Enhanced expression of iNOS intratumorally and at the immunization site after immunization with IFNγ-secreting rat glioma cells

Journal of Neuroimmunology 123 (2002) 135 – 143 www.elsevier.com/locate/jneuroim

Enhanced expression of iNOS intratumorally and at the immunization site after immunization with IFNg-secreting rat glioma cells Anna C. Johansson a,b,*, Pontus Hegardt a,c, Shorena Janelidze a,c, Edward Visse a,c, Bengt Widegren a,b, Peter Siesjo¨ c a

Section for Tumor Immunology, Department of Cell and Molecular Biology, University of Lund, BMC I12, 221 84 Lund, Sweden b Institute of Genetics, Department of Molecular Genetics, University of Lund, So¨lvegatan 29, 223 59 Lund, Sweden c Section of Neurosurgery, Department of Clinical Neuroscience, University Hospital, 221 85 Lund, Sweden Received 11 May 2001; received in revised form 29 August 2001; accepted 2 November 2001

Abstract Nitric oxide (NO) can modulate both tumor growth and antitumor immune responses. In order to elucidate the mechanism of curative therapeutic immunization with IFNg-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. D 2002 Elsevier Science B.V. All rights reserved. Keywords: NO; iNOS; IFNg; Therapeutic immunization; Rat glioma; Immunohistochemistry

1. Introduction One objective of immune therapy against cancer is to induce a systemic immune response where immune cells, after they have encountered a tumor antigen at sites of immune activation, are able to recognize and kill tumor cells at distant sites. Active immune therapy against different forms of cancer, in the absence of defined tumor antigens, can be achieved using whole tumor cells genetically modified to express immune stimulatory molecules such as different cytokines and/or costimulatory molecules (Aruga et al., 1997; Hurwitz et al., 1998). Although the therapeutic effects of immunization site cytokine-secreting tumor cells are well established, and despite the demonstration of quantitative changes as increased cellularity at the immuni-

Abbreviations: MHC, major histocompatibility complex; TAP, transport-associated protein; LPS, lipopolysaccharide; JAK, Janus kinase; STAT, signal transducers and activators of transcription; IFN, interferon; TNF, tumor necrosis factor; IL, interleukin. * Corresponding author. Section for Tumor Immunology, Department of Cell and Molecular Biology, University of Lund, BMC I12, 221 84 Lund, Sweden. Tel.: +46-46-222-4363; fax: +46-46-222-4606. E-mail address: [email protected] (A.C. Johansson).

zation site, in the draining lymph nodes, and at the tumor site, little is known about the mechanisms leading to tumor rejection (Simons et al., 1999; Chang et al., 2000; Visse et al., 2000). We have previously achieved complete regression of preestablished brain tumors in >40% of rats receiving therapeutic immunizations with tumor cells engineered to secrete IFNg (Visse et al., 1999), and we have also confirmed these results with another rat brain tumor induced in our laboratory, N29 (Visse et al., manuscript in preparation). The IFNg secreted from transfected tumor cells could have several targets involving autocrine effects on the tumor cells, effects on immune cells at the immunization site, or systemic effects. IFNg is known to modulate the expression of more than 200 genes, of which several regulate immune function. The induction of MHC, TAP and the immune proteasome, as well as the inducible form of nitric oxide synthase (iNOS), are considered the most important in the net effect of immune function (Boehm et al., 1997; Tannenbaum and Hamilton, 2000). While up-regulation of MHC, TAP and proteasome subunits mainly affects the tumor cells and nonprofessional antigen presenting cells, the induction of iNOS and the consequent release of nitric oxide (NO) from macrophages/

