All animal studies were approved by the University of North Carolina Institutional Animal Care and Use Committee.
1. Culturing Cortical Astrocytes from Neonatal Mice
2. Culturing Neural Stem Cells from Neonatal Mice
3. Orthotopic Injection of Recombined Cells into the Brain of Recipient Iimmunocompetent Mice
We developed an nGEM model system with cortical astrocytes and NSC harvested from neonatal GEM harboring floxed conditional oncogenic alleles that can be phenotypically characterized in vitro and in vivo (Figure 1). In order to investigate the consequences of oncogenic mutations specifically in cortical astrocytes in vitro, it is critical to first enrich for astrocytes. Cortical astrocyte harvests contain a mixture of microglia, astrocytes, oligodendrocytes, OPC, and neurons, but mechanical and genetic methods can aid in astrocyte enrichment. Whereas neurons die under normal culture conditions, microglia and oligodendrocytes can be removed by shaking overnight41,43. Additionally, genetic recombination of floxed, oncogenic alleles targeted to GFAP+ astrocytes using an Ad5GFAPCre can enrich for astrocytes. We used this method to infect cortical harvests from Rb1loxP/loxP GEM pups with floxed Nf1loxP/loxP and/or PtenloxP/loxP alleles. Ad5GFAPCre infection caused a proliferative advantage in the recombined GFAP+ astrocytes, which increased astrocyte purity from 59% to > 90% after 5 – 9 passages in culture (Figures 2A and 2B). Alternatively, we enriched for astrocytes by expressing T121 under control of the GFAP promoter to induce a proliferative advantage in astrocytes harvested from TgGZT121 mice (Figure 2C). While cortical astrocytes grow adherent to the culture dish (Figure 2C), NSC grow as non-adherent neurospheres in vitro (Figure 3A). Both WT NSC and NSC with recombined, floxed oncogenic alleles – TgGZT121 (T), KrasG12D (R), and homozygous Pten deletion (P-/-), referred to as TRP-/- – express the NSC marker Sox2, while only NSC harvested from GEM containing TgGZT121 express T121 after Cre-mediated recombination (Figure 3B).
To determine how G1/S (Rb), MAPK and/or PI3K pathway alterations affect growth of GFAP+ astrocytes in vitro, proliferation was examined using two methods. MTS and cell counting showed that expression of T121 and KrasG12D increased the proliferation of cortical astrocytes (Figures 4A and 4B). Similarly, homozygous Rb1 (Rb) and Nf1 (N), deletion combined with heterozygous Pten (P+/-) deletion also increased cortical astrocyte proliferation (Figure 4C). Transformed cells have unlimited proliferative capacity. The transforming effects of mutations can be measured in vitro using colony formation assays. We therefore tested the transforming effects of T121 and KrasG12D expression and homozygous Pten deletion in TRP-/- cortical astrocytes by colony formation assay as previously described44. Whereas WT astrocytes did not form colonies, T astrocytes formed colonies at 1.03% efficiency. Notably, TRP-/- astrocytes had the highest colony formation efficiency at 6.40% (Table 1). In order to be suitable for preclinical drug testing, it is important that in vitro tumor models be easily manipulated genetically. Additional genetic alterations can be stably introduced into cortical astrocytes by plasmid or viral vectors. As an example, we stably transduced the luciferase gene into TRP-/- astrocytes (TRP-/- luc) using a VSV-pseudotyped pMSCV retroviral vector. Expression of luciferase increased luminescence ~1,000 fold compared to parental cells (Figure 4D). Additionally, GEM-derived cortical astrocytes can be used to determine the efficacy of targeted inhibitors in vitro. We have shown previously that treatment with low dose PI-103, a dual mTOR/PI3K inhibitor, inhibited PI3K signaling without affecting cell viability30. However, the cytostatic/cytotoxic effects of higher PI-103 doses were not determined. Thus, we tested whether PI-103 can reduce TRP-/- astrocyte growth in vitro. PI-103 caused a maximal 88% reduction in TRP-/- astrocyte growth (Figure 4E). We have previously utilized orthotopic allografts of TRP-/- astrocytes to test other clinically relevant therapeutics in vivo (Schmid et al., manuscript submitted)45,46. These data suggest that transformed cortical astrocytes harvested from conditional GEM may provide a flexible model system in which to perform preclinical drug testing in immune competent mice.
