Orthotopic transplantation of v-src–expressing glioma cell lines into immunocompetent mice: establishment of a new transplantable in vivo model for malignant glioma

Restricted access

Object

The aim of this study was to develop and characterize a new orthotopic, syngeneic, transplantable mouse brain tumor model by using the cell lines Tu-9648 and Tu-2449, which were previously isolated from tumors that arose spontaneously in glial fibrillary acidic protein (GFAP)-v-src transgenic mice.

Methods

Striatal implantation of a 1-μl suspension of 5000 to 10,000 cells from either clone into syngeneic B6C3F1 mice resulted in tumors that were histologically identified as malignant gliomas. Prior subcutaneous inoculations with irradiated autologous cells inhibited the otherwise robust development of a microscopically infiltrating malignant glioma. Untreated mice with implanted tumor cells were killed 12 days later, when the resultant gliomas were several millimeters in diameter. Immunohistochemically, the gliomas displayed both the astroglial marker GFAP and the oncogenic form of signal transducer and activator of transcription–3 (Stat3). This form is called tyrosine-705 phosphorylated Stat3, and is found in many malignant entities, including human gliomas. Phosphorylated Stat3 was particularly prominent, not only in the nucleus but also in the plasma membrane of peripherally infiltrating glioma cells, reflecting persistent overactivation of the Janus kinase/Stat3 signal transduction pathway. The Tu-2449 cells exhibited three non-random structural chromosomal aberrations, including a deletion of the long arm of chromosome 2 and an apparently balanced translocation between chromosomes 1 and 3. The GFAP-v-src transgene was mapped to the pericentromeric region of chromosome 18.

Conclusions

The high rate of engraftment, the similarity to the high-grade malignant glioma of origin, and the rapid, locally invasive growth of these tumors should make this murine model useful in testing novel therapies for human malignant gliomas.

Abbreviations used in this paper:FISH = fluorescence in situ hybridization; GBM = glioblastoma multiforme; GFAP = glial fibrillary acidic protein; IL-6 = interleukin-6; PNST = peripheral nerve sheath tumor; pY-Stat3 = tyrosine-705 phosphorylated signal transducer and activator of transcription–3; SSC = standard saline citrate; TBS = Tris-buffered saline.

Article Information

Address reprint requests to: Henry M. Smilowitz, Ph.D., Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06030. email: smilowitz@nso1.uchc.edu.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    Photomicrographs showing sections used for histopathological examination of formalin-fixed, paraffin-embedded malignant gliomas, which was performed 12 days after implantation of 10,000 cells of the clone Tu-9648 or Tu-2449 (shown) into the striatum of syngeneic B6C3F1 mice. A: Transplanted tumor located in the striatum. Immunohistochemical labeling (red) for Stat3α is shown. The tumor nodule (red area), which is immunoreactive for Stat3, is not sharply delineated and has several small satellites. Bar = 250 μm. B: Axon bundles of the internal capsule, which are immuno-reactive for neurofilaments (red areas), are diffusely infiltrated by the implanted tumor. Bar = 150 μm. C: Diffusely infiltrating, polymorphic cells of a transplanted tumor. H & E, bar = 100 μm. D: The tumor cells show moderate to intense cytoplasmic GFAP immunoreactivity. Bar = 50 μm. E: Distinct, plasma membrane–associated immunoreactivity for activated pY-Stat3 of neoplastic cells is seen at the margin of a transplanted tumor (arrows). Nuclear pY-Stat3 labeling is minor to undetectable in this image's field, but was strong in other tumors derived from the transplanted cell lines (not shown). Bar = 50 μm. F: Margin of a glioma that spontaneously developed in a GFAP-v-src transgenic mouse. As in the transplanted tumors, the neoplastic cells show distinct pY-Stat3 immunoreactivity of the plasma membrane (arrows). In addition, nuclear labeling of neoplastic cells is prominent in this field. Bar = 50 μm.

