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Rapid target gene validation in complex cancer mouse models using re-derived embryonic stem cells.

Huijbers IJ, Bin Ali R, Pritchard C, Cozijnsen M, Kwon MC, Proost N, Song JY, de Vries H, Badhai J, Sutherland K, Krimpenfort P, Michalak EM, Jonkers J, Berns A - EMBO Mol Med (2014)

Bottom Line: In our newly developed approach for the fast generation of tumor cohorts we have overcome this obstacle, as exemplified for three GEMMs; two lung cancer models and one mesothelioma model.Three elements are central for this system; (i) The efficient derivation of authentic Embryonic Stem Cells (ESCs) from established GEMMs, (ii) the routine introduction of transgenes of choice in these GEMM-ESCs by Flp recombinase-mediated integration and (iii) the direct use of the chimeric animals in tumor cohorts.As proof-of-principle, we demonstrate that MycL1 is a key driver gene in Small Cell Lung Cancer.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

ABSTRACT
Human cancers modeled in Genetically Engineered Mouse Models (GEMMs) can provide important mechanistic insights into the molecular basis of tumor development and enable testing of new intervention strategies. The inherent complexity of these models, with often multiple modified tumor suppressor genes and oncogenes, has hampered their use as preclinical models for validating cancer genes and drug targets. In our newly developed approach for the fast generation of tumor cohorts we have overcome this obstacle, as exemplified for three GEMMs; two lung cancer models and one mesothelioma model. Three elements are central for this system; (i) The efficient derivation of authentic Embryonic Stem Cells (ESCs) from established GEMMs, (ii) the routine introduction of transgenes of choice in these GEMM-ESCs by Flp recombinase-mediated integration and (iii) the direct use of the chimeric animals in tumor cohorts. By applying stringent quality controls, the GEMM-ESC approach proofs to be a reliable and effective method to speed up cancer gene assessment and target validation. As proof-of-principle, we demonstrate that MycL1 is a key driver gene in Small Cell Lung Cancer.

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Luciferase imaging of SCLC in chimeras.In vivo imaging of a invCAG-Luc;Rb1F/F;Trp53F/F chimeric mouse injected intrathoracically with Ad5-Cre. Tumor growth was monitored weekly by bioluminescence imaging.Luciferase activity emitted from the thorax of 10 chimeric invCAG-Luc;Rb1F/F;Trp53F/F mice. Each line represents measurements of an individual mouse. The chimeric mouse with the lowest coat-color chimerism (○, 20%) did not develop a tumor, while the second lowest chimera (□, 35%) did develop SCLC though with a long latency. One chimera (♦, 962975) failed to show any Luciferase activity but did develop SCLC. Analysis of the tumor revealed a lack of Cre-mediated switching of the invCag-Luc transgene (supplementary Fig S7).Survival curves of chimeric Rb1F/F;Trp53F/F mice containing either the invCag-Luc (black line) or the invCag-MycL1-Luc (red line) transgene, intratracheally injected with Ad5-Cre. Median survival indicated by the dotted line was 250 and 167 days, respectively.Survival curves of F1 Rb1F/F;Trp53F/F mice containing either the invCag-Luc (black line) or the invCag-MycL1-Luc (red line) transgene, intratracheally injected with Ad5-Cre. Median survival indicated by the dotted line was 235 and 140 days, respectively.Luciferase activity emitted from the thorax of 11 F1 invCAG-MycL1-Luc;Rb1F/F;Trp53F/F mice. Each line represents measurements of an individual mouse.MycL1 copy number in SCLC tumors from three different genotypes determined by real-time PCR and aCGH. Each circle represents a primary SCLC tumor. All tumors with more than four copies (dotted line) were considered positive for MycL1 amplification. Note that overexpression of MycL1 by the transgene significantly reduces the frequency of genomic MycL1 amplifications in tumors as compared to the Rb1F/F;Trp53F/F control ( P = 0.002 Fisher's Exact Test) and the invCAG-Luc;Rb1F/F;Trp53F/F control ( P = 0.035 Fischer's Exact Test).
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fig04: Luciferase imaging of SCLC in chimeras.In vivo imaging of a invCAG-Luc;Rb1F/F;Trp53F/F chimeric mouse injected intrathoracically with Ad5-Cre. Tumor growth was monitored weekly by bioluminescence imaging.Luciferase activity emitted from the thorax of 10 chimeric invCAG-Luc;Rb1F/F;Trp53F/F mice. Each line represents measurements of an individual mouse. The chimeric mouse with the lowest coat-color chimerism (○, 20%) did not develop a tumor, while the second lowest chimera (□, 35%) did develop SCLC though with a long latency. One chimera (♦, 962975) failed to show any Luciferase activity but did develop SCLC. Analysis of the tumor revealed a lack of Cre-mediated switching of the invCag-Luc transgene (supplementary Fig S7).Survival curves of chimeric Rb1F/F;Trp53F/F mice containing either the invCag-Luc (black line) or the invCag-MycL1-Luc (red line) transgene, intratracheally injected with Ad5-Cre. Median survival indicated by the dotted line was 250 and 167 days, respectively.Survival curves of F1 Rb1F/F;Trp53F/F mice containing either the invCag-Luc (black line) or the invCag-MycL1-Luc (red line) transgene, intratracheally injected with Ad5-Cre. Median survival indicated by the dotted line was 235 and 140 days, respectively.Luciferase activity emitted from the thorax of 11 F1 invCAG-MycL1-Luc;Rb1F/F;Trp53F/F mice. Each line represents measurements of an individual mouse.MycL1 copy number in SCLC tumors from three different genotypes determined by real-time PCR and aCGH. Each circle represents a primary SCLC tumor. All tumors with more than four copies (dotted line) were considered positive for MycL1 amplification. Note that overexpression of MycL1 by the transgene significantly reduces the frequency of genomic MycL1 amplifications in tumors as compared to the Rb1F/F;Trp53F/F control ( P = 0.002 Fisher's Exact Test) and the invCAG-Luc;Rb1F/F;Trp53F/F control ( P = 0.035 Fischer's Exact Test).

