Bystander Killing of Malignant Glioma by Bone Marrow–derived Tumor-Infiltrating Progenitor Cells Expressing a Suicide Gene
Authors: Hrvoje Miletic, Yvonne Fischer, Sara Litwak, Tsanan Giroglou, Yannic Waerzeggers, Alexandra Winkeler, Huongfeng Li, Uwe Himmelreich, Claudia Lange, Werner Stenzel, Martina Deckert, Harald Neumann, Andreas H Jacobs, Dorothee von Laer
Journal: Molecular Therapy (2007)

Adult stem cells are promising cellular vehicles for therapy of malignant gliomas as they have the ability to migrate into these tumors and even track infiltrating tumor cells. However, their clinical use is limited by a low passaging capacity that impedes large-scale production. In the present study, a bone marrow–derived, highly proliferative subpopulation of mesenchymal stem cells (MSCs)—here termed bone marrow–derived tumor-infiltrating cells (BM-TICs)—was genetically modified for the treatment of malignant glioma. Upon injection into the tumor or the vicinity of the tumor, BM-TICs infiltrated solid parts as well as the border of rat 9L glioma. After intra-tumoral injection, BM-TICs expressing the thymidine kinase of herpes simplex virus (HSV-tk) and enhanced green fluorescent protein (BM-TIC-tk-GFP) were detected by non-invasive positron emission tomography (PET) using the tracer 9-[4-[18F]fluoro-3-hydroxymethyl)butyl]guanine ([18F]FHBG). A therapeutic effect was demonstrated in vitro and in vivo by BM-TICs expressing HSV-tk through bystander-mediated glioma cell killing. Therapeutic efficacy was monitored by PET as well as by magnetic resonance imaging (MRI) and strongly correlated with histological analysis. In conclusion, BM-TICs expressing a suicide gene were highly effective in the treatment of malignant glioma in a rat model and therefore hold great potential for the therapy of malignant brain tumors in humans. Adult stem cells are promising cellular vehicles for therapy of malignant gliomas as they have the ability to migrate into these tumors and even track infiltrating tumor cells. However, their clinical use is limited by a low passaging capacity that impedes large-scale production. In the present study, a bone marrow–derived, highly proliferative subpopulation of mesenchymal stem cells (MSCs)—here termed bone marrow–derived tumor-infiltrating cells (BM-TICs)—was genetically modified for the treatment of malignant glioma. Upon injection into the tumor or the vicinity of the tumor, BM-TICs infiltrated solid parts as well as the border of rat 9L glioma. After intra-tumoral injection, BM-TICs expressing the thymidine kinase of herpes simplex virus (HSV-tk) and enhanced green fluorescent protein (BM-TIC-tk-GFP) were detected by non-invasive positron emission tomography (PET) using the tracer 9-[4-[18F]fluoro-3-hydroxymethyl)butyl]guanine ([18F]FHBG). A therapeutic effect was demonstrated in vitro and in vivo by BM-TICs expressing HSV-tk through bystander-mediated glioma cell killing. Therapeutic efficacy was monitored by PET as well as by magnetic resonance imaging (MRI) and strongly correlated with histological analysis. In conclusion, BM-TICs expressing a suicide gene were highly effective in the treatment of malignant glioma in a rat model and therefore hold great potential for the therapy of malignant brain tumors in humans. IntroductionMalignant gliomas still have a dismal prognosis despite advances in neurosurgery and adjuvant radio- and chemotherapy. One major reason is their infiltrative growth pattern, with single tumor cells migrating distantly from the solid tumor mass and infiltrating normal brain tissue, where they can cause recurrent tumors. Novel therapies should aim to target these infiltrating cells, too.One very promising therapeutic approach is the intra-tumoral delivery of adult stem cells that express therapeutic genes. Stem cells are suitable cellular vehicles for glioma therapy as they have the ability to migrate to a pathologic focus. Several groups of researchers have shown that neural and mesenchymal stem cells (NSCs and MSCs) migrate specifically to and into malignant gliomas and even track infiltrating tumor cells.1Aboody KS Brown A Rainov NG Bower KA Liu S Yang W et al.Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas.Proc Natl Acad Sci USA. 2000; 97: 12846-12851Crossref PubMed Scopus (1007) Google Scholar,2Nakamizo A Marini F Amano T Khan A Studeny M Gumin J et al.Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas.Cancer Res. 2005; 65: 3307-3318Crossref PubMed Scopus (915) Google Scholar,3Nakamura K Ito Y Kawano Y Kurozumi K Kobune M Tsuda H et al.Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model.Gene Ther. 2004; 11: 1155-1164Crossref PubMed Scopus (516) Google Scholar However, there are two important limitations in terms of large-scale production of these cells for clinical application. NSCs can be isolated only from fetal or adult brain or generated from embryonic stem cells. In addition, they have a very limited passaging capacity in vitro. MSCs, although easy accessible from the bone marrow, also have a low passaging capacity, with a maximum of up to 5–50 population doublings (PDs) in vitro.4Hamada H Kobune M Nakamura K Kawano Y Kato K Honmou O et al.Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene therapy.Cancer Sci. 2005; 96: 149-156Crossref PubMed Scopus (203) Google Scholar,5Colleoni S Donofrio G Lagutina I Duchi R Galli C Lazzari G Establishment, differentiation, electroporation, viral transduction, and nuclear transfer of bovine and porcine mesenchymal stem cells.Cloning Stem Cells. 2005; 7: 154-166Crossref PubMed Scopus (80) Google Scholar So far, these stem cells can be expanded efficiently only after introduction of a growth-promoting gene. Examples are MSCs that are transfected with the human telomerase reverse transcriptase gene4Hamada H Kobune M Nakamura K Kawano Y Kato K Honmou O et al.Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene therapy.Cancer Sci. 2005; 96: 149-156Crossref PubMed Scopus (203) Google Scholar or the murine NSC line C17.2,6Snyder EY Deitcher DL Walsh C Arnold-Aldea S Hartwieg EA Cepko CL Multipotent neural cell lines can engraft and participate in development of mouse cerebellum.Cell. 1992; 68: 33-51Abstract Full Text PDF PubMed Scopus (753) Google Scholar genetically modified to express c-myc.An attractive option for clinical use is a highly proliferative subpopulation of bone marrow–derived MSCs called bone marrow–derived multipotent adult progenitor cells (MAPCs), which can be expanded for more than 100 PDs without exogenous immortalization. MAPCs are pluripotent and differentiate into most somatic tissues in vivo when injected into an early blastocyst.7Jiang Y Jahagirdar BN Reinhardt RL Schwartz RE Keene CD Ortiz-Gonzalez XR et al.Pluripotency of mesenchymal stem cells derived from adult marrow.Nature. 2002; 418: 41-49Crossref PubMed Scopus (5128) Google Scholar In our study, we derived bone marrow cells according to the original protocol published by Jiang et al.7Jiang Y Jahagirdar BN Reinhardt RL Schwartz RE Keene CD Ortiz-Gonzalez XR et al.Pluripotency of mesenchymal stem cells derived from adult marrow.Nature. 2002; 418: 41-49Crossref PubMed Scopus (5128) Google Scholar As described for MAPCs, the isolated cells were negative for CD44, CD90, and major histocompatibility complex (MHC) class I antigens,7Jiang Y Jahagirdar BN Reinhardt RL Schwartz RE Keene CD Ortiz-Gonzalez XR et al.Pluripotency of mesenchymal stem cells derived from adult marrow.Nature. 2002; 418: 41-49Crossref PubMed Scopus (5128) Google Scholar whereas all three markers are found on MSCs.8Mareschi K Ferrero I Rustichelli D Aschero S Gammaitoni L Aglietta M et al.Expansion of mesenchymal stem cells isolated from pediatric and adult donor bone marrow.J Cell Biochem. 2006; 97: 744-754Crossref PubMed Scopus (260) Google Scholar,9Stute N Holtz K Bubenheim M Lange C Blake F Zander AR Autologous serum for isolation and expansion of human mesenchymal stem cells for clinical use.Exp Hematol. 