Xenografts of established human cell lines and PDX require immunodeficient hosts. In contrast to PDX, many established human GBM cell lines do not recapitulate the histopathological features of GBM. For example, U87MG orthotopic xenografts formed circumscribed tumors that did not invade normal brain (Figure 5A). In contrast, injection of TRP-/- astrocytes into the brains of immune competent, syngeneic mice yielded invasive tumors that recapitulated the histological features of their human counterparts, particularly invasion of normal brain (Figure 5B). To longitudinally quantify TRP-/- allograft growth, mice were sacrificed every 5 days after cell injection and tumor burden was determined by quantifying tumor area on H&E-stained brain sections. Tumor area increased exponentially over time (Figure 5C). Orthotopic injection of 105 TRP-/- astrocytes into recipient brains led to neurological morbidity, with a median survival of 22 days (Figure 5D). In addition to cortical astrocytes, we performed orthotopic injections using TRP-/- NSC. TRP-/- NSC-derived tumors were T121 positive, proliferative, and maintained expression of the NSC marker Sox2 (Figure 6). Of note, injection of cortical astrocytes and NSC harvested from WT C57Bl/6 mice as well as phenotypically WT cortical astrocytes or NSC with unrecombined, floxed oncogenic alleles from conditional GEM failed to elicit tumorigenesis during this time frame (data not shown). Longitudinal imaging in vivo has been used to monitor tumor growth kinetics in response to drug treatments40. Thus, TRP-/- luc astrocytes were injected into the brains of immune competent syngeneic littermates and tumor growth was determined by serial bioluminescence imaging. Bioluminescence increased 15 fold over 16 days (Figures 7A and 7B). We have demonstrated that cortical astrocytes and NSC harvested from conditional GEM can be genetically modified and phenotypically characterized in vitro and in vivo for definition of the genetics and cell biology of astrocytoma pathogenesis and potentially used for preclinical drug development.
Figure 1. Schematic of cortical astrocyte and NSC harvest from nGEM. Phenotypically WT cortical astrocytes and NSC were harvested from GEM with conditional oncogenic alleles. Genetic recombination was induced in vitro with an AdCre vector. Transformed cells were phenotypically characterized in vitro by various methods and in vivo by orthotopic injection into the brains of immune-competent, syngeneic littermates.
Figure 2. Enrichment of GFAP+ astrocytes upon serial passaging in vitro. Representative immunofluorescence images showing GFAP+ astrocytes harvested from Rb1loxP/loxP GEM pups with Nf1loxP/loxP and/or PtenloxP/loxP in which genetic recombination was induced with Ad5GFAPCre and the cells passaged X times (PX) (A). Quantification of enrichment of GFAP+ astrocytes in panel A. Bars represent standard error from 3-24 replicates (B). Representative immunofluorescence (top) and phase contrast (bottom) images of adherent cortical astrocytes harvested from TgGZT121 GEM pups that express T121 from the GFAP promoter at passage 9 after recombination with Ad5CMVCre in vitro (C). Astrocytes were quantified as the number of GFAP+ (green) cells divided by the total number of DAPI-stained nuclei (blue) at every other passage. Images were taken at 10X original magnification (A,C). Scale bars represent 100 µm (A,C). Please click here to view a larger version of this figure.
Figure 3. WT and recombined NSC harvested from TRP-/- GEM. Representative phase contrast images of phenotypically WT (top) and TRP-/- (bottom) NSC grown as neurospheres in vitro (A). Representative immunofluorescence staining for T121 (green) and Sox2 (red) expression in WT (top) and TRP-/- (bottom) neurospheres (B). Scale bars represent 100 µm (A,B). Please click here to view a larger version of this figure.
