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The study was a non-randomized controlled phase I-II trial to study were to ascertain the safety, feasibility and efficacy of immunotherapy with autologous IFN-γ transfected tumour cells in patients with glioblastoma multiforme. Autologous tumour cells harvested during surgery were cultured and transduced with the human IFN-γ gene. Irradiated cells were administered as intradermal immunizations every third week. Endpoints for safety were records of toxicity and adverse events, for feasibility the per cent of treated patients out of eligible patients and time to treatment and for clinical efficacy overall survival (OS) and progress free survival (PFS). Eight eligible patients, between 50 and 69 years, were immunized between 8 and 14 times after treatment with surgery and radiotherapy without adverse events or toxicity. Neurological status and quality of life were unchanged during immunotherapy. The immunized patients had a significantly (p

The prognostic impact of insulin-like growth factor binding protein 7 (IGFBP7) in breast cancer is unclear. Host factors, including lifestyle, anthropometry and metabolic profile, might influence tumor-specific IGFBP7. This study aimed to investigate whether IGFBP7 levels and messenger ribonucleic acid (mRNA) expression are associated with the patient and tumor characteristics and prognosis in breast cancer. Patients with primary breast cancer in Lund, Sweden, were included preoperatively in the study between 2002 and 2012 (n = 1018). Tumor-specific IGFBP7 protein levels were evaluated with immunohistochemistry using tissue microarrays in tumors from 878 patients. IGFBP7 mRNA expression and its corresponding clinical data were obtained from The Cancer Genome Atlas and analyzed for 809 patients. Tumor-specific IGFBP7 protein levels were categorized based on Histo 300 scores into IGFBP7low (6.2%), IGFBP7intermediate (75.7%) and IGFBP7high (18.1%). Both low IGFBP7 protein levels and mRNA expression were associated with less aggressive tumor characteristics. Overall, IGFBP7low conferred low recurrence risk. The prognostic impact of IGFBP7high varied according to any alcohol consumption and tamoxifen treatment. IGFBP7high was associated with low recurrence risk in alcohol consumers but high recurrence risk in alcohol abstainers (Pinteraction= 0.039). Moreover, the combination of IGFBP7high and estrogen receptor-positive tumors was associated with low recurrence risk only in tamoxifen-treated patients (Pinteraction= 0.029). To conclude, IGFBP7low might be a good, independent prognosticator in breast cancer. The prognostic impact of IGFBP7high depends on host factors and treatment. IGFBP7 merits further investigation to confirm whether it could be a suitable biomarker for treatment selection., We investigated the prognostic impact of tumor-specific IGFBP7 levels in breast cancer. Overall, low IGFBP7 conferred good prognosis, while high IGFBP7 in ER+ tumors conferred good prognosis in tamoxifen-treated patients. IGFBP7 merits further investigation as a biomarker for treatment selection., Graphical Abstract

Previous reports of mortality and morbidity in pediatric severe traumatic brain injury (TBI) vary considerably, with few population-based studies. Mortality rates from 3-33 % and varying morbidity have been reported, most commonly using the extended Glasgow outcome scale (eGOS). The Lund concept is a treatment algorithm for severe TBI aiming at controlling intracranial pressure (ICP) by reducing cerebral perfusion pressure (CPP). The aim of the present study was to retrospectively assess mortality and morbidity in a population of pediatric TBI treated with the Lund concept. All cases of pediatric TBI (18 years) in the southern region of Sweden during 19 years were assessed. Patients were treated according to the Lund concept. Mortality, eGOS, ICP, CPP, time in NICU, drugs delivered and surgical procedures were recorded. Data were analysed both by dichotomised categories and by ordinal statistics. 136 cases of severe TBI18 years of age were recorded (incidence 2.0/100000) and 86 patients were admitted to the tertiary NICU. Mortality including all cases was 36% (incidence 0.7/100000) and in NICU 10%. Outcome was good in 60%, moderate in 25%, unfavourable in 15% with none in a vegetative status. In both dichotomized and ordinal analyses, CPP40 mmHg and ICP15 were associated with poor outcome, supporting current guidelines. However, high CPP was also associated with increased mortality and morbidity, supporting that elevated CPP might increase cerebral edema. The Lund concept resulted in low mortality and a favourable outcome in a majority of severe pediatric TBI patients.

