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"Glowing head" mice: a genetic tool enabling reliable preclinical image-based evaluation of cancers in immunocompetent allografts.

Day CP, Carter J, Weaver Ohler Z, Bonomi C, El Meskini R, Martin P, Graff-Cherry C, Feigenbaum L, Tüting T, Van Dyke T, Hollingshead M, Merlino G - PLoS ONE (2014)

Bottom Line: Imageable reporters are essential for accurately tracking tumor growth and response, particularly for metastases.Here we assessed the value of reporter-tolerized GEMs as allograft recipients by targeting minimal expression of a luciferase-GFP fusion reporter to the anterior pituitary gland (dubbed the "Glowing Head" or GH mouse).Moreover, reporter expression can also alter the tumor response to chemotherapy or targeted therapy in a context-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, Maryland, United States of America.

ABSTRACT
Preclinical therapeutic assessment currently relies on the growth response of established human cell lines xenografted into immunocompromised mice, a strategy that is generally not predictive of clinical outcomes. Immunocompetent genetically engineered mouse (GEM)-derived tumor allograft models offer highly tractable preclinical alternatives and facilitate analysis of clinically promising immunomodulatory agents. Imageable reporters are essential for accurately tracking tumor growth and response, particularly for metastases. Unfortunately, reporters such as luciferase and GFP are foreign antigens in immunocompetent mice, potentially hindering tumor growth and confounding therapeutic responses. Here we assessed the value of reporter-tolerized GEMs as allograft recipients by targeting minimal expression of a luciferase-GFP fusion reporter to the anterior pituitary gland (dubbed the "Glowing Head" or GH mouse). The luciferase-GFP reporter expressed in tumor cells induced adverse immune responses in wildtype mouse, but not in GH mouse, as transplantation hosts. The antigenicity of optical reporters resulted in a decrease in both the growth and metastatic potential of the labeled tumor in wildtype mice as compared to the GH mice. Moreover, reporter expression can also alter the tumor response to chemotherapy or targeted therapy in a context-dependent manner. Thus the GH mice and experimental approaches vetted herein provide concept validation and a strategy for effective, reproducible preclinical evaluation of growth and response kinetics for traceable tumors.

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Generation of the rGH-ffLuc-eGFP (“Glowing Head”) genetically engineered mouse.A, Structure of the expression vector for generation of Glowing Head (GH) transgenic mice. Expression of a firefly luciferase-eGFP fusion gene (ffLuc-eGFP) was targeted to the mouse anterior pituitary gland by using the rat growth hormone promoter (rGH) and human growth hormone gene sequences, which include a polyadenylylation site (hGHpA)20. B, Optical expression pattern of transgene in GH mice as visualized by BL imaging. Reporter activity was detected in the anterior pituitary gland of both genders and the testes of male mice. C, Serum levels of growth hormone from age-matched GH mice and wildtype (WT) c-Brd mice was assessed by ELISA (mean ± SE). Blood was withdrawn at the same time of day. No significant differences in circulating growth hormone levels between the GH and WT mice were found. D, ffLuc-eGFP-labeled LLC tumors were subcutaneously transplanted into WT, GH, and NOD-SCID mice. Blood was withdrawn to prepare sera when tumors reached 500 mm3, and the serum levels of anti-GFP antibody were analyzed by ELISA. The levels of anti-GFP antibody in WT mice are significantly higher than those in GH and NOD-SCID mice (p<0.005), but no difference was found between those in GH and NOD-SCID mice (p = 0.19). The sera from healthy mice without tumor transplantation served as controls to define zero point.
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pone-0109956-g002: Generation of the rGH-ffLuc-eGFP (“Glowing Head”) genetically engineered mouse.A, Structure of the expression vector for generation of Glowing Head (GH) transgenic mice. Expression of a firefly luciferase-eGFP fusion gene (ffLuc-eGFP) was targeted to the mouse anterior pituitary gland by using the rat growth hormone promoter (rGH) and human growth hormone gene sequences, which include a polyadenylylation site (hGHpA)20. B, Optical expression pattern of transgene in GH mice as visualized by BL imaging. Reporter activity was detected in the anterior pituitary gland of both genders and the testes of male mice. C, Serum levels of growth hormone from age-matched GH mice and wildtype (WT) c-Brd mice was assessed by ELISA (mean ± SE). Blood was withdrawn at the same time of day. No significant differences in circulating growth hormone levels between the GH and WT mice were found. D, ffLuc-eGFP-labeled LLC tumors were subcutaneously transplanted into WT, GH, and NOD-SCID mice. Blood was withdrawn to prepare sera when tumors reached 500 mm3, and the serum levels of anti-GFP antibody were analyzed by ELISA. The levels of anti-GFP antibody in WT mice are significantly higher than those in GH and NOD-SCID mice (p<0.005), but no difference was found between those in GH and NOD-SCID mice (p = 0.19). The sera from healthy mice without tumor transplantation served as controls to define zero point.

Mentions: Our results suggested that immunogenicity of xenobiotic reporter gene products is largely responsible for their inconsistency in the context of a fully functional immune system. To circumvent this issue, we generated C57BL/6- and FVB/N-based GEM models recognizing ffLuc and eGFP proteins as self. For its high specificity, rGH gene sequences [17] (Fig. 2A) were employed to target expression of an ffLuc-eGFP fusion gene to the anterior pituitary gland of the mouse, thereby avoiding interfering signaling from the most common metastatic sites.