2004; 32: 1212-1225Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar Our study focused on the tumor-infiltrating ability of this MAPC-like cell population rather than on their capacity to differentiate into different lineages. Therefore, we here name these MAPC-like cells bone marrow–derived tumor-infiltrating cells (BM-TICs). BM-TICs were compared with various cell lines that can be expanded to high cell numbers for their migratory behavior in malignant glioma in vivo. We demonstrated that BM-TICs have a migratory capacity similar to other stem cell lines, whereas hematopoietic cell lines and fibroblast control cells were unable to migrate specifically. BM-TICs were transduced with a retroviral vector expressing the thymidine kinase of herpes simplex virus (HSV-tk) and the green fluorescent protein (BM-TIC-tk-GFP) and traced by non-invasive positron emission tomography (PET). A bystander-mediated therapeutic effect of BM-TIC-tk-GFP for glioma cells was demonstrated in vitro and in vivo.ResultsPhenotypic characterization of rat BM-TICs and MSCsBM-TICs were obtained from the bone marrow of adult Fischer rats according to the protocol for isolation and expansion of MAPCs.7Jiang Y Jahagirdar BN Reinhardt RL Schwartz RE Keene CD Ortiz-Gonzalez XR et al.Pluripotency of mesenchymal stem cells derived from adult marrow.Nature. 2002; 418: 41-49Crossref PubMed Scopus (5128) Google Scholar The expression of different cell-surface markers was analyzed by flow cytometry. BM-TICs were negative for CD31, CD44, CD45, CD90, CD105, CD106, and MHC class I, whereas MSCs expressed CD44, CD90, and MHC class I, as described in the literature (Table 1).7Jiang Y Jahagirdar BN Reinhardt RL Schwartz RE Keene CD Ortiz-Gonzalez XR et al.Pluripotency of mesenchymal stem cells derived from adult marrow.Nature. 2002; 418: 41-49Crossref PubMed Scopus (5128) Google Scholar,8Mareschi K Ferrero I Rustichelli D Aschero S Gammaitoni L Aglietta M et al.Expansion of mesenchymal stem cells isolated from pediatric and adult donor bone marrow.J Cell Biochem. 2006; 97: 744-754Crossref PubMed Scopus (260) Google Scholar,9Stute N Holtz K Bubenheim M Lange C Blake F Zander AR Autologous serum for isolation and expansion of human mesenchymal stem cells for clinical use.Exp Hematol. 2004; 32: 1212-1225Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar BM-TICs were thus phenotypically distinct from the classical MSC population.Table 1Phenotypic characterization of rBM-TIC and rMSCSurface markersaCell-surface expression of the indicated proteins was determined by flow cytometry.BM-TICMSCCD31−−CD44−+CD45−−CD90−+CD105−−CD106−−MHCI−+Abbreviations: BM-TIC, bone marrow–derived tumor-infiltrating cell; MSC, mesenchymal stem cell; MHCI, major histocompatibility complex class I.a Cell-surface expression of the indicated proteins was determined by flow cytometry. Open table in a new tab Migratory capacity of BM-TICs and hematopoietic cell lines in 9L glioma in vivoCell lines with a high passaging capacity, which are suitable for genetic modification and large-scale production, were tested for migration in a rat glioma model. In this study, BM-TICs were used at PDs of 15–70. No major influence of the passage number on the migratory behavior described below was observed.We investigated whether BM-TIC, K562, Jurkat, or Raji cell lines infiltrate established malignant glioma. The NSC line C17.2, for which migration has been shown in a previous study,1Aboody KS Brown A Rainov NG Bower KA Liu S Yang W et al.Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas.Proc Natl Acad Sci USA. 2000; 97: 12846-12851Crossref PubMed Scopus (1007) Google Scholar was used as positive control, and 3T3 as negative control. To rule out the interference of an immune (Supplementary Figure S1) response against allogeneic cell lines with their migration ability, syngeneic Fischer rat fibroblasts Rat-1 were used as a second negative control. Glioma cells (9L glioma cell line) transduced with the red fluorescence gene DsRed (9LDsRed) were implanted into the striatum of Fischer rats, and 5 days thereafter enhanced GFP (eGFP)–transduced cell lines were injected into established tumors or in the vicinity of tumors. At 3 and 7 days after implantation of the distinct cell lines, rats were killed and brain sections were analyzed by fluorescence microscopy. Within 3 days after injection, BM-TICs had migrated from the injection site to the tumor border (Figure 1a) as well as into the solid tumor areas (Figure 1b). At day 7, BM-TICs had infiltrated the majority of the solid tumor mass (Figure 1c) and were also found at the tumor border, even tracking tumor cells infiltrating the normal tissue (Figure 1d). Importantly, BM-TICs did not migrate into normal brain tissue without having been triggered by a tumor cell. The NSC line C17.2 showed a similar migratory behavior (Figure 1e). In contrast, 3T3 cells remained at the injection site and were well demarcated from the tumor tissue (Figure 1f). 