A genetically engineered mouse for imaging of apoptosis in a tissue specific manner

CENTER for MOLECULAR IMAGING

Stefanie Galbán1, Yong Hyun Jeon2, Lisa M. Sharkey2, Ben Hoff2, Craig J. Galbán2, Brian D. Ross2 and Alnawaz Rehemtulla1

Departments of Radiation Oncology1 and Radiology2 University of Michigan, Ann Arbor, MI

Abstract #: 2434

Conclusions

MethodsSubcloning of Caspase-3/7 GloSensor into pEFThe bioluminescent reporter was subcloned from Promega’s CP vector into pEF by PCR amli�cation and insertion into MCS at SalI and EcoRI restriction sites. Transfection and clone selection of Caspase -3/7 GloSensor expressing MDA-MB231/1833 cellsD54-MG or MDA-MB231/1833 cells were transfected with Caspase-3/7 GloSensor by Fugen reagent following standard protocol and placed in appropriate selection media 48 hrs post transfection. Single clones were selected and tested for reporter expression by western blotting with luciferase antibody (Promega) and for bioluminescence. Clones with similar bioluminescence activity and re-porter expression were selected for experiments.Bioluminescence assay in cell cultureGlioma or breast cancer cell lines stably expressing the reporter or di�erent reporter versions were seeded in a 96-well assay plate 24 hrs prior to treatment. Cells were treated with 200 ng/ml TRAIL and imaged at indicated timepoints. Live-cell luminescent imaging was performed by adding 100 ug/ml of D-luciferin to the assay media. Photon counts were aquired at di�erent timepoints prior-and post-reatment using the Envison luminometer (Perkin Elmer).High throughput screening1833 reporter cells were seeded in a 96-well plates at a density of 10.000 cells per well by using automated pippetors. 48 hrs post seed-ing media was changed to CO2 independent media containing 1% GloSensor cAMP reagent ( ref.1&2)(Promega) and incubated with a �nal compound concentration of 10 uM. Addition of media and compound library was performed using a Titertek Mulidrop Micro-plate Dispensor (Thermo Fisher Scienti�c, Watham, MA). 1280 Lopac compound library (Sigma) were used at above indicated concen-trations. Relative Luminescence was calculated by normalizing values of compound treated wells to untreated wells. The Z-factor was calculated as previously described: Zhang et al, Biomol Screen 1999; 4:67-73.Caspase-3/7 GloSensor transgenic mouse generationFor the purpose of imaging apoptosis non-invasively in animal models of cancer, we generated a transgenic reporter mouse by pro-nuclear microinjection of a transgene containing the apoptosis reporter (GloSensor Caspase -3/7) into fertilized eggs obtained from FVB/N females. Transgenic mice were generated by the t ransgenic animal model core at the University of Michigan. The schematic in �gure 4 and 5 depicts the transgene. In brief, constitutively active CAG promoter drives expression of EGFP in the absence of Cre ex-pression. A polyA sequence with strong termination signal ensures transcriptional inhibition of the bioluminescent reporter (Caspase-3/7 Glosensor) located 3’ of the EGFP cDNA. loxP sites (triangles) are �anking the EGFP cDNA. Cre-mediated excision of the �oxed EGFP-stop cassette results in transcription of the bioluminescent reporter (GloSensor caspase-3/7). Establishment of xenograft and GEM models and in vivo bioluminescence assayFor breast cancer bone metastasis model 100.000 MDA-MB231/1833 cells stably expressing the Caspase-3/7 GloSensor reporter were implanted into the tibia of the mouse. Tumor growth was followed by MRI and treatment initiated when tumor reached 5-15 mm^3. For in vivo bioluminescence, mice were anasthetized using a 2% iso�uorane/air mixture and injected with a single dose of 150 mg/kg D-luciferin itraperitoneally. Consecutive images were acquired before and 6 hrs post treatment or as indicated in �gures by using the IVIS imaging system (Caliper Life Sciences, Hopkinton, MA). Fold induction of bioluminescence activation was calculated by normaliz-ing post treatment values to pre treatment values of each individual animal.References(1) Fan, F. et al. (2008) Novel genetically encoded biosensors using �re�y luciferase. ACS Chem. Biol. 3(6), 346-51. (2) Binkowski, B.F., Fan, F. and Wood, K.V. (2009) Engineered luciferases for molecular sensing in living cells. Curr. Opin. Biotech. 20, 14-8.(3) Coppola, J. et al. (2008) Noninvasive imaging of apoptosis and its application in cancer therapeutics. Clin Cancer Res.Apr 15;14(8):2492-501.