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monocytes and neutrophils modulates the local immune response by, e.g., inhibition of T-cell proliferative responses, suppression of production of certain cytokines, induction of T-cell apoptosis and suppression of cytolytic responses (Albina et al., 1991; Bauer et al., 1997; MacMicking et al., 1997; Allione et al., 1999; Medot Pirenne et al., 1999; Gahm et al., 2000). iNOS can be induced in response to various cytokines such as IL-1, TNFa and IFNg, or bacterial products such as LPS, but in vivo IFNg is the most potent and prevailing inducer of iNOS (Nathan and Xie, 1994). As the main sources of IFNg production in vivo are T-lymphocytes and NK-cells, the iNOS induction is mediated by these unless IFNg is produced artificially as from transfected cells or by systemic delivery. The effects of NO on the immune response are pleiotrophic and still unclear as discussed by Nathan (Nathan, 1997). While NO can function as a cytotoxic agent in the first line of defense against microbes or arising tumors, it can also protect the organism from immunologically mediated tissue damage by several immunosuppressive mechanisms, encompassing the direct inhibition of T-cell function through disruption of the Jak3/STAT-5 pathway and inhibition of cytokine release (Bingisser et al., 1998; Berendji et al., 1999; Bogdan et al., 2000). As reviewed by Dix et al. (1999), several putative immunosuppressive factors have been identified in patients harboring malignant brain tumors, such as TGF-b (Transforming Growth Factor b), IL-10, and yet unidentified factors directly or indirectly released from tumor cells. Evidence of immune suppression in experimental animals bearing brain tumors are more scarce but has been reported to be mediated by NO, TGF-b, and yet unidentified factors (Fakhrai et al., 1996; Munz et al., 1999; Hegardt et al., 2000). To clarify the role of NO in the antitumor effect induced by immunizations with IFNg-secreting tumor cells, we have examined the expression of iNOS at the immunization site, in draining lymph nodes, in the spleen and in the intracerebral tumors of rats immunized with IFNg-releasing N32 glioma cells.

2. Materials and methods

2.2. Tumor induction/tumor transfection The rat glioma N32 (N32-wt) was induced by the transplacental administration of ethyl-N-nitrosourea (ENU) (Siesjo et al., 1993). In order to avoid induction of antigenic changes in the tumor cells, early passages grown less than 2 months in vitro were utilized in all experiments. The generation of the N32-IFNg cells and the N32-pLXSN cells (N32-empty vector) used in this study has previously been described (Visse et al., 1999). 2.3. Cell culture All tumor cells were maintained in RPMI 1640 (Gibco) supplemented with 10% Fetal Calf Serum, 2 mM L-Glutamine, 10 mM HEPES, 0.5 mM Pyruvate and 0.096% NaHCO3. In vitro cultured tumor cells were detached with trypsin – EDTA, washed three times in serum-free RPMI and then suspended in RPMI with 1% syngeneic Fisher rat serum before being used in transplantation or immunization. 2.4. Fluorescence labeling of tumor cells Cells were stained with 5 ml of DiO-Vybrantk CellLabeling Solution (Molecular Probes, Eugene, OR, USA) in a cell suspension of 1  106 cells/ml serum-free RPMI for 30 min at 37C in the dark. The cells were washed three times in serum-free RPMI and then suspended in RPMI with 1% syngeneic Fisher rat serum before immunization. 2.5. Tumor cell inoculation and immunization For intradermal immunization, 1  10 6 cells were injected into the right thigh of the rats. The different cell lines used were: N32-IFNg (n = 5), N32-empty vector (n = 3) and N32-wt (n = 3). In a second experiment, 1  106 fluorescence labeled tumor cells of the cell lines N32-IFNg (n = 2) and N32-wt (n = 2) were injected into the right thigh of the rats. In a third experiment, animals were immunized intraperitoneally with N32-IFNg cells (n = 8), N32-wt cells (n = 4) or were sham immunized using media only (n = 4), 10 days after intracerebral challenge with 1  104 N32-wt cells/5 ml. For intracerebral challenge, cells were stereotactically injected into the right nucleus caudatus of the rat brains. All cells used for immunizations were irradiated with 80 Gy prior to injection.

2.1. Animals and animal procedures 2.6. Preparation of tissue sections Syngeneic Fischer 344 male rats were used in all experiments. The rats were bred at the animal facility of the Wallenberg laboratory by continuous brother – sister matings. The rats were kept in an environment controlled for temperature and humidity and all animal procedures were performed according to the practices of the Swedish Board of Animal Research and were approved by the Committee of Animal Ethics in Lund-Malmo¨.

