT Tpn, and that this region of Tpn features chaperone activity facilitating peptide binding of MHC-I.
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http://dx.doi.org/10.1002/eji.200939364DOI Listing
October 2009

Identification of proteins involved in neural progenitor cell targeting of gliomas.

BMC Cancer 2009 Jun 26;9:206. Epub 2009 Jun 26.

CNS Gene Therapy Unit, Dept Experimental Medical Science, Lund University, Lund, Sweden.

Background: Glioblastoma are highly aggressive tumors with an average survival time of 12 months with currently available treatment. We have previously shown that specific embryonic neural progenitor cells (NPC) have the potential to target glioma growth in the CNS of rats. The neural progenitor cell treatment can cure approximately 40% of the animals with malignant gliomas with no trace of a tumor burden 6 months after finishing the experiment. Furthermore, the NPCs have been shown to respond to signals from the tumor environment resulting in specific migration towards the tumor. Based on these results we wanted to investigate what factors could influence the growth and progression of gliomas in our rodent model.

Methods: Using microarrays we screened for candidate genes involved in the functional mechanism of tumor inhibition by comparing glioma cell lines to neural progenitor cells with or without anti-tumor activity. The expression of candidate genes was confirmed at RNA level by quantitative RT-PCR and at the protein level by Western blots and immunocytochemistry. Moreover, we have developed in vitro assays to mimic the antitumor effect seen in vivo.

Results: We identified several targets involved in glioma growth and migration, specifically CXCL1, CD81, TPT1, Gas6 and AXL proteins. We further showed that follistatin secretion from the NPC has the potential to decrease tumor proliferation. In vitro co-cultures of NPC and tumor cells resulted in the inhibition of tumor growth. The addition of antibodies against proteins selected by gene and protein expression analysis either increased or decreased the proliferation rate of the glioma cell lines in vitro.

Conclusion: These results suggest that these identified factors might be useful starting points for performing future experiments directed towards a potential therapy against malignant gliomas.
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http://dx.doi.org/10.1186/1471-2407-9-206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2713262PMC
June 2009

Immunizations with IFNgamma secreting tumor cells can eliminate fully established and invasive rat gliomas.

J Immunother 2009 Jul-Aug;32(6):593-601

Glioma Immunotherapy Group, Lund University, Lund, Sweden.

Immunotherapy of malignant primary brain tumors holds the potential to improve the dismal prognosis after current clinical therapy. Although immunotherapy of experimental gliomas has been demonstrated to have the capacity to cure intracerebral tumors no convincing effects of immunotherapy have been shown in clinical trials. One reason for this could be that some of the models used do not display full features of human glioblastomas. The N29 rat gliomas exhibited all the histologic features of human glioblastoma multiforme including nuclear atypia, mitotic figures, necrosis, and diffuse infiltration into the normal brain tissue. Surprisingly, immunotherapy with autologous interferon gamma producing tumor cells against preestablished intracerebral N29 tumors yielded a higher cure rate than immunotherapy against less invasive tumors. Furthermore, when immunizations were postponed until day 5 after tumor establishment 50% of the animals survived. When immunizations were postponed until day 11 after tumor establishment no glioma-bearing animals were cured but survival was significantly prolonged. The superior effect of immunotherapy in the invasive N29 model compared with the less invasive tumors could depend on combined effects of up-regulation of major histocompatibility complex I and induction of major histocompatibility complex II plus CD80 after transfection and irradiation of the tumor cells used for immunizations. This study demonstrates that immunotherapy against experimental brain tumors indeed is feasible even against highly invasive and established tumors. These results strengthen the translational potential of immunotherapy against malignant brain tumors.
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http://dx.doi.org/10.1097/CJI.0b013e3181a95148DOI Listing
October 2009

CD133+ and nestin+ tumor-initiating cells dominate in N29 and N32 experimental gliomas.

Int J Cancer 2009 Jul;125(1):15-22

Department of Clinical Sciences, Division of Neurosurgery,The Rausing Laboratory, BMC D14, Lund University, Lund, Sweden.

The current study was designed to critically evaluate the notion that cancer stem cell (CSC)-like cells constitute a subpopulation of cells within experimental gliomas. Virtually all cells within the N29 and N32 rat glioma models homogenously expressed CD133, the stem/progenitor marker nestin as well as the neural lineage markers glial fibrillary acidic protein, betaIII-tubulin, and CNPase in vitro. The phenotype was largely retained on exposure to conditions promoting differentiation in vitro and after intracranial implantation of tumor cells into syngeneic hosts. Unsorted adherently grown cells displayed very high clonogenicity in vitro and robust tumorigenicity in vivo. Single N29 and N32 tumor cells invariably formed clones in vitro, and intracerebral inoculation of as few as 10 adherently growing N29 and N32 tumor cells, respectively, gave rise to a tumor. These results provide an alternative view on CSC-like cells in glioma models: sphere-formation is not a prerequisite for accumulation of tumorigenic cells, and CSC-like cells do not reside within a rare subpopulation of cells in these glioma models. N29 and N32 gliomas may accordingly be used for the development of treatment strategies directed specifically against a practically pure population of brain tumor-initiating CSC-like cells.
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http://dx.doi.org/10.1002/ijc.24306DOI Listing
July 2009

Tumor eradication after cyclophosphamide depends on concurrent depletion of regulatory T cells: a role for cycling TNFR2-expressing effector-suppressor T cells in limiting effective chemotherapy.

Cancer Immunol Immunother 2009 Aug 4;58(8):1219-28. Epub 2008 Dec 4.

National Centre for Asbestos Related Diseases, School of Medicine and Pharmacology, University of Western Australia, Perth, Australia.

Tumor cell death potentially engages with the immune system. However, the efficacy of anti-tumor chemotherapy may be limited by tumor-driven immunosuppression, e.g., through CD25+ regulatory T cells. We addressed this question in a mouse model of mesothelioma by depleting or reconstituting CD25+ regulatory T cells in combination with two different chemotherapeutic drugs. We found that the efficacy of cyclophosphamide to eradicate established tumors, which has been linked to regulatory T cell depletion, was negated by adoptive transfer of CD25+ regulatory T cells. Analysis of post-chemotherapy regulatory T cell populations revealed that cyclophosphamide depleted cycling (Ki-67(hi)) T cells, including foxp3+ regulatory CD4+ T cells. Ki-67(hi) CD4+ T cells expressed increased levels of two markers, TNFR2 and ICOS, that have been associated with a maximally suppressive phenotype according to recently published studies. This suggest that cyclophosphamide depletes a population of maximally suppressive regulatory T cells, which may explain its superior anti-tumor efficacy in our model. Our data suggest that regulatory T cell depletion could be used to improve the efficacy of anti-cancer chemotherapy regimens. Indeed, we observed that the drug gemcitabine, which does not deplete cycling regulatory T cells, eradicates established tumors in mice only when CD25+ CD4+ T cells are concurrently depleted. Cyclophosphamide could be used to achieve regulatory T cell depletion in combination with chemotherapy.
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http://dx.doi.org/10.1007/s00262-008-0628-9DOI Listing
August 2009