Background Some glioblastoma (GBM) patients survive more than five years with hitherto no clearly established epidemiological or molecular causes. Since varying rates of GBM five-year survival have been reported our aim was to assess the true prevalence of five-year survivors in Sweden from 1958 to 1999, before the introduction of concomitant temozolomide treatment.Methods After screening the Swedish Cancer Registry and the Cause of Death Register 736 out of 12765 patients with high-grade glioma were defined as five-year survivors. The full text pathology report was reviewed in 585 patients. Data on epidemiology and treatment were retrieved from the medical records of 556 patients.Results 77 five-year survivors with primary GBM were identified which corresponded to 0.60 % of the initial population. During 1990 to 1999 GBM five-year survival was 0. 90%. Younger age, Karnofsky score > 70 and non-eloquent tumour location were found in most but not all five-year survivors.Conclusion GBM five-year survival was exceedingly rare in Sweden until 2004, comprising less than 1 % of registered HGGs. Relying on registry data without reviewing the pathology report will overestimate the accurate number of five-year survivors. To our knowledge, this is the only nationwide population-based study of five-year survival in GBM patients.

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 IL2rγnull (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.

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 VEGFAhighIL-7highIL-17AlowTNF-βlow (Group A) and VEGFAlowIL-7lowIL-17AhighTNF-βhigh (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. (Less)

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.

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.

The knowledge of response to radiation in the immuno-microenvironment of high grade gliomas is sparse. In vitro results have indicated an inflammatory response of myeloid cells after irradiation. Therefore, microdialysis was used to verify whether this is operative in tumor tissue and brain adjacent to tumor (BAT) after clinical radiotherapy of patients with high grade glioma. Stereotactic biopsies and implantation of microdialysis catheters in tumor tissue and BAT were performed in eleven patients with high-grade glioma. The patients were given daily radiation fractions of 2–3.4 Gy. Microdialysis samples were collected before radiotherapy and during the first five days of radiation. Cytokines, glucose metabolites, glutamate and glycerol were analyzed. Immunohistochemistry was performed to detect macrophages (CD68) and monocytes (CD163) as well as IL-6, IL-8 and MCP-1. A significant increase of IL-8, MCP-1 and MIP-1a were detected in tumor tissue already after the first dose of radiation and increased further during 5 days of radiation. IL-6 did also increase but after five fractions of radiation. In BAT, the cytokine response was modest with significant increase of IL-8 after third dose of radiation. We found a positive correlation between baseline IL-8 and IL-6 microdialysis levels in tumor tissue and survival. Glucose metabolites or glycerol and glutamate did not change during radiation. In all tumors staining for macrophages was demonstrated. IL-6 was found in viable tumor cells while MCP-1 was demonstrated in macrophages or tumor matrix. Our findings suggest that radiation induces a rapid enhancement of the prevailing inflammation in high-grade glioma tissue. The microdialysis technique is feasible for this type of study and could be used to monitor metabolic changes after different interventions. Electronic supplementary material The online version of this article (doi:10.1007/s11060-016-2271-1) contains supplementary material, which is available to authorized users.

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.

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.

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.

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.