Animals were sacrificed at days 1, 3, 7 and 10 after intradermal immunizations. Following sacrifice, the draining lymph nodes, the spleen and the immunization site were taken out. From each brain, a 0.5-cm thick slice, including the injection site, was cut out at day 24 after intracerebral challenge. The tissues were snap-frozen in liquid nitrogen-cooled isopentane at 55 C before being

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stored at 80 C until required for sectioning. Six-micrometer thin sections were cut with a cryostat (Leica, Wetzlar, Germany), mounted on standard glass slides and then airdried for 30 min. Slides were then wrapped in aluminum foil and placed in the freezer for storage at 80 C until further processing. Prior to staining, the glass slides were thawed for 30 min and fixed in acetone at room temperature for 10 min. Following rehydration in TBS (Trisbuffered saline, 50 mM Tris, 145 mM NaCl pH 7.6) for 5 min, the slides were processed for further immunohistochemical procedures. 2.7. Primary antibodies The following primary antibodies were used: rabbit antimouse iNOS cat# KAP-NO001 (StressGen Biotechnologies, Victoria, BC, Canada); mouse antirat mononuclear phagocyte, clone 1C7 (Pharmingen, San Diego, CA, USA); mouse antirat granulocytes, clone HIS48 (Pharmingen). To confirm the specificity of the iNOS antibody, we screened different antibodies in spleen of normal and LPS-treated rats. The antibody used for iNOS detection in this study gave a distinct staining with minimal background, both in immunohistochemistry and western blots (data not shown). The antibody used as negative control was a polyclonal FAS-L (N-20) antibody cat# sc-834 (Santa Cruz Biotechnology, CA, USA). 2.8. Immunohistochemistry Briefly, the sections were treated with 0.03% H2O2 in TBS supplemented with 0.20% sodium azide for 30 min to block endogenous peroxidase. Sections were blocked for 20 min with 5% donkey serum (Jackson Immunoresearch, West Grove, PA, USA) and then incubated with the primary antibody for 60 min. As negative controls, the primary antibody was omitted or an immunostaining was performed with an unrelated antibody prepared in the same way as the primary antibody. Sections were then incubated for 30 min with a biotinylated secondary antibody (donkey antirabbit biotin, cat# 711-066-152 (Jackson Immunoresearch), followed by incubation for 30 min with streptavidin horseradish peroxidase (Jackson Immunoresearch). Between all steps sections were washed for 5 min with TBS. Finally, the sections were stained for 5– 10 min with 3-amino-9-ethylcarbazole (AEC+) (DAKO, Glostrup, Denmark) and rinsed in running tap water before counter-stained with Hematoxylin, Gill’s formula (Vector Laboratories, Burlingame, CA, USA). Slides were mounted wet in HistotecR (Serotec, Oxford, UK) permanent aqueous mounting media without cover slips and dried at room temperature. 2.9. Immunoflourescence double staining Sections were blocked for 20 min with 5% donkey serum (Jackson Immunoresearch) and incubated with an

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anti-iNOS antibody for 60 min. Sections were incubated with a biotinylated secondary antibody (donkey antirabbit biotin, Jackson Immunoresearch) followed by streptavidin conjugated Alexa Flourk 594 (Molecular Probes; cat# S11227) for 30 min each. Sections were then incubated with the second primary antibody, antigranulocytes or antimononuclear phagocyte for 60 min, and further incubated for 30 min with goat antimouse IgG conjugated with Alexa Flourk 488 (Molecular Probes, cat# A-11029). Between all steps, sections were washed with TBS for 5 min. Sections were counter-stained DAPI (Molecular Probes, cat# D-1306) and mounted in ProLongk antifading reagent (Molecular Probes, cat# P-7481). For experiments where tumor cells were labeled with the green fluorescent dye DiO, the mononuclear phagocytes were labeled with antimouse IgG conjugated with Alexa Flourk 350 (Molecular Probes, cat# A-21049). Nuclear staining was not performed. 2.10. Digital analysis of skin biopsy sections Immunohistochemically stained sections were analyzed by the use of a light microscope (Tx-60, Olympus America, Melville, NY, USA), and scanned using a three-channel system (3CCD) red –green –blue (RGB) Practica Colorscan (Schneider Feinwertechnik, Dresden, Germany). The whole skin biopsy area was scanned in a single image using 40  magnification at a resolution of 100 dpi (dots per inch). Images were captured using Adobe Photoshopk 5.0 software for Macintosh (Adobe Systems, Mountain View, CA,