Mentions: The chimeric animals with the new Luciferase reporter constructs were treated with Ad5-Cre to induce tumor formation. The majority of the invCAG-Luc;Rb1F/F;Trp53F/F chimeras developed SCLC with similar latency as presented earlier (Figs 4A–C and 2E). One mouse with the lowest coat-color chimerism failed to develop a tumor after 375 days, possibly reflecting insufficient contribution of GEMM-ESCs to lung epithelium for reliable use in experimental cohorts (Fig 4B). Bioluminescence imaging of luciferase activity revealed tumor initiation at variable time points, ranging between 140 and 320 days, after which the majority of tumors displayed exponential growth until animals had to be sacrificed because of respiratory distress. In the mesothelioma model the results were less pronounced. Here, all but one chimera developed mesothelioma with thoracic Luciferase expression; however, the increase in Luciferase expression over time was limited and in some cases leveled off after an initial increase (supplementary Fig S6). This occurred for both reporter constructs, but was most often observed for the reporter construct carrying the EF1a promoter. The underlying cause for this behavior remains speculative and could have multiple reasons. It might be due to quenching of the luminescence signal by pleural effusion, i.e. accumulated liquid in the pleural cavity. Also, the immunogenicity of the Luciferase protein might trigger an immune response against Luciferase-expressing tumor cells, leading to selective outgrowth of tumor cells with low or no luciferase expression (Jeon et al, 2007). Thirdly, the CAG and EF1a promoters might be silenced by methylation. We have indications that at least the latter event occurs, as treatment of cultured primary mesothelioma cells derived from a chimeric animal with the demethylating agent, 5-aza-2dC, resulted in a marked increase in Luciferase expression (supplementary Fig S6E). Promoter silencing is likely due to the presence of bacterial DNA of the plasmid integrated in the Col1A1 locus (Tasic et al, 2011). Still, the promoter silencing appears to be model or cell type dependent, as tumors in the SCLC model showed robust Luciferase expression judged by the exponential increase in luminescence signals measured in the majority of the invCAG-Luc;Rb1F/F;Trp53F/F chimeras (Fig 4B). In the few cases where no Luciferase expression was observed in SCLC tumors, the invCag-Luc transgene had failed to recombine after Cre expression (supplementary Fig S7).