We were the first to demonstrate that combined immunotherapy with GM-CSF producing GL261 cells and recombinant IFNgamma of preestablished GL261 gliomas could cure 90% of immunized mice. To extend these findings and to uncover the underlying mechanisms, the ensuing experiments were undertaken. We hypothesized that immunizations combining both GM-CSF and IFNgamma systemically would increase the number of immature myeloid cells, which then would mature and differentiate into dendritic cells (DCs) and macrophages, thereby augmenting tumor antigen presentation and T-cell activation. Indeed, the combined therapy induced a systemic increase of both immature and mature myeloid cells but also an increase in T regulatory cells (T-regs). Cytotoxic anti-tumor responses, mirrored by an increase in Granzyme B-positive cells as well as IFNgamma-producing T-cells, were augmented after immunizations with GM-CSF and IFNgamma. We also show that the combined therapy induced a long-term memory with rejection of intracerebral (i.c.) rechallenges. Depletion of T-cells showed that both CD4(+) and CD8(+) T-cells were essential for the combined GM-CSF and IFNgamma effect. Finally, when immunizations were delayed until day 5 after tumor inoculation, only mice receiving immunotherapy with both GM-CSF and IFNgamma survived. We conclude that the addition of recombinant IFNgamma to immunizations with GM-CSF producing tumor cells increased the number of activated tumoricidal T-cells, which could eradicate established intracerebral tumors. These results clearly demonstrate that the combination of cytokines in immunotherapy of brain tumors have synergistic effects that have implications for clinical immunotherapy of human malignant brain tumors. (c) 2008 Wiley-Liss, Inc. (Less)

High-grade gliomas are one of the most aggressive human tumors with

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.

Dendritic cells (DCs) are highly specialized antigen-presenting cells that play a key role in the initiation and regulation of immune responses. The ability of DCs to process antigens and the outcome of their interaction with T cells are largely dependent on phenotype as well as maturation state of DCs. In this study, we generated DCs from rat bone marrow precursors. Bone marrow cells cultured in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4, and Flt-3 ligand (FL) produced immature DCs that expressed intermediate levels of major histocompatibility complex (MHC) class II, low levels of CD80 and CD86 molecules and displayed a high capacity of endocytosis. Bone marrow-derived DCs (BMDCs) matured in the presence of lipopolysaccharide (LPS) upregulated expression of MHC class II, CD80 and CD86, while their phagocytic capacity was dramatically reduced. Mature BMDCs stimulated vigorous proliferation of purified allogeneic CD4(+) T cells in a primary mixed leukocyte reaction (MLR) and elicited a mixed cytokine profile in allogeneic CD4(+) T cells: DCs activated CD4(+) T cells to produce interferon (IFN)-gamma, IL-4, and IL-10. Thus, rat BMDCs effectively internalize antigens and stimulate T cell proliferation but fail to induce an unidirectional polarization of T helper (T-H) cells and in this respect differ from both human and mouse DCs. (c) 2005 Elsevier B.V. All rights reserved. (Less)

To mimic the two-signal requirements for T cell activation mediated by ligands, we exposed the superantigens SEA or SEE (signal 1) to T cells incubated with HLA-DR/LFA-3 or HLA-DR/B7-1-CHO transfected cells (signal 2). LFA-3 costimulation was able to induce T cell proliferation as well as IFN-gamma and IL-4 production at similar levels as in cells induced by B7-1. Analysis of the CD28RE of the IL-2 promoter showed specific transcription factor recruitment at the CD28RE element upon induction by B7-1/SEE. Further functional studies with an IL-2 enhancer-promoter carrying either wild type or mutated versions of the CD28RE site revealed that this element is necessary for full activation upon B7-1 costimulation. While both CD28/B7-1 and CD2/LFA-3 costimulation resulted in the up-regulation of IL-4 and IFN-gamma promoters, IL-2 promoter activity and production of IL-2 were only seen after B7-1 costimulation. However, contrary to what has been previously proposed, we show that costimulation with either B7-1 or LFA-3 further enhanced the ERK-2 activity and strongly activated the p38 MAPK pathway, but only B7-1 costimulation induced high levels of JNK-1 activity. These data suggest that the differential effect of CD28 vs. CD2 can be related to the difference in the ability of the two pathways to induce JNK-1 activity.