Figure 4. Characterization of cortical astrocytes in vitro. Growth of WT and TR cortical astrocytes was determined by counting cells on days 1-7 (A). Relative optical density (O.D.) of WT and TR cortical astrocytes was determined by MTS (B). Relative growth of WT and recombined Rb1-/-;Nf1-/-;Pten+/- (RbNP+/-) astrocytes was determined by MTS (C). Luminescence of parental TRP-/- and luciferase expressing TRP-/- astrocytes (TRP-/- luc) (D). Relative O.D. of TRP-/- astrocytes treated with PI103 for 5 days as determined by MTS (E). Proliferation and dose response was calculated using the exponential growth equation in GraphPad Prism 5. Bars represent standard error from 6 replicates per condition. Please click here to view a larger version of this figure.
Figure 5. In vivo gliomagenesis. Representative H&E stained section of a U87MG xenograft shows discrete tumor margins (A). Representative H&E stained section of a TRP-/- allograft shows diffuse invasion of normal brain (B). Scale bars in left and right H&E images represent 1 mm and 100 µm, respectively. Tumor area was determined by analyzing H&E stained sections of formalin fixed, paraffin embedded brains from immune-competent, syngeneic littermates injected with 105 TRP-/- astrocytes and sacrificed at 5 day intervals post-injection. (C). Kaplan–Meier survival analysis of TRP-/- allograft mice aged to morbidity shows a median survival of 22 days (D).
Figure 6. Immunofluorescence of TRP+/- NSC allografts. Representative immunofluorescence images show that TRP+/- NSC injected into the brains of immune-competent, syngeneic littermates express T121 (green) and Sox2 (white) and proliferate, as determined by EdU (red) incorporation. Mice were perfused with EdU and sacrificed 6 weeks post-injection and their brains perfused with paraformaldehyde. Scale bar represents 20 µm. Please click here to view a larger version of this figure.
Figure 7. Longitudinal imaging of TRP-/- luc allografts. Representative bioluminescence images (A) and quantification of luciferase flux over time (B) shows growth of TRP-/- allografts in immune-competent, syngeneic mice injected with 105 TRP-/- luc cortical astrocytes and imaged at the indicated days post-injection. Please click here to view a larger version of this figure.
Genotype | Plating efficiency (%) | SEM |
WT | 0 | 0 |
T | 1.03 | 0.15 |
TRP-/- | 6.40 | 0.83 |
Table 1. Colony formation of cortical astrocytes. WT, T and TRP-/- cortical astrocytes were plated in triplicate at 4,000, 2,000, and 250 cells/well respectively. Colonies were stained with crystal violet 14 days after plating, imaged, and counted using ImageJ. Plating efficiency was calculated as the number of colonies divided by the number of cells plated.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
Dulbecco's Modified Eagle Medium (DMEM) (1X) | Invitrogen | 11995065 | DMEM can also be purchased from other suppliers including GIBCOSigma and Cellgro |
Fetal Bovine Serum, Regular | Cellgro | 35-010-CV | |
Penicillin-Streptomycin | Invitrogen | 15140122 | |
Sharp-Pointed Dissecting Scissors | Fisher Scientific | 08-395 | |
Cartilage Thumb Forceps, Curved | Fisher Scientific | 1631 | |
Miltex 17-301 Style 1 Jeweler Style Forceps, Fine, 4 | Miltex | 17-301 | |