  • View in gallery

    Karyotype of a G-banded metaphase cell from a Tu-2449 lesion. Upper: Structurally rearranged chromosomes that could not be confidently identified using G-band analysis are indicated by the letter A. Lower: Fluorescence in situ hybridization with Cambio Mouse Rainbow FISH paint set 1 (whole chromosome paint probes 1–7) to a metaphase cell from a Tu-2449 lesion. Translocation between chromosomes 1 and 3 is indicated by blue and red arrows, respectively. Deletion of chromosome 2 (2A1) is marked with a green arrow.

  • View in gallery

    An FISH study showing mapping of the GFAP-v-src transgene to the chromosome 18A1 region. Cohybridization of Cambio whole chromosome mouse paint for chromosome 18 and the GFAP-v-src transgene to Tu-2449 metaphase chromosome is shown. Red arrows point to chromosome 18, which is labeled with avidin and detected with avidin–Texas Red. Green arrows point to the v-src transgene probes labeled with digoxigenin and detected with antidigoxigenin fluorescein. Metaphase chromosomes are blue.

  • View in gallery

    Kaplan–Meier plots showing the fraction of surviving mice according to days after intracerebral implantation of tumor cell lines. The Tu-9648 (upper) and Tu-2449 (lower) tumor cells were implanted with and without prior immunotherapy (immunized and nonimmunized). The B6C3F1 hybrid mice were challenged with either 5000 cultured Tu-9648 or 10,000 cultured Tu-2449 cells in the left striatum (Day 0). Immunized mice had received a series of four weekly subcutaneous injections of irradiated (50 Gy) Tu-9648 or Tu-2449 cells before the intracerebral challenge.

References

1

Amalfitano GChatel MPaquis PMichiels JF: Fluorescence in situ hybridization study of aneuploidy of chromosomes 7, 10, X, and Y in primary and secondary glioblastomas. Cancer Genet Cytogenet 116:692000

2

Angers-Loustau AHering RWerbowetski TEKaplan DRDel Maestro RF: Src regulates actin dynamics and invasion of malignant glial cells in three dimensions. Mol Cancer Res 2:5956052004

3

Band CJMounier CPosner BI: Epidermal growth factor and insulin-induced deoxyribonucleic acid synthesis in primary rat hepatocytes is phosphatidylinositol 3-kinase dependent and dissociated from protooncogene induction. Endocrinology 140:562656341999

4

Benn PPerle MChromosome staining and banding techniques. Rooney DECzepulkowski BH: Human Cytogenetics: A Practical Approach New YorkOxford University Press1992. 91117

5

Bigner SHVogelstein B: Cytogenetics and molecular genetics of malignant gliomas and medullobalstoma. Brain Pathol 1:12181990

6

Bromberg JFHorvath CMBesser DLathem WWDarnell JE Jr: Stat3 activation is required for cellular transformation by v-src. Mol Cell Biol 18:255325581998

7

Bromberg JFWrzeszczynska MHDevgan GZhao YPestell RGAlbanese C: Stat3 as an oncogene. Cell 98:2953031999

8

Frame MC: Src in cancer: deregulation and consequences for cell behavior. Biochem Biophys Acta 1602:1141302002

9

Fung YKCrittenden LBFadly AMKung HJ: Tumor induction by direct injection of cloned v-src DNA into chickens. Proc Natl Acad Sci U S A 80:3533571983

10

Gelman IHHanafusa H: src-specific immune regression of Rous sarcoma virus-induced tumors. Cancer Res 53:9159201993

11

Holland EC: Mouse models of human cancer as tools in drug development. Cancer Cell 6:1971982004

12

Hosmer DW JrLemeshow S: Applied Survival Analysis: Regression Modeling of Time to Event Data New YorkJohn Wiley & Sons1999

13

Kim DHMohapatra GBollen AWaldman FMFeuerstein BG: Chromosomal abnormalities in glioblastoma multiforme tumors and glioma cell lines detected by comparative genomic hybridization. Int J Cancer 60:8128191995

14

Lassman AB: Molecular biology of gliomas. Curr Neurol Neurosci Rep 4:2282332004

15

Loeffler SFayard BWeis JWeissenberger J: Interleukin-6 induces transcriptional activation of vascular endothelial growth factor (VEGF) in astrocytes in vivo and regulates VEGF promoter activity in glioblastoma cells via direct interaction between STAT3 and Sp1. Int J Cancer 115:2022132005