Based upon earlier experimental work by our group, we have started a human immuno-gene therapy study. The goal is to study the effects of immunisation with autologous tumour cells expressing gene sequences for human interferon-gamma For more than two decades we have sought for efficient treatment against malignant gliomas. Our most successful treatment in the animal models is immuno-gene therapy where murine genes for the cytokines IFN-gamma, IL-7 and B7-1 were chosen for their ability to stimulate different stages of the pathway for cytotoxic T lymphocyte (CTL) activation. Rats of the syngeneic inbred strain Fischer 344 had rat glioma cells of the N32 line inoculated in the right caudate nucleus, and 1 or 3 days later N32 cells transfected with either IFN-gamma, IL-7 or B7-1 genes were injected subcutaneously (and in some studies intraperitoneally). This treatment was repeated three to four times with 7- to 14-day interval and resulted in significantly improved survival compared with treatment with wild-type rat glioma cells (e.g. not transfected with the cytokine genes). The continued work concentrated on treatment with IFN-gamma secreting tumour cells of both the N32 line and also a newly developed ENU-induced rat glioma cell line called N29. This work proved the effectiveness of the technique. Cure was achieved in 72% of the animals treated with the IFN-gamma cells. Tumour-infiltrating leukocytes from N32-IFN-gamma-immunised animals showed a significantly stronger infiltration by CD8+ T-cells, significantly more NK cells, and an increased number of CD25-expressing T-cells. These results confirmed the possible usefulness of IFN-gamma-transfected tumour cells in the immune-therapy of rat brain tumours. The animal experiments have motivated us to start a human immuno-gene therapy study including 20 patients with glioblastoma multiforme (GBM), where >80% of the tumour can be surgically removed. The goal is to ascertain whether immunisation with autologous tumour cells expressing gene sequences for human interferon-gamma is safe for the patients, gives rise to an immunological response, and adds any beneficial effect to conventional therapy (tumour growth, prolonged survival). Hitherto, nine patients have been included in the study, two of which have received 6 and 10 immunisations, respectively. Two patients have died from their disease before cells have been ready for immunisation; in two cases no malignant cells have appeared in the cell cultures and three patients are ready to start their immunisation shortly. The immunisation takes place in the dermis of the upper arm. Seven days after each immunisation, a skin biopsy is taken from the centre of one of the injection sites. The composition of the cellular infiltration in the skin is studied by markers for T lymphocytes (CD3); helper cells, subset of T cells (CD4); killer cells, subset of T cells (CD8); natural killer cells (CD16) and B lymphocytes, B cells (CD20). Also the expression of cytokines for functional T cell subsets are studied: IL-2, IL-4, IL-10, IL-12, IL-18, TNF-alpha and IF-gamma and TGF-beta(1,2 and 3-) Peripheral blood is sampled both before and after operation and also after each immunisation event. Co-culture of this blood with tumour cells from the patient allows for a selection of T-cells that can recognise tumour-specific antigens. The results from the first human treatments are presented. (C) 2002 Published by Elsevier Science B.V. (Less)

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.

We have previously reported that immunizations with mutagen-induced immunogenic variants of a weakly immunogenic rat glioma could protect against isografts of the original tumor cells. In this study we show that prolonged survival and cures of rats with established gliomas in their brains can be achieved by therapeutic immunizations with tumor cell mutants, combined with in vitro and in vivo interferon (IFN)-gamma (adjuvant) treatment, or tumor cells admixed with semipurified syngeneic dendritic cells. Cure of rats with established intracerebral gliomas was possible when immunizations were initiated up to 5 days after intracerebral isografting of original tumor cells. Unexpectedly, immunizations combined with in vitro and in vivo IFN-gamma treatment or with admixed semipurified dendritic cells equalized the immunogenic potential of the original tumor cells and that of mutagen-induced immunogenic cell variants (tum-). This demonstrates that effective immunizations against a weakly immunogenic brain tumor can be achieved by different adjuvant concepts. The therapeutic effect of immunizations with tumor cells admixed with semipurified dendritic cells was highly significant in female rats, whereas only occasional cures and prolonged survival were recorded in male rats. The overall results show that therapeutic immunizations can indeed be effective against an established and growing intracerebral tumor.