Rapid target gene validation in complex cancer mouse models using re-derived embryonic stem cells.

Huijbers IJ, Bin Ali R, Pritchard C, Cozijnsen M, Kwon MC, Proost N, Song JY, de Vries H, Badhai J, Sutherland K, Krimpenfort P, Michalak EM, Jonkers J, Berns A - EMBO Mol Med (2014)

Luciferase imaging of SCLC in chimeras.In vivo imaging of a invCAG-Luc;Rb1F/F;Trp53F/F chimeric mouse injected intrathoracically with Ad5-Cre. Tumor growth was monitored weekly by bioluminescence imaging.Luciferase activity emitted from the thorax of 10 chimeric invCAG-Luc;Rb1F/F;Trp53F/F mice. Each line represents measurements of an individual mouse. The chimeric mouse with the lowest coat-color chimerism (○, 20%) did not develop a tumor, while the second lowest chimera (□, 35%) did develop SCLC though with a long latency. One chimera (♦, 962975) failed to show any Luciferase activity but did develop SCLC. Analysis of the tumor revealed a lack of Cre-mediated switching of the invCag-Luc transgene (supplementary Fig S7).Survival curves of chimeric Rb1F/F;Trp53F/F mice containing either the invCag-Luc (black line) or the invCag-MycL1-Luc (red line) transgene, intratracheally injected with Ad5-Cre. Median survival indicated by the dotted line was 250 and 167 days, respectively.Survival curves of F1 Rb1F/F;Trp53F/F mice containing either the invCag-Luc (black line) or the invCag-MycL1-Luc (red line) transgene, intratracheally injected with Ad5-Cre. Median survival indicated by the dotted line was 235 and 140 days, respectively.Luciferase activity emitted from the thorax of 11 F1 invCAG-MycL1-Luc;Rb1F/F;Trp53F/F mice. Each line represents measurements of an individual mouse.MycL1 copy number in SCLC tumors from three different genotypes determined by real-time PCR and aCGH. Each circle represents a primary SCLC tumor. All tumors with more than four copies (dotted line) were considered positive for MycL1 amplification. Note that overexpression of MycL1 by the transgene significantly reduces the frequency of genomic MycL1 amplifications in tumors as compared to the Rb1F/F;Trp53F/F control ( P = 0.002 Fisher's Exact Test) and the invCAG-Luc;Rb1F/F;Trp53F/F control ( P = 0.035 Fischer's Exact Test).
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fig04: Luciferase imaging of SCLC in chimeras.In vivo imaging of a invCAG-Luc;Rb1F/F;Trp53F/F chimeric mouse injected intrathoracically with Ad5-Cre. Tumor growth was monitored weekly by bioluminescence imaging.Luciferase activity emitted from the thorax of 10 chimeric invCAG-Luc;Rb1F/F;Trp53F/F mice. Each line represents measurements of an individual mouse. The chimeric mouse with the lowest coat-color chimerism (○, 20%) did not develop a tumor, while the second lowest chimera (□, 35%) did develop SCLC though with a long latency. One chimera (♦, 962975) failed to show any Luciferase activity but did develop SCLC. Analysis of the tumor revealed a lack of Cre-mediated switching of the invCag-Luc transgene (supplementary Fig S7).Survival curves of chimeric Rb1F/F;Trp53F/F mice containing either the invCag-Luc (black line) or the invCag-MycL1-Luc (red line) transgene, intratracheally injected with Ad5-Cre. Median survival indicated by the dotted line was 250 and 167 days, respectively.Survival curves of F1 Rb1F/F;Trp53F/F mice containing either the invCag-Luc (black line) or the invCag-MycL1-Luc (red line) transgene, intratracheally injected with Ad5-Cre. Median survival indicated by the dotted line was 235 and 140 days, respectively.Luciferase activity emitted from the thorax of 11 F1 invCAG-MycL1-Luc;Rb1F/F;Trp53F/F mice. Each line represents measurements of an individual mouse.MycL1 copy number in SCLC tumors from three different genotypes determined by real-time PCR and aCGH. Each circle represents a primary SCLC tumor. All tumors with more than four copies (dotted line) were considered positive for MycL1 amplification. Note that overexpression of MycL1 by the transgene significantly reduces the frequency of genomic MycL1 amplifications in tumors as compared to the Rb1F/F;Trp53F/F control ( P = 0.002 Fisher's Exact Test) and the invCAG-Luc;Rb1F/F;Trp53F/F control ( P = 0.035 Fischer's Exact Test).
Mentions: The chimeric animals with the new Luciferase reporter constructs were treated with Ad5-Cre to induce tumor formation. The majority of the invCAG-Luc;Rb1F/F;Trp53F/F chimeras developed SCLC with similar latency as presented earlier (Figs 4A–C and 2E). One mouse with the lowest coat-color chimerism failed to develop a tumor after 375 days, possibly reflecting insufficient contribution of GEMM-ESCs to lung epithelium for reliable use in experimental cohorts (Fig 4B). Bioluminescence imaging of luciferase activity revealed tumor initiation at variable time points, ranging between 140 and 320 days, after which the majority of tumors displayed exponential growth until animals had to be sacrificed because of respiratory distress. In the mesothelioma model the results were less pronounced. Here, all but one chimera developed mesothelioma with thoracic Luciferase expression; however, the increase in Luciferase expression over time was limited and in some cases leveled off after an initial increase (supplementary Fig S6). This occurred for both reporter constructs, but was most often observed for the reporter construct carrying the EF1a promoter. The underlying cause for this behavior remains speculative and could have multiple reasons. It might be due to quenching of the luminescence signal by pleural effusion, i.e. accumulated liquid in the pleural cavity. Also, the immunogenicity of the Luciferase protein might trigger an immune response against Luciferase-expressing tumor cells, leading to selective outgrowth of tumor cells with low or no luciferase expression (Jeon et al, 2007). Thirdly, the CAG and EF1a promoters might be silenced by methylation. We have indications that at least the latter event occurs, as treatment of cultured primary mesothelioma cells derived from a chimeric animal with the demethylating agent, 5-aza-2dC, resulted in a marked increase in Luciferase expression (supplementary Fig S6E). Promoter silencing is likely due to the presence of bacterial DNA of the plasmid integrated in the Col1A1 locus (Tasic et al, 2011). Still, the promoter silencing appears to be model or cell type dependent, as tumors in the SCLC model showed robust Luciferase expression judged by the exponential increase in luminescence signals measured in the majority of the invCAG-Luc;Rb1F/F;Trp53F/F chimeras (Fig 4B). In the few cases where no Luciferase expression was observed in SCLC tumors, the invCag-Luc transgene had failed to recombine after Cre expression (supplementary Fig S7).