Fig. 1. Computerized image analysis of immunohistochemical staining of iNOS production in skin biopsies of rats immunized with N32-IFNg cells (n = 5) or control groups; N32-pLXSN cells (n = 3), N32-wt cells (n = 3). Results are shown as the means ( F S.E.) within each group. The significance of the difference was evaluated by the Mann – Whitney U-test. P < 0.05 at days 1, 3, 7 and 10 indicates a significant difference in iNOSpositive cells of animals immunized with N32-IFNg as compared with control groups.

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Fig. 2.

Fig. 3.

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USA) and then saved in TIFF format (Tagged Image File Format) for analysis by Image-Pro Plusk 3.0 software (Media Cybernetics, Silver Spring, MD, USA). The ratio of stained area compared to area of interest was calculated and expressed as percent stained area. Sections stained double immunofluorescence were analyzed by the use of a fluorescence microscope (Tx-60, Olympus) equipped with a mercury lamp and filters for fluorescence (U-MWG, UMWB, U-MWU, Olympus). Images were taken at 400  magnification using a Cool-SNAP Pro 36-bit Color Digital Camera (Media Cybernetics), and captured using the ImagePro Plusk 4.1 software (Media Cybernetics). Pseudo-colored images of the fluorescence double staining were created by the use of the Fluoro-Prok Plug In (Media Cybernetics). 2.11. Statistics Statistical differences between groups were determined by using the nonparametric Mann –Whitney U-test, P < 0.05 being considered statistically significant. The test was performed using StatViewR software (Abacus Concepts, Berkeley, CA, 1996).

3. Results 3.1. iNOS expression in skin biopsies, draining lymph nodes and spleen To examine the presence of iNOS expression at different stages and sites of immune activation, we examined skin biopsies, lymph nodes and spleens at days 1, 3, 7 and 10 after intradermal immunizations with N32-IFNg and compared the results with those obtained in control animals immunized with N32-pLXSN and N32-wt cells. At all days investigated, the iNOS expression in skin biopsies from animals immunized with N32IFNg was significantly higher than in skin biopsies from control animals (Fig. 1). The iNOS expression started on day 1, peaked on day 7 and then declined on day 10, whereas in control animals the iNOS expression was the same irrespective of the day investigated. Immunohistochemical staining of iNOS-positive cells in skin biopsies of animals immunized with N32-IFNg and N32-wt are shown in Fig. 2A and B, respectively. No iNOS-positive cells were found in draining lymph nodes or the spleen in any of the groups, or days investigated. Representative sections from the spleen and lymph nodes of animals

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immunized with N32-IFNg are shown in Fig. 2C and D, respectively. 3.2. iNOS expression in brain tumors We also studied the iNOS expression in tumors of animals with preestablished brain tumor, receiving peripheral immunizations with N32-IFNg. These were compared with control animals (N32-wt and sham immunization). To avoid bias from inflammatory changes induced by the injection of tumor cells in the brain we used a protocol with postponed immunizations (see Visse et al., 2000). In this setting, rats were immunized 10 days after intracerebral inoculation and the iNOS expression was evaluated on day 24. Immunohistochemical staining of brain tumors in animals immunized with N32-IFNg showed patches of iNOS positive cells spread over the complete tumor area, whereas only very few positive cells were detected in control animals (Fig. 3A and B). The distribution of iNOS positive cells correlates to a likewise patchy distribution of ED1 positive cells (Visse et al., 2000). Furthermore, the staining pattern also reflects that not all ED1 positive cells were iNOS positive. No iNOS staining was detected in the brain tissue surrounding the tumors. 3.3. Cellular origin of the iNOS expression in skin biopsies and in the brain tumor Immunofluorescence double staining was performed with different leucocyte cell surface markers to define the cells expressing iNOS. Mononuclear phagocytes were found to express the major part of the iNOS detected in both skin biopsies and in brain tumors of animals immunized with N32-IFNg (Fig. 4). However, some granulocytes also stained for iNOS (data not shown). 3.4. iNOS expression and phagocytosis of tumor cells By labeling the tumor cells with a fluorescent dye, the engulfment of tumor cells by mononuclear phagocytes was clearly demonstrated in the skin biopsies at all days investigated. Simultaneous staining of iNOS in this system showed that the mononuclear phagocytes expressing iNOS were associated with areas of intact or phagocytised tumor cells on day 1, while on day 7 the iNOS-expressing cells were separated from areas of engulfed tumor cells (Fig. 5). At this time point, most of the tumor cells were engulfed. Mononuclear phagocytes with engulfed tumor cells could also be detected in the draining lymph nodes,