Previously established immunogenic (tum-) clones of an ENU (ethyl-N-nitrosourea)-induced rat glioma, N32, were compared to the original tumor concerning their capacity to induce T lymphocyte responses after in vivo immunization and in vitro restimulation of responder spleen cells in mixed lymphocyte tumor culture (MLTC) assays. Quite unexpectedly, original N32 (tum+) in vivo primed spleen cells proliferated to the same extent in vitro in response to tum+ stimulator cells as did tum- in vivo primed spleen cells. However, flow-cytometric analysis of parallel cultures showed a greatly increased proportion of CD3+CD8+ lymphocytes in the proliferating responder cell population from tum- immunized hosts, contrary to a CD3+CD4+ lymphocyte dominance after tum+ immunization. Although the original tum+ N32 tumor cells are not capable of inducing a clearly demonstrable isograft rejection response, they induce a strong T cell response readily detectable in MLTC assays. We propose that the increased CD8+ lymphocyte proliferation could be an essential feature of the isograft rejection response induced by tum- tumor variants. Possible mechanisms of the augmented CD8+ T cell response are discussed.

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.

The ethyl-N-nitrosourea-induced rat glioma N32 was treated with the mutagenic compound N-methyl-N'-nitro-N-nitrosoguanidine and the surviving cells cloned by limited dilution. Out of 20 clones tested 8 did not produce tumors subcutaneously even after challenge doses 3 log units above the minimal tumor dose for N32. All of 5 clones grew in a retarded manner intracerebrally but produced tumors in some animals. Preimmunizations with three of the rejected clones (tum-) gave protection against subcutaneous and intracerebral isografts of the unmutated N32. This effect could be enhanced if the cells used for immunizations were pretreated with interferon gamma (IFN gamma) for 48 h. If immunizations were started subsequent to challenge, only immunization with one of two tested tum- clones pretreated with IFN gamma induced significant rejection against intracerebral N32 isografts. Both N32 and its tum- clones were MHC class I positive and MHC class II negative. IFN gamma treatment enhanced the MHC class I expression with 20%-90% on the tum- clones and with 40% on N32. MHC class II expression could be induced on N32 cells after 7 days of IFN gamma treatment but not on any of the tum- clones tested. We conclude that the enhancing effect of IFN gamma treatment on tumor isograft rejection may depend on up-regulation of MHC class I but not of MHC class II. This investigation demonstrates that it is possible to induce rejection of weakly immunogenic intracerebral brain tumors by immunization with selected highly immunogenic tumor cell mutants. In conjunction with relevant cytokines, the cross-protective effect of these tum- variants might be further enhanced and serve as a model for immunotherapy against malignant human brain tumors.

Interferon gamma (IFN-gamma) has successfully been used in immunotherapy of different experimental tumours. Mechanistically, IFN-gamma has extensive effects on the immune system including release of nitric oxide (NO) by upregulation of the inducible nitric oxide synthase (iNOS). NO has putative immunosuppressive effects but could also play a role in killing of tumour cells. Therefore, the aim of the present study was to clarify whether inhibition of iNOS in rats immunized with glioma cells (N32) producing IFN-gamma (N32-IFN-gamma), could enhance the anti-tumour immune response. Initially, both a selective iNOS, L-N-6-(I-Iminoethyl)-L-lysine (L-NIL), and non-selective, N-nitro-L-arginine methyl ester (L-NAME), inhibitor of NOS were tested in vitro. After polyclonal stimulation with LPS and SEA, both L-NIL and L-NAME enhanced proliferation and production of IFN-gamma from activated rat splenocytes and this effect was inversely correlated to the production of NO. However, L-NIL had a broader window of efficacy and a lower minimal effective dose. When rats were immunized with N32-IFN-gamma), and administered NOS inhibitors by intraperitoneal (i.p.) mini-osmotic pumps, only splenocytes of rats treated with L-NIL, but not L-NAME, displayed an enhanced proliferation and production of IFN-gamma when re-stimulated with N32 tumour cells. Based on these findings, L-NIL was administered concurrently with N32-IFN-gamma cells to rats with intracerebral (i.c.) tumours resulting in a prolonged survival. These results show that inhibition of iNOS can enhance an IFN-gamma-based immunotherapy of experimental i.c. tumours implying that NO released after immunization has mainly immunosuppressive net effects. (Less)