Here we describe a new cell death surrogate marker for use in High throughput screening of therapeutics and for in vivo imaging. This reporter, which is based on the split luciferase technology, was evaluated in a breast cancer and glioma cell line and showed a high signal to noise ratio and a wide dynamic range. Robust fold inductions of over 50 fold in vitro to stimuli engaging the receptor mediated or mitochondrial apoptotic pathway were equally translatable to in vivo use. In vivo utility was demonstrated by the use of cancer cell xenografts and the development of a new transgenic mouse model, wherein activation of the Caspase-3/7 GloSensor was achieved by Cre recombination in a tissue dependent manner. The reporter was further found to be highly applicable in HTS and for imaging of cancer cell subpopulations. In summary, we predict that this Caspase biosensor will prove its usefullness in discovering novel drugs and signaling molecules which impinge on Caspase activation in cells and living animals.

Utility of GloSensor caspase-3/7 in HTS

Figure 2: High-throughput screen of chemical library. A, Stable clones of the breast cancer cell line MDA-MB231 subclone 1833 expressing GloSensor 3/7 were treated with TRAIL and imaged every hour for 10 consecutive min. Fold induction was calculated by normalizing data to pre treatment values per single well. B, Z factors were calculated for every timepoint and an average Z factor of 0.82 su�ced assay suitablitiy for HTS. C, and D, Stable clones of the breast cancer cell line MDA-MB231 subclone 1833 (C) or glioma cell line D54 (D) expressing GloSensor caspase-3/7 were used to screen a library of 1,280 pharmacologically active compounds (LOPAC). 10.000 cells/well were treated with vehicle control ( DMSO) or 10 uM of each compound in a 96-well format and imaged every hour for a 24 hr time period. Fold induction of bioluminescence signal intensity over values obtained from vehicle treated cells was plotted at maximal induction (mean ± SEM).

AbstractThe importance of the apoptotic machinery is exemplified in a number of disease processes. For example, inhibi-tion of apoptosis can result in a number of cancers, autoimmune diseases, inflammatory diseases, and viral infec-tions. Cancer is characterized by dysregulated cell proliferation and altered cell death, which constitutes a common basis for neoplastic evolution. The most implicit and clinically attractive anticancer strategies, therefore, consist of eliminating tumor cells by preventing their expansion and ultimately inducing apoptotic cell death. We have developed a transgenic mouse model wherein Caspase 3 mediated cell death can be imaged in real time and non-invasively utilizing a luciferase based biosensor. Upon Cre-mediated recombination, transgene ex-pression containing the Caspase 3 biosensor is achieved in a cell or tissue specific manner. Here we show that ex-pression of the Caspase 3 biosensor can be achieved in several tissues. In addition, we demonstrate in mouse models of cancer, that the ability to dynamically and sensitively image the activation of the Caspase 3 provides an opportunity to understand the dynamics of cell death in response to specific drugs or combination therapies. We also provide results demonstrating the utility of the technology in evaluating the efficacy of cancer thera-peutics in cancer stem cells, a population that is rare and transient.:

Imaging of cell death in breast bone metastasis

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Figure 3: Breast bone metastases expressing GloSensor caspase-3/7. A, and B, Breast cancer cells 1833 (subclone of MDA-MB 231) stably expressing the Caspase-3/7 GloSensor were implanted intra tibial (I.T.) into nude mice. Tumor volume was measured by MRI and treatment was initiated once tumors reached a volume of 10 mm3. Tumor speci�c bioluminescence activity was measured before treatment and at various time points following a onetime dose of TRAIL (8 mg/kg). Fold induction of bioluminescence signal intensity over pretreatment values was plotted as mean ± SEM for each group. A, Representative bioluminescence images of animals with intra tibial xenografts are shown before treatment or at various time points post TRAIL administration.