3T3 cells did not migrate toward the border of the tumor or infiltrating tumor cells. The cell line K562 was detected in the brain adjacent to the tumor mass surrounding the tumor (Figure 1g). The same was observed for Jurkat and Raji cells (data not shown). Cells of these lines were not found in the tumor mass. However, individual cells were detected in normal brain parenchyma adjacent to the tumor (Figure 1h). The cell line Rat-1, too, did not migrate into the tumor mass but stayed in the brain adjacent to the tumor mass (Figure 1i).Gap junction formation between BM-TICs and 9L cellsThe formation of gap junctions between therapeutic cells and tumor cells is critical in a suicide gene therapy approach, as gap junctions are responsible for the bystander effect. Gap junction communication between cells can be measured by dye-transfer experiments and flow cytometry.10Czyz J Irmer U Schulz G Mindermann A Hulser DF Gap-junctional coupling measured by flow cytometry.Exp Cell Res. 2000; 255: 40-46Crossref PubMed Scopus (60) Google Scholar In these experiments, one cell population contains a non-membrane-permeable dye that is taken up by another cell population upon incubation. Here, 9L cells were labeled with the membrane dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) and incubated together with BM-TICs containing the non-membrane-permeable dye calcein-AM. As shown in (Figure 2a), 9L/DiI and BM-TIC/calcein-AM cells form two distinct populations of either red- or green-fluorescent cells immediately after mixing. After 3 hours of incubation, however, 9L/DiI cells have taken up green-fluorescent calcein-AM and appear as a population of double-positive red- and green-fluorescent cells (Figure 2b), whereas BM-TICs have lost intensity of green fluorescence owing to transfer of calcein-AM and taken up more DiI. DiI cannot be transferred from one cell to another as it is a membrane-bound protein. However, DiI can be set free from apoptotic cells (during the incubation period, by trypsinization, before or during flow cytometry) and thereafter can bind to the membrane of living cells (9L cells and BM-TICs), which might explain the shift in both populations. In contrast, cytoplasmatic or free calcein-AM can be taken up by a living cell only via gap junctions. Therefore, the shift of 9L/DiI in the direction of calcein-AM can be explained only by gap-junctional communication.Figure 2Gap junction formation between bone marrow–derived tumor-infiltrating cells (BM-TICs) and 9L cells. 9L cells were stained with the membrane dye DiI (red fluorescent) and BM-TICs were stained with the cytoplamic dye calcein-AM (green fluorescent), which can be transferred from cell to cell only via gap junctions. Flow cytometry measurements of dye transfer between 9L/DiI and BM-TIC/calcein-AM was assessed (a) immediately after mixing and (b) after 3 hours' incubation at 37°C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In vitro bystander killing of 9L glioma cells by BM-TICs expressing HSV-tkBM-TICs were transduced with a retroviral vector expressing the HSV-tk suicide gene (BM-TIC-tk; see Materials and Methods). The bystander killing effect of BM-TIC-tk on 9L-DsRed glioma cells was examined by co-culture experiments. After addition of ganciclovir (GC) to the medium, BM-TIC-tk exerted a strong and dose-dependent bystander effect on glioma cells (Figure 3). Killing of 90% of glioma cells was achieved in cultures containing 50% or even only 10% BM-TIC-tk. Killing was statistically significant with GC concentrations ranging from 10−3 to 103 