Glioblastoma multiforme is the most common malignant primary brain tumor and also one of the most therapy-resistant tumors. Because of the dismal prognosis, various therapies modulating the immune system have been developed in experimental models. Previously, we have shown a 37-70% cure in a rat glioma model where rats were peripherally immunized with tumor cells producing IFNgamma. On the basis of these results, we wanted to investigate whether a combination of GM-CSF and IFNgamma could improve the therapeutic effect in a mouse glioma model, GL261 (GL-wt). Three biweekly intraperitoneal (i.p.) immunizations with irradiated GM-CSF-transduced GL261 cells (GL-GM) induced a 44% survival in mice with intracranial glioma. While treatment of GL-wt and GL-GM with IFNgamma in vitro induced upregulation of MHC I and MHC II on the tumor cells, it could not enhance survival after immunization. However, immunizations with GL-GM combined with recombinant IFNgamma at the immunization site synergistically enhanced survival with a cure rate of 88%. Tumors from mice receiving only 1 immunization on Day 10 after tumor inoculation were sectioned on Day 20 for analysis of leukocyte infiltration. Tumor volume was reduced and the infiltration of macrophages was denser in mice immunized with GL-GM combined with IFNgamma compared with that of both wildtype and nonimmunized mice. To our knowledge, this is the first study to demonstrate a synergy between GM-CSF and IFNgamma in experimental immunotherapy of tumors, by substantially increasing survival as well as inducing a potent anti-tumor response after only 1 postponed immunization.

Nitric oxide (NO) can modulate both tumor growth and antitumor immune responses. In order to elucidate the mechanism of curative therapeutic immunization with IFNgamma-producing glioma cells, we examined the expression of inducible nitric oxide synthase (iNOS) in tissue sections from immunized animals. There was a significantly enhanced iNOS expression both intratumorally and at the immunization site. Although the mechanisms behind this dual expression of iNOS most probably are different, our results suggest a role for NO in both the induction and execution of the antitumor response.

The purpose of the present study was to establish a rapid and reproducible method for quantification of tissue-infiltrating leukocytes using computerized image analysis. To achieve this, the staining procedure, the image acquisition, and the image analysis method were optimized. Because of the adaptive features of the human eye, computerized image analysis is more sensitive to variations in staining compared with manual image analysis. To minimize variations in staining, an automated immunostainer was used. With a digital scanner camera, low-magnification images could be sampled at high resolution, thus making it possible to analyze larger tissue sections. Image analysis was performed by color thresholding of the digital images based on values of hue, saturation, and intensity color mode, which we consider superior to the red, green, and blue color mode for analysis of most histological stains. To evaluate the method, we compared computerized analysis of images with a ×100 or a ×12.5 magnification to assess leukocytes infiltrating rat brain tumors after peripheral immunizations with tumor cells genetically modified to express rat interferon-γ (IFN-γ) or medium controls. The results generated by both methods correlated well and did not show any significant differences. The method allows efficient and reproducible processing of large tissue sections that is less time-consuming than conventional methods and can be performed with standard equipment and software. (J Histochem Cytochem 49:1073–1079, 2001)