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Figure 1: Noninvasive imaging of caspase-3 utilizing a split-luciferase reporter strategy. A, Schematic representation of the GloSensor caspase-3/7 reporter (Promega). The apoptosis imaging reporter constitutes the split luciferase (C-Luc and N-Luc) domains fused with an intervening caspase-3 cleavage motif (DEVD). B, Upon induction of apoptosis, the reporter molecule is proteolytically cleaved by caspase-3 at the DEVD motif. This cleavage enables interaction between C-Luc and N-Luc, thus reconstituting luciferase activity.

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D54 cells

Figure 4: Caspase-3/7 transgene for in vivo use. A, Schematic of the CLE GloSensor Caspase-3/7 transgene. Constitutively active CAG promoter drives expression of EGFP in the absence of Cre expression. A polyA sequence with strong termination signal ensures transcriptional inhibition of the bioluminescent reporter (Caspase-3/7 Glosensor) located 3’ of the EGFP cDNA. loxP sites (triangles) are �anking the EGFP cDNA. B, CLE Caspase-3/7 GloSensor mouse was crossed with a CMV-Cre deleter mouse strain to initiate recombination within multiple organs in bitransgenic mice. Cre-mediated excision of the �oxed EGFP-stop cassette results in transcription of the bioluminescent reporter (GloSensor caspase-3/7). C, Representative bioluminescence images of bitransgenic (pCLE358(V2) tg/+; CMV-Cre tg/+; left and right) or monotransgic (pCLE358(V2) tg/+; CMV-Cre +/+; middle) animals are shown.

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Figure 5: Cre dependable activation of bioluminescent apoptosis reporter in pancreatic tissue.B, Schematic of the CLE apoptosis BLI reporter transgene. The CLE apoptosis reporter mouse was crossed with a p48-Cre deleter mouse resulting in transcription of the bioluminescent reporter in the pancreas. B, Representative bioluminescence images of bi-transgenic (pCLE apoptosis reporter tg/+; p48-Cre ki/+) or monotransgenic (pCLE apoptosis reporter tg/+; p48-Cre +/+) animals are shown pre and post cerulein injection (50 mg/kg, multiple injections). C, Quanti�cation of BLI signal induction upon cerulein induced apoptosis. D, Representative bioluminescent and �uorescent (GFP) ex vivo images of pancreata from mono or bitransgenic animals.

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GloSensor caspase-3/7 schematicFigure 6: Application of caspase-3/7 GloSensor in assessing drug sensitivity of rare subpopulationsA, FACS pro�le of dissociated D54-cells stably expressing the Caspase -3/7 GloSensor. 200.000 cells were stained for CD133 using anti-CD133 and CY5 �uoresent labeled secondary antibody, and sorted into CD133+ and CD133− populations by �ow cytometry. P3 repre-sent CD133 + cell population. B, through D, 10.000 c/ well of CD133+ and CD133− sorted D54 cells both expressing the Caspase-3/7 Glo-Sensor were incubated with 200 ng/ml TRAIL (B), 50 uM MNS (C), 50 uM MK886 (D) or 12.5 um GW7647 (E) and bioluminescence was mea-sured at indicated times and plotted as fold induction over values obtained from vehicle treated cells. Experiments were performed in triplicates and plotted as mean ± SEM.

GloSensor caspase-3/7 use in cancer stem cells

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