Sofia Eberstål

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

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 cyclooxygease-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 E2 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.

Cure of established GL261 mouse gliomas after combined immunotherapy with GM-CSF and IFNgamma is mediated by both CD8(+) and CD4(+) T-cells.

We were the first to demonstrate that combined immunotherapy with GM‐CSF producing GL261 cells and recombinant IFNγ 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 IFNγ 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 IFNγ‐producing T‐cells, were augmented after immunizations with GM‐CSF and IFNγ. 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 IFNγ effect. Finally, when immunizations were delayed until day 5 after tumor inoculation, only mice receiving immunotherapy with both GM‐CSF and IFNγ survived. We conclude that the addition of recombinant IFNγ 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.

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

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 (Th1) 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.

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

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.

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

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.

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

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.

Identification of two distinct mesenchymal stromal cell populations in human malignant glioma.

Gene profiling has revealed that malignant gliomas can be divided into four distinct molecular subtypes, where tumors with a mesenchymal gene expression are correlated with short survival. The present investigation was undertaken to clarify whether human malignant gliomas contain endogenous mesenchymal stromal cells (MSC), fulfilling consensus criteria defined by The International Society for Cellular Therapy, recruited from the host. We found that MSC-like cells can be isolated from primary human malignant gliomas. Two distinct MSC-like cell populations, differing in their expression of the CD90 surface marker, were discovered after cell sorting. RNA sequencing revealed further genetic differences between these two cell populations and MSC-like cells lacking CD90 produced higher amounts of VEGF and PGE2 compared to cells with the true MSC phenotype, implying that the CD90− MSC-like cells most probably are more active in tumor vascularization and immunosuppression than their CD90+ counterpart. The results highlight the CD90− subpopulation as an important tumor component, however, its functional effects in glioma remains to be resolved. Using the protocols presented here, it will be possible to isolate, characterize and analyze brain tumor-derived MSC-like cells in more detail and to further test their functions in vitro and in in vivo xenograft models of glioma.

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

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.

Low-dose irradiated mesenchymal stromal cells break tumor defensive properties in vivo : Therapeutic potential of mesenchymal stromal cells in cancer

Solid tumors, including gliomas, still represent a challenge to clinicians and first line treatments often fail, calling for new paradigms in cancer therapy. Novel strategies to overcome tumor resistance are mainly represented by multi‐targeted approaches, and cell vector‐based therapy is one of the most promising treatment modalities under development. Here, we show that mouse bone marrow‐derived mesenchymal stromal cells (MSCs), when primed with low‐dose irradiation (irMSCs), undergo changes in their immunogenic and angiogenic capacity and acquire anti‐tumoral properties in a mouse model of glioblastoma (GBM). Following grafting in GL261 glioblastoma, irMSCs migrate extensively and selectively within the tumor and infiltrate predominantly the peri‐vascular niche, leading to rejection of established tumors and cure in 29% of animals. The therapeutic radiation dose window is narrow, with effects seen between 2 and 15 Gy, peaking at 5 Gy. A single low‐dose radiation decreases MSCs inherent immune suppressive properties in vitro as well as shapes their immune regulatory ability in vivo. Intra‐tumorally grafted irMSCs stimulate the immune system and decrease immune suppression. Additionally, irMSCs enhance peri‐tumoral reactive astrocytosis and display anti‐angiogenic properties. Hence, the present study provides strong evidence for a therapeutic potential of low‐dose irMSCs in cancer as well as giving new insight into MSC biology and applications.

Abstract 2409: Enhanced cell-based immunotherapy by IFNγ, IL-7 or COX-2 inhibition in a rat glioma model

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC 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.