We have shown previously that rejection of preinduced rat brain tumours is possible following therapeutic immunizations with interferon-gamma (IFN gamma)-transfected glioma cells (N32-IFN gamma). In the present study we have used the same model to evaluate whether quantitative differences in tumour-infiltrating lymphocytes can be detected between animals receiving therapeutic immunizations with either IFN gamma-transfected glioma cells, wild-type glioma cells or no treatment. Since leucocyte transpedesis into the tumour can be anticipated to depend on the state of vascularization, we have mapped the development of microvessels in the tumour in parallel with the leucocyte infiltration. Our results show that microvessels start to form at day 7 and then gradually increase in number and size, indicating the establishment of an extensive vascularization by day 24. Leucocyte infiltration displays a biphasic pattern after tumour grafting. We have therefore studied the infiltration kinetics after an early immunization (1 day after intracerebral isografting) and compared the effects with those of a late immunization (10 days after intracerebral isografting) with N32-IFN gamma or wild-type N32. Our results show (1) an early infiltration of granulocytes 3 days after isografting; (2) a T-cell-receptor-positive (TCR+) T-cell infiltration starting on day 10; (3) a macrophage infiltration starting on day 13; (4) a CD8+ cell infiltration starting on day 13. The proportions of TCR+ T cells, CD8+ cells and natural killer cells differs significantly between animals immunized with N32-IFN gamma and those receiving wild-type N32, when analysed 14 days after immunization at day 10. This difference can only be detected when animals are immunized at later stages of tumour growth. We propose that this could depend on an early-immunization-independent leucocyte infiltration during tumour establishment. This has to be considered when evaluating studies of leucocyte infiltration in experimental tumours.

Blood vessels and adjacent cells form perivascular stem cell niches in adult tissues. In this perivascular niche, a stem cell with mesenchymal characteristics was recently identified in some adult somatic tissues. These cells are pericytes that line the microvasculature, express mesenchymal markers and differentiate into mesodermal lineages but might even have the capacity to generate tissue-specific cell types. Here, we isolated, purified and characterized a previously unrecognized progenitor population from two different regions in the adult human brain, the ventricular wall and the neocortex. We show that these cells co-express markers for mesenchymal stem cells and pericytes in vivo and in vitro, but do not express glial, neuronal progenitor, hematopoietic, endothelial or microglial markers in their native state. Furthermore, we demonstrate at a clonal level that these progenitors have true multilineage potential towards both, the mesodermal and neuroectodermal phenotype. They can be epigenetically induced in vitro into adipocytes, chondroblasts and osteoblasts but also into glial cells and immature neurons. This progenitor population exhibits long-term proliferation, karyotype stability and retention of phenotype and multipotency following extensive propagation. Thus, we provide evidence that the vascular niche in the adult human brain harbors a novel progenitor with multilineage capacity that appears to represent mesenchymal stem cells and is different from any previously described human neural stem cell. Future studies will elucidate whether these cells may play a role for disease or may represent a reservoir that can be exploited in efforts to repair the diseased human brain.

Introduction: Based on our previous experimental result we have performed a clinical trial at the Lund University Hospital, with immunotherapy using IFN transduced autologous tumor cells for intradermal (i.d.) immunizations in adult patients with glioblastoma multiforme. Eight patients have been treated with an increase in overall survival by more than six months as compared to age-matched controls. The trial has shown that the therapy has no side effects and that an immune activation can be detected. Current experimental results show that by changing the route of immunization from i.d. to sub-cutaneous (s.c.) immunization, a substantial increase in survival time of animals is seen. The mechanisms behind this are now under investigation. Experimental Procedures: We are currently investigating the therapeutic efficacy of i.d. or s.c. administerd IFN transduced irradiated tumor cells (N32-IFNγ) against intra cerebral tumors in the rat glioma model (N32) syngeneic with the Fisher 344 rat. The rats are immunized day 1, 15 and day 29 after tumor inoculation. The induction of the immune response is studied by tracing irradiated CFDA-SE stained tumor cells from the immunization site (i.d. or s.c.) to secondary lymphoid organs using immuno-histochemistry. The immune response is studied at different time-points after the first and after the third immunization. Preliminary Results: S.c. immunization with N32-IFNγ cured 53% of the tumor bearing animals compared to i.d. immunization where only 6% of the rats survived for 100 days, when the study was finalized. After the first immunization, tumor cell debris can be traced to inguinal lymph nodes of i.d. immunized animals, but not of s.c. immunized animals. After the third immunization tumor debris is found in inguinal lymph nodes both after i.d. and s.c. immunization. No other secondary lymphoid tissues tested contain tumor debris. Mononuclear phagocytes expressing MHC class II are responsible for engulfing tumor cells at the immunization site of both i.d. and s.c. immunized animals. Cells that strongly express the enzyme cyclooxygenase-2, the rate-limiting enzyme for production of prostaglandine E2, are seen after s.c. immunization but not after i.d. immunization. Further studies are ongoing to reveal possible differences in kinetics and antigen presenting cells. Conclusions: To our knowledge, no systematic comparison has been performed between different immunization routes using whole tumor cells as immunogen. If these results are further confirmed, the route of immunization will be altered from i.d. to s.c. in future clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-321.