16

Loeper SRomeike BFHeckmann NJung VHenn WFeiden W: Frequent mitotic errors in tumor cells of genetically micro-heterogeneous glioblastomas. Cytogenet Cell Genet 94:182001

17

Lund CVNguyen MTNOwens GCPakchoian AJShaterian AKruse CA: Reduced glioma infiltration in Src-deficient mice. J Neurooncol 78:19292006

18

Mulholland PJFiegler HMazzanti CGorman PSasienti PAdams J: Genomic profiling identifies discrete deletions associated with translocations in glioblastoma multiforme. Cell Cycle 5:7837912006

19

Niu GWright KLHuang MSong LHaura ETurkson J: Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis. Oncogene 21:200020082002

20

Paz KSocci NDVan Nimwegen EViale ADarnell JE: Transformation fingerprint: induced STAT3-c, v-Src and Ha-Ras cause small initial changes but similar established profiles in mRNA. Oncogene 23:845584632004

21

Pohl UWick WWeissenberger JSteinbach JPDichgans JAguzzi A: Characterization of Tu-2449, a glioma cell line derived from a spontaneous tumor in GFAP-v-src-transgenic mice: comparison with established murine glioma cell lines. Int J Oncol 15:8298341999

22

Rao VKWangsa DRobey RWHuff LHonjo YHung J: Characterization of ABCG2 gene amplification manifesting as extrachromosomal DNA in mitoantrone-selected SF295 human glioblastoma cells. Cancer Genet Cytogenet 160:1261332005

23

Romer JCurran T: Targeting medulloblastoma: Small-molecule inhibitors of the Sonic Hedgehog pathway as potential cancer therapeutics. Cancer Res 65:497549782005

24

Smilowitz HMJoel DDSlatkin DNMicca PLNawrocky MMYoungs K: Long-term immunological memory in the resistance of rats to transplantable intracerebral 9L gliosarcoma (9LGS) following subcutaneous immunization with 9LGS cells. J Neurooncol 46:1932032000

25

Stettner MRWang WNabors LBBharara SFlynn DCGrammer JR: Lyn kinase activity is the predominant cellular SRC kinase activity in glioblastoma tumor cells. Cancer Res 65:553555432005

26

Theurillat JPHainfellner JMaddalena AWeissenberger JAguzzi A: Early induction of angiogenetic signals in gliomas of GFAP-v-src transgenic mice. Am J Pathol 154:5815901999

27

Turkson J: STAT proteins as novel targets for cancer drug discovery. Expert Opin Ther Targets 8:4094222004

28

Turkson JBowman TGarcia RCaldenhoven EDe Groot RPJove R: Stat3 activation by src induces specific gene regulation and is required for cell transformation. Mol Cell Biol 18:254525521998

29

Weissenberger JLoeffler SKappeler AKopf MLukes AAfanasieva TA: IL-6 is required for glioma development in a mouse model. Oncogene 23:330833162004

30

Weissenberger JSteinbach JPMalin GSpada SRulicke TAguzzi A: Development and malignant progression of astrocytomas in GFAP-v-src transgenic mice. Oncogene 14:200520131997

31

Wemmert SKetter RRahnenfuhrer JBeerenwinkel NStrowitzki MFriden W: Patients with high-grade gliomas harboring deletions of chromosomes 9p and 10q benefit from temozolomide treatment. Neoplasia 7:8838932005

32

Wilde ABeattie ECLem LRiethof DALiu SHMobley WC: EGF receptor signaling stimulates SRC kinase phosphorylation of clathrin, influencing clathrin redistribution and EGF uptake. Cell 96:6776871999

33

Wisner TWEngland JMPan DYStoker AWHalpern MS: Tumor immunity generated in the course of regression of v-src induced sarcomas. J Virol 65:702070241991

34

Yu HJove R: The STATs of cancer—new molecular targets come of age. Nat Rev Cancer 4:971052004

TrendMD

Cited By

Metrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 88 88 9
Full Text Views 126 126 5
PDF Downloads 28 28 1
EPUB Downloads 0 0 0

PubMed

Google Scholar