Fig. 2. Representative immunohistochemical staining of iNOS positive cells at the immunization site in the draining lymph nodes and the spleen 7 days after immunization. (A) N32-IFNg cells (skin biopsy). (B) N32-wt cells (skin biopsy). (C) N32-IFNg cells (spleen). (D) N32-IFNg cells (lymph node). Fig. 3. Representative immunohistochemical staining of iNOS-positive cells in intracerebral tumors 24 days after inoculation and 10 days after (peripheral) immunization with N32-IFNg (A) and N32-wt (B) tumor cells. Normal surrounding brain tissue is visible in the lower right corners.

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The early up-regulation of iNOS at the site of immunization could be triggered directly by IFNg secretion from the tumor cells. At later stages, the iNOS expression most probably was induced by release of IFNg from activated immune cells. This explanation is supported by the findings that iNOS producing mononuclear phagocytes are gathered around intact tumor cells at day 1, whereas cells with phagocytised tumor debris are separated from iNOSexpressing cells at day 7. The cytotoxic effects of NO produced by iNOS are well documented and the tissue damage caused by NO has been shown to correlate with increased iNOS expression (Kolb and

Fig. 4. Immunofluorescence double staining of iNOS (red) and mononuclear phagocytes (green) in skin biopsies. Nuclei were counter-stained with DAPI (blue). (A) iNOS positive cells (red). (B) Mononuclear phagocytes (green). (C) Nuclear staining depicting distribution of all cells (blue). (D) Double-positive cells have a yellow/orange staining.

where no iNOS staining could be detected (data not shown).

4. Discussion In this study, we have shown a significant increase of iNOS expression at the immunization site and in brain tumors of animals immunized with rat glioma cells engineered to produce IFNg. No iNOS production was detected in draining lymph nodes or spleens. Mainly mononuclear phagocytes were responsible for the iNOS expression, but some granulocytes were also found to express iNOS.

Fig. 5. Immunofluorescence double staining of iNOS (red)/DiO stained tumor cells (green)/mononuclear phagocytes (blue) in skin biopsies of rats immunized with IFNg-producing N32 tumor cells. Pink/red cells indicate double staining of iNOS-producing mononuclear phagocytes. Green dots inside pink/red cells indicate iNOS-producing mononuclear phagocytes with engulfed tumor cells debris. Green cells or green dots indicate intact or engulfed tumor cell debris, respectively. (A) Day 1, (B) Day 7.