mu;mol/lfor 50% BM-TIC-tk and from 10−2 to 103 μmol/l for 10% BM-TIC-tk. In contrast, 9LDsRed cells mixed with native BM-TICs were resistant to GC up to high concentrations (Figure 3). These experiments show that BM-TIC-tk can be used to deliver toxic molecules, such as phosphorylated GC, to glioma cells.Figure 3Bystander effect of bone marrow–derived tumor-infiltrating cells expressing the thymidine kinase (BM-TIC-tk) on 9L glioma cells in vitro. Mixed cultures of 9L-DsRed cells containing either 50 or 10% BM-TIC or BM-TIC-tk, respectively, were incubated with ganciclovir (GC) at increasing concentrations. After 3 days, cells were lysed and the fluorescence intensity of DsRed in the lysates, corresponding to the number of living 9L glioma cells, was quantified with an automated 96-well plate fluorescence reader. The graph shows mean fluorescence and standard deviations from three independent experiments. P refers to levels of statistical significance between BM-TIC 50% and BM-TIC-tk 50% (indicated above single values) and BM-TIC 10% and BM-TIC-tk 10% (indicated underneath single values). Student's t-test for non-discrete grade values. *P< 0.05, **P< 0.01, ***P< 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In vivo bystander killing of 9L glioma cells by BM-TIC-tk-GFPTo analyze the therapeutic effect of BM-TICs in vivo, BM-TICs transduced with HSV-tk and GFP (BM-TIC-tk-GFP) were injected into 9L tumors 5 days after tumor implantation. As a control, non-migrating Rat-1 transduced with HSV-tk and GFP (Rat-1-tk-GFP) were implanted. From day 4 after cell injection, one group of BM-TIC-implanted rats (n = 9), one group of Rat-1-implanted rats (n = 8), and one control group implanted with 9L only (n = 8) received daily intraperitoneal injections of 30 mg per kg GC for 10 days. Two control groups of BM-TIC-tk-GFP- (n = 8) or Rat-1-tk-GFP-implanted rats (n = 8) were not treated with GC. Kaplan–Meier survival analysis showed more than 60% long-term survivors in the BM-TIC-tk-GFP plus GC–treated group surviving for 110 days (Figure 4a). Two out of nine animals died at day 28, and one died at day 54 after tumor implantation. All animals in the control groups died within the observation period. Thus, treated animals showed a prolonged survival compared with both control groups that was statistically highly significant (P< 0.001). In contrast, Rat-1-tk-GFP and GC–treated animals showed a significantly reduced survival compared with BM-TIC-tk-GFP and GC–treated animals (P< 0.01), but also a significantly prolonged survival compared with control groups (P< 0.05).Figure 4Bystander effect of bone marrow–derived tumor-infiltrating cells (BM-TICs) expressing the thymidine kinase (BM-TIC-tk) and green fluorescent protein (BM-TIC-tk-GFP) on 9L glioma in vivo. BM-TIC-tk-GFP or Rat-1-tk-GFP cells were injected into 9L tumors (groups BM-TIC-tk-GFP, BM-TIC-tk-GFP + ganciclovir (GC), Rat-1-tk-GFP, and Rat-1-tk-GFP + GC) 5 days after tumor cell implantation in adult rats. One group (GC) received 9L cells only. Four days after BM-TIC-tk-GFP or Rat-1-tk-GFP implantation, the groups GC, BM-TIC-tk-GFP + GC, and Rat-1-tk-GFP + GC were treated with GC for 10 days. (a) In total 66.67% of rats survived 110 days in the BM-TIC-tk-GFP plus GC–treated group. The graph shows the Kaplan–Meier survival curve. The difference in survival between the treated group and control groups was statistically significant (P< 0.001; log-rank test). The Rat-1-tk-GFP + GC–treated group survived significantly shorter compared with the BM-TIC-tk-GFP + GC–treated group (P < 0.01; log rank test). (b) Histological analysis of brains of long-term survivors in the BM-TIC-tk-GFP + GC–treated group revealed a cavity and scar formation at the initial site without tumor cells. Hematoxylin and eosin staining, original magnification ×25. (c) CD45+ leukocytes infiltrate the scar tissue. Anti-CD45-staining, original magnification ×25.