Ethanol (200 proof) | Decon Labs | 2710 | |
Razor Blades | VWR | 55411-050 | |
Hanks' Balanced Salt Solution (HBSS) (1X), liquid | Invitrogen | 14175-095 | |
TrypLE Express (1X), Phenol Red | Invitrogen | 12605-010 | Other Trypsin solutions are also suitable for cortical astrocyte havest and culture |
CULTURE DISH, 60 x 15 mm | Thomas Scientific | 9380H77 | |
15 ml tubes | BD Biosciences | 352096 | |
50 ml tubes | BD Biosciences | 352070 | |
Adenovirus stock | Gene Transfer Vector Core, U. Iowa | Ad5CMVCre | Store in 5 µl aliquots at -80 C. Hazardous. Use Bsl2 safetyy precautions |
Sodium sulfate (Na2SO4) | Sigma-Aldrich | 238597 | |
Potassium sulfate (K2SO4) | Sigma-Aldrich | 221325 | |
Magnesium chloride (MgCl2) | Sigma-Aldrich | M8266 | |
Calcium chloride (CaCl2) | Sigma-Aldrich | 746495 | |
HEPES potassium salt | Sigma-Aldrich | H0527 | |
D-(+)- Glucose | Sigma-Aldrich | G8270 | |
Phenol Red | Sigma-Aldrich | P3532 | |
Sodium hydroxide (NaOH) | Sigma-Aldrich | S5881 | |
Papain | Worthington | LS003127 | |
L-Cysteine-HCl | Sigma-Aldrich | C1276 | |
Syringe filter (0.22 µm pore size) | Millipore | SLGP033NS | |
Neurocult proliferation kit, mouse | Stemcell Technologies | 5702 | This kit contains the NeuroCult NSC Basal Medium and NeuroCult NSC supplement needed for NSC culture |
0.2% Heparin solution | Stemcell Technologies | 7980 | |
EGF | Invitrogen | PMG8041 | |
bFGF | Invitrogen | PHG0261 | |
Hanks' Balanced Salt Solution (HBSS) (10X) | Invitrogen | 14185-052 | |
Magnesium sulfate (MgSO4) | Sigma-Aldrich | M7506 | |
Sodium bicarbonate (NaHCO3) | Sigma-Aldrich | S5761 | |
E-64 | Sigma-Aldrich | E3132 | Make 10 mM stock in DMSO, store at -20 °C |
6-well plates | Fisher Scientific | 07-200-83 | |
Cell strainer (40 µm pore size) | Corning | 352340 | |
Stem cell dissociation solution | Stemcell Technologies | 5707 | Alternatively, use gentle enzyme solutions such as Accutase |
Methyl cellulose 15 cP | Sigma-Aldrich | M7027 | |
Dulbecco's Modified Eagle's Media 2X | Millipore | SLM-202-B | For making 5% methyl cellulose solution |
1.7mL Snap Cap Microcentrifuge Tube | Corning | 3620 | |
Hamilton syringe, 250 µl LT no needle | Fisher Scientific | 14-815-92 | |
PB600-1 Antigen Dispenser | Hamilton | 83700 | |
Disposable 18 ga needles | Fisher Scientific | NC9015638 | |
27 ga 1/2" luer tip needle | Fisher Scientific | 14-826-48 | |
2,2,2-Tribromoethanol (Avertin) | Sigma-Aldrich | T48402 | |
Betadine | Fisher Scientific | NC9386574 | |
Puralube Opthalmic Ointment | Fisher Scientific | NC9689910 | |
Model 900 Stereotaxic frame | Kopf Instruments | ||
VETBOND | Fisher Scientific | NC9259532 | Tissue adhesive |
Lidocaine | ShopMedVet | RXLIDO-EPI | |
CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) | Promega | G3580 | |
Anti-Sox2 | Millipore | AB5603 | |
Polyclonal Rabbit Anti-Glial Fibrillary Acidic Protein (GFAP) | Dako | Z0334 | |
IVIS Kinetic | PerkinElmer | For in vivo imaging | |
D-Luciferin – K+ Salt Bioluminescent Substrate | PerkinElmer | 122796 | For in vivo bioluminescence imaging |
EdU Imaging Kit | Invitrogen | C10340 | |
MSCV Luciferase PGK-hygro | Addgene | 18782 |
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.