Introduction: Glioblastoma multiforme (GBM) is the most common brain tumor in adults and the median survival time after diagnosis is less than a year despite extensive surgical resection and adjuvant radio- and chemotherapy. Consequently, there is a need for new therapies, and immunotherapy represents a promising treatment approach. We have earlier described the regression of experimental gliomas in response to peripheral immunization with genetically modified tumor cells and a clinical trial is finalized using interferon-gamma (IFNγ) transduced tumor cells in adult patients with GBM. We are currently improving the therapy by adding different cytokines as well as targeting the immune suppression. Further on, to avoid the time consuming step of tumor cell transduction, the present study was undertaken to investigate the effect of immunization using wild-type (wt) tumor cells together with recombinant cytokines and inhibitors of immune suppression. Material and Methods: An experimental rat glioma model, N32 (established by transplacental ENU induction) was used in all experiments. Briefly, N32 rat glioma cells were injected into the right striatum in brains of syngeneic male Fischer 344 rats using a stereotactic frame. The rats were immunized subcutaneously in the thigh with irradiated N32 wt-cells (w/o recombinant IFNγ) or N32 cells genetically modified to produce IFNγ and/or interleukin-7 (N32-IFNγ, N32-IL7). Mini-osmotic pumps were used for the administration of the selective COX-2 inhibitor parecoxib. The immune response was measured in blood and lymph nodes using flow cytometry and ELISA. Results and conclusion: In the present survival study of glioma bearing rats we report that a combination of N32-IFNγ and N32-IL7 is superior compared to each monotherapy, demonstrating a better outcome when multiple steps of the immune activation is enhanced (i.e. antigen-presentation and maturation of T-cells). We also show that COX-2 inhibition results in significantly enhanced survival when combined with N32-IFNγ immunization. Continous parecoxib administration (day 1-28) was superior to intermittent administration (day 7-13 and 17-23), indicating that absence of PGE2 during immune activation improves the therapeutic effect. The COX-2 inhibition, when combined with N32-IFNγ immunization, resulted in a significant increase of IFNγ levels in plasma, which might explain the enhanced cure rate. In addition we show that only wt-immunization is not enough to protect any animals against tumor challenge, but when combined with recombinant IFNγ or COX-2 inhibition animals are cured. In conclusion, we demonstrate the ability to optimize cell-based immunotherapy by stimulating immune activation and inhibiting the immune suppression. These findings may have clinical applications in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2409.

Glioblastoma has remained the deadliest primary brain tumor while its current therapy offers only modest survival prolongation. Immunotherapy has failed to record notable benefits in routine glioblastoma treatment. Conventionally, immunotherapy relies on T cells as tumor-killing agents; however, T cells are outnumbered by macrophages in glioblastoma microenvironment. In this study, we explore the effect of AF16, a peptide from the endogenous antisecretory factor protein, on the survival of glioma-bearing mice, the tumor size, and characteristics of the tumor microenvironment with specific focus on macrophages. We elucidate the effect of AF16 on the inflammation-related secretome of human and murine macrophages, as well as human glioblastoma cells. In our results, AF16 alone and in combination with temozolomide leads to cure in immunocompetent mice with orthotopic GL261 gliomas, as well as prolonged survival in immunocompromised mice. We recorded decreased tumor size and changes in infiltration of macrophages and T cells in the murine glioma microenvironment. Human and murine macrophages increased expression of proinflammatory markers in response to AF16 treatment and the same effect was seen in human primary glioblastoma cells. In summary, we present AF16 as an immunomodulatory factor stimulating pro-inflammatory macrophages with a potential to be implemented in glioblastoma treatment protocols.