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Tumor cells were not detected on serial histological sections in the brains of BM-TIC-tk-GFP plus GC–treated long-term survivors. A cavity and scar tissue resulting from the initial tumor mass, which had been successfully treated, was observed in long-term survivors and was infiltrated by CD45-positive inflammatory cells (Figure 4b and c).Detection of HSV-1-tk-GFP-transduced BM-TICs and therapeutic follow-up by PET and MRIFor clinical application of therapeutic cells, it is essential to be able to locate these cells using non-invasive methods. For this purpose, BM-TIC-tk-GFP cells were injected into 9LDsRed tumors, and 9-[4-[18F]fluoro-3-hydroxymethyl)butyl]guanine ([18F]FHBG) PET scans for detection of BM-TIC-tk-GFP were applied 6–7 days later. [18F]FHBG is the specific substrate for HSV-tk and therefore confirms HSV-tk expression in vivo. In addition, methyl-[11C]-l-methionine ([11C]MET) PET scans and magnetic resonance imaging (MRI) scans for detection of the 9LDsRed tumor were performed up to 22 days. BM-TIC-tk-GFP cells were detected in the tumor (Figure 5a and b), and fluorescence microscopy of histological sections confirmed eGFP-positive cells at the border and in solid tumor parts (Figure 5e). The relative expression level of HSV-tk was 0.113 ± 0.025% of the injected dose/g tissue (Figure 5f). In contrast, animals that had received glioma cells only (control group) had a significantly lower [18F]FHBG uptake in the tumor (0.053 ± 0.006% of injected dose/g; P < 0.05; Figure 5c–d) and f).Figure 5Detection of bone marrow–derived tumor-infiltrating cells (BM-TICs) expressing the thymidine kinase and green fluorescent protein (BM-TIC-tk-GFP) by positron emission tomography (PET) imaging. BM-TIC-tk-GFP cells were injected into 9LDsRed tumors, and 6–7 days later 9-[4-[18F]fluoro-3-hydroxymethyl)butyl]guanine ([18F]FHBG) PET scans were performed. In addition, methyl-[11C]-l-methionine [11C]MET PET scans and magnetic resonance imaging (MRI) scans for detection of the 9LDsRed tumor were performed on different days. On day 7 after BM-TIC-tk-GFP implantation, animals were perfused and fluorescence microscopy was applied for detection of BM-TIC-tk-GFP. (a) [18F]FHBG PET scan of a BM-TIC-tk-injected animal showing HSV-tk expression in the tumor area with a maximum of 0.14% of injected dose/g. (b) [11C]MET PET scan of the same animal detecting 9LDsRed tumor 7 days after tumor implantation. (c) [18F]FHBG PET scan of a control animal (9LDsRed only) showing low [18F]FHBG uptake in the tumor area with a maximum of 0.05% of injected dose/g. (d) [11C]MET PET scan of the same animal detecting 9LDsRed tumor 7 days after tumor implantation. (e) Enhanced GFP (eGFP)–positive BM-TIC-tk-GFP cells are detected at the tumor border and the solid tumor part. Overlay of green (BM-TIC-tk-GFP) and red (9LDsRed tumor) fluorescence. Magnification ×50. (f) Histogram showing relative HSV-tk expression level. [18F]FHBG uptake in BM-TIC-tk-GFP-injected animals (0.113 ± 0.025% of the injected dose/g; n = 3) was significantly higher (P < 0.05) than in the control group (0.053 ± 0.006% of injected dose/g; n = 3). Shown are the mean values of each group. P refers to levels of statistical significance between the BM-TIC-tk-GFP and 9LDsRed-only groups. Student's t-test for non-discrete grade values. *P < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)For treatment with GC, the animals were divided into three groups as described above for the survival study. On day 6 and day 7 or 8 after tumor implantation and before GC treatment, tumors were detected by MRI and [11C]MET PET scans, respectively (Figure 6a–f). In animals without GC treatment, implanted BM-TIC-tk-GFP cells were detected by [18F]FHBG PET scans 7 days after implantation (Figure 6h). In contrast, animals with 3 days of GC treatment had an uptake similar to control animals that received 9LDsRed cells and GC (Figure 6g and i), indicating loss of HSV-tk activity as a result of the treatment. Five days after initiation of GC treatment, the tumor size of treated animals was reduced compared with both control groups, as shown on MRI and [11C]MET PET scans (Figure 6j–o). As determined by semi-quantitative estimations based on the size of the hyperintense lesion in gadopentetic acid–enhanced (Gd-DTPA) three-dimensional MRI, the tumor size in the treated animals was 20–40% that of both controls on day 13. Three to five days after termination of GC application, animals with a good response to treatment exhibited a low contrast-enhancing lesion in MRI (Figure 6p) and only a very weak signal in [11C]MET PET (Figure 6q). The hyperintense lesion for individual animals increased from day 7–8 to day 13 by a factor of 1.7 ± 0.3 for the treated animals and by a factor of 6.1 ± 0.5 for both control groups. The lesion size in the treated animals did not change significantly between day 13 (during GC treatment) and day 21 (after GC treatment; change by a factor of 0.95 ± 0.1). Compared with day 13, lesion appearance in the final MRIs was very heterogeneous, indicating the possible presence of cystic and necrotic compartments. By histology, only residual tumor cells were detected, surrounded and intermingled with CD45-positive inflammatory cells (Figure 7a, d and e). The latter consisted predominantly of CD8+ T cells. In addition, CD4+ T cells, ED1+ macrophages, and CD161+ natural killer cells were seen (Figure 7f–i). In both control groups, animals became terminally ill during the treatment and were killed. Histology revealed large tumor masses (Figure 7b and c) that contained less prominent leukocytic infiltrations compared with the treated group (Figure 7j–o).Figure 6Magnetic resonance imaging (MRI) and positron emission tomography (PET) imaging for therapeutic follow-up. Representative three-dimensional MRI (T1-weightened FLASH, echo time = 5 ms, repetition time = 70 ms, 60° pulse, resolution 121 × 121 × 242 μm, post-administration of gadopentetic acid (Gd-DTPA), [18C] methyl-[18C]-l-methionine MET PET and [18F] 9-[4-[18F]fluoro-3-hydroxymethyl)butyl]guanine FHBG PET scans. Time points after tumor implantation: (a–c) 6 days, MRI before ganciclovir (GC) treatment; (d–f) 7–8 days, [18C]MET PET before GC treatment; (g–i) 12 days, [18F]FHBG PET during GC treatment; (j–l) 13 days, MRI during GC treatment; (m–o) 14 days, [18C]MET PET during GC treatment; (p) 21 days, MRI after GC treatment; (q) 22 days [18C]MET PET after GC treatment. The three different groups were treated as described for the survival study: (a, d, g, j, m, p, q) bone marrow–derived tumor-infiltrating cells expressing the thymidine kinase of green fluorescent protein (BM-TIC-tk-GFP) injection with GC treatment; (b, e, h, k, n) BM-TIC-tk-GFP injection without GC treatment; (c, f, i, l, o) 9LDsRed only with GC treatment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Histology and immunohistochemistry. Animals were killed and perfused when they became terminally ill or, in the therapeutic group, 4–8 days after end of ganciclovir (GC) treatment. (a–c) Macroscopic photographs of transversal brain sections. Hematoxylin and eosin (H&E) staining. (a) Bone marrow–derived tumor-infiltrating cells (BM-TICs) expressing the thymidine kinase and green fluorescent protein (BM-TIC-tk-GFP) injected and GC–treated animal with a small lesion. Control animals show large tumor masses. (b) BM-TIC-injected animal. (c) Control animal with 9LDsred only and GC treatment. (d–i) Animal shown in a at higher magnification (×25) and immunohistochemistry. (d) Residual tumor cells infiltrated by inflammatory cells. H&E staining. (e) Inflammatory cells are identified as CD45+ leukocytes, which consist of (f) CD4+ T cells, (g) CD8+ T cells, (h) ED1+ macrophages and microglia, and (i) CD161+ natural killer cells. (j–o) Control animal shown in b at higher magnification (×25) and immunohistochemistry. (j) Large tumor mass with necrotic areas and no obvious immune cell infiltration. H&E staining. (k) CD45+ leukocytes are detected around necrotic areas and at the tumor border and are less prominent compared with the therapeutic group. Leukocytes consist of (l) CD4+ T cells, (m) CD8+ T cells, (n) ED1+ macrophages and microglia, and (o) few CD161+ NK cells. Original magnification ×25.View Large Image Figure ViewerDownload Hi-res image Download (PPT)These data demonstrate that PET imaging is a suitable method to detect therapeutic cells in vivo. Fur