Bottom Line: In our newly developed approach for the fast generation of tumor cohorts we have overcome this obstacle, as exemplified for three GEMMs; two lung cancer models and one mesothelioma model.Three elements are central for this system; (i) The efficient derivation of authentic Embryonic Stem Cells (ESCs) from established GEMMs, (ii) the routine introduction of transgenes of choice in these GEMM-ESCs by Flp recombinase-mediated integration and (iii) the direct use of the chimeric animals in tumor cohorts.As proof-of-principle, we demonstrate that MycL1 is a key driver gene in Small Cell Lung Cancer.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

ABSTRACT
Human cancers modeled in Genetically Engineered Mouse Models (GEMMs) can provide important mechanistic insights into the molecular basis of tumor development and enable testing of new intervention strategies. The inherent complexity of these models, with often multiple modified tumor suppressor genes and oncogenes, has hampered their use as preclinical models for validating cancer genes and drug targets. In our newly developed approach for the fast generation of tumor cohorts we have overcome this obstacle, as exemplified for three GEMMs; two lung cancer models and one mesothelioma model. Three elements are central for this system; (i) The efficient derivation of authentic Embryonic Stem Cells (ESCs) from established GEMMs, (ii) the routine introduction of transgenes of choice in these GEMM-ESCs by Flp recombinase-mediated integration and (iii) the direct use of the chimeric animals in tumor cohorts. By applying stringent quality controls, the GEMM-ESC approach proofs to be a reliable and effective method to speed up cancer gene assessment and target validation. As proof-of-principle, we demonstrate that MycL1 is a key driver gene in Small Cell Lung Cancer.

Show MeSH
Related in: MedlinePlus