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Kolb Bachofen, 1998). The direct cytotoxic effects of NO on tumor cells at the immunization site or in the growing tumor could be favorable for the mode of antigen presentation. Higher concentrations of NO have been associated with necrotic and apoptopic cell death (Kroncke et al., 1997), both shown to be important for antigen presentation and maturation of dendritic cells (Bonnotte et al., 2000; Hoffmann et al., 2000; Jenne et al., 2000; Sauter et al., 2000). NO can modulate IL-12 production in early immune activation and, depending on concentration, it can both enhance and suppress immune activation, specifically Th1 responses (Niedbala et al., 1999). Thus, one explanation for the effect of NO in our system could be that it modulates the systemic antitumor immune response by affecting IL-12 production. The lack of iNOS expression in draining lymph nodes or the spleen could be because cells expressing iNOS do not migrate from the immunization site or because they down regulate their expression after migration. Preliminary results clearly show that no intact tumor cells could be found in the lymph nodes, hence, induction of iNOS by tumor cellderived IFNg is less likely (Johansson et al., manuscript in preparation). The remaining possible source of IFNg secretion in lymph nodes is activated T-lymphocytes or recirculating T-lymphocytes of memory phenotype, both entering through efferent lymph (Young, 1999). This IFNg secretion could be insufficient to induce detectable iNOS expression and thereby protect lymphocytes from deleterious amounts of NO. Expression of iNOS in the brain has been reported in both experimental (Feinstein et al., 1994) and human brain tumors (Ludwig et al., 2000). We have generally measured low or undetectable levels of iNOS in several rat gliomas studied (unpublished results). Different cytokines such as IFN-g, TNF-a and IL-1b often act in synergistic combinations to induce iNOS, but the induction of iNOS from each cytokine is probably both species- and cell-specific as discussed by Kwon and George (1999). Although we have detected both TNFa and IL-1b in the brain tumors, their expression was unrelated to the specific immunization (Johansson et al., unpublished results), thus, it seems likely that these cytokines did induce iNOS. If we disregard from the two more unlikely explanations for the enhanced iNOS expression in the brain tumors of immunized rats; systemic IFNg levels or homing of macrophages, the most plausible explanation of the enhanced iNOS expression in brain tumors of immunized animals is through release of IFNg produced by infiltrating leukocytes, as there is no other cellular source of IFNg production. This assumption is supported by our previous results, showing a significant increase in T cells, CD8+ cells and NK cells in rats immunized with IFNg transfected tumor cells compared to control animals (Visse et al., 2000). The NO production in the brain tumors of the N32-IFNg immunized animals could have multiple functions. iNOSmediated NO production could exert direct cytotoxic effects

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on tumor cells (Cobbs et al., 1995; Bakshi et al., 1998) but could also modulate antigen processing and presentation as discussed earlier. However, whether the latter also has any impact on the effector phase of the anti tumor response is not known (Plautz et al., 2000). Our results show that similar types of mononuclear phagocytes express iNOS both at the immunization site and in the brain tumor. It is to be noted that brain-resident microglia (identified by morphological criteria and OX42 staining, data not shown), both within the tumor and in the surrounding brain tissue, did not express iNOS. This strengthens the previously discussed assumption that iNOS was induced by infiltrating immune cells within the tumor. These findings also demonstrate the lack of inflammation in the surrounding brain tissue and, thus, the strict specificity of the evoked immune response. We propose that the early induction of iNOS expression seen locally at the immunization site might contribute to a better immune activation. However, the increased NO production could also have suppressive effects on the immune activation at later stages. Several lines of evidence have demonstrated that both a growing tumor and immunotherapeutic interventions can induce immune suppression mediated by overproduction of NO (Koblish et al., 1998; Medot Pirenne et al., 1999; Hegardt et al., 2000). It has furthermore been shown that this systemic production of NO can be paralleled by a diminished capacity for NO production from intratumoral macrophages (Alleva et al., 1994; Dinapoli et al., 1996). Thus, there is no clear-cut evidence that intratumoral macrophages could exert a suppressive effect by NO overproduction. In order to distinguish the net effects of NO production, both locally and systemically, we have initiated trials with NOS inhibitors and therapeutic immunizations. To fully ascertain the role of NO released from immune cells this will have to include administration of these compounds both at a systemic level and locally. Practically the latter would most probably require a sustained local release of NOS inhibitors. In conclusion, the distinct expression of iNOS in skin biopsies and brain tumors after immunization with N32IFNg might indicate that the local production of NO is important for the antitumor immune response since this also correlates to the therapeutic effect demonstrated in our previous studies. The net effect of systemic suppressive effects of NO on lymphocytes induction and proliferation vis a` vis the enhancing effects on immune induction and effector function have to be elucidated in further studies.

Acknowledgements We thank Hans-Olov Sjo¨gren for the review of the manuscript and Eva Gynnstam for her excellent technical assistance. This work was supported by grants from the Children’s Cancer Foundation of Sweden, the Crafoord Fo-

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undation, the Jonas Foundation, the Minerva Foundation, the Skane Region Funds and the Hedvig Foundation.

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