"Glowing head" mice: a genetic tool enabling reliable preclinical image-based evaluation of cancers in immunocompetent allografts.

Day CP, Carter J, Weaver Ohler Z, Bonomi C, El Meskini R, Martin P, Graff-Cherry C, Feigenbaum L, Tüting T, Van Dyke T, Hollingshead M, Merlino G - PLoS ONE (2014)

Generation of the rGH-ffLuc-eGFP (“Glowing Head”) genetically engineered mouse.A, Structure of the expression vector for generation of Glowing Head (GH) transgenic mice. Expression of a firefly luciferase-eGFP fusion gene (ffLuc-eGFP) was targeted to the mouse anterior pituitary gland by using the rat growth hormone promoter (rGH) and human growth hormone gene sequences, which include a polyadenylylation site (hGHpA)20. B, Optical expression pattern of transgene in GH mice as visualized by BL imaging. Reporter activity was detected in the anterior pituitary gland of both genders and the testes of male mice. C, Serum levels of growth hormone from age-matched GH mice and wildtype (WT) c-Brd mice was assessed by ELISA (mean ± SE). Blood was withdrawn at the same time of day. No significant differences in circulating growth hormone levels between the GH and WT mice were found. D, ffLuc-eGFP-labeled LLC tumors were subcutaneously transplanted into WT, GH, and NOD-SCID mice. Blood was withdrawn to prepare sera when tumors reached 500 mm3, and the serum levels of anti-GFP antibody were analyzed by ELISA. The levels of anti-GFP antibody in WT mice are significantly higher than those in GH and NOD-SCID mice (p<0.005), but no difference was found between those in GH and NOD-SCID mice (p = 0.19). The sera from healthy mice without tumor transplantation served as controls to define zero point.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4219677&req=5

pone-0109956-g002: Generation of the rGH-ffLuc-eGFP (“Glowing Head”) genetically engineered mouse.A, Structure of the expression vector for generation of Glowing Head (GH) transgenic mice. Expression of a firefly luciferase-eGFP fusion gene (ffLuc-eGFP) was targeted to the mouse anterior pituitary gland by using the rat growth hormone promoter (rGH) and human growth hormone gene sequences, which include a polyadenylylation site (hGHpA)20. B, Optical expression pattern of transgene in GH mice as visualized by BL imaging. Reporter activity was detected in the anterior pituitary gland of both genders and the testes of male mice. C, Serum levels of growth hormone from age-matched GH mice and wildtype (WT) c-Brd mice was assessed by ELISA (mean ± SE). Blood was withdrawn at the same time of day. No significant differences in circulating growth hormone levels between the GH and WT mice were found. D, ffLuc-eGFP-labeled LLC tumors were subcutaneously transplanted into WT, GH, and NOD-SCID mice. Blood was withdrawn to prepare sera when tumors reached 500 mm3, and the serum levels of anti-GFP antibody were analyzed by ELISA. The levels of anti-GFP antibody in WT mice are significantly higher than those in GH and NOD-SCID mice (p<0.005), but no difference was found between those in GH and NOD-SCID mice (p = 0.19). The sera from healthy mice without tumor transplantation served as controls to define zero point.
Mentions: Our results suggested that immunogenicity of xenobiotic reporter gene products is largely responsible for their inconsistency in the context of a fully functional immune system. To circumvent this issue, we generated C57BL/6- and FVB/N-based GEM models recognizing ffLuc and eGFP proteins as self. For its high specificity, rGH gene sequences [17] (Fig. 2A) were employed to target expression of an ffLuc-eGFP fusion gene to the anterior pituitary gland of the mouse, thereby avoiding interfering signaling from the most common metastatic sites.

Bottom Line: Imageable reporters are essential for accurately tracking tumor growth and response, particularly for metastases.Here we assessed the value of reporter-tolerized GEMs as allograft recipients by targeting minimal expression of a luciferase-GFP fusion reporter to the anterior pituitary gland (dubbed the "Glowing Head" or GH mouse).Moreover, reporter expression can also alter the tumor response to chemotherapy or targeted therapy in a context-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, Maryland, United States of America.

ABSTRACT
Preclinical therapeutic assessment currently relies on the growth response of established human cell lines xenografted into immunocompromised mice, a strategy that is generally not predictive of clinical outcomes. Immunocompetent genetically engineered mouse (GEM)-derived tumor allograft models offer highly tractable preclinical alternatives and facilitate analysis of clinically promising immunomodulatory agents. Imageable reporters are essential for accurately tracking tumor growth and response, particularly for metastases. Unfortunately, reporters such as luciferase and GFP are foreign antigens in immunocompetent mice, potentially hindering tumor growth and confounding therapeutic responses. Here we assessed the value of reporter-tolerized GEMs as allograft recipients by targeting minimal expression of a luciferase-GFP fusion reporter to the anterior pituitary gland (dubbed the "Glowing Head" or GH mouse). The luciferase-GFP reporter expressed in tumor cells induced adverse immune responses in wildtype mouse, but not in GH mouse, as transplantation hosts. The antigenicity of optical reporters resulted in a decrease in both the growth and metastatic potential of the labeled tumor in wildtype mice as compared to the GH mice. Moreover, reporter expression can also alter the tumor response to chemotherapy or targeted therapy in a context-dependent manner. Thus the GH mice and experimental approaches vetted herein provide concept validation and a strategy for effective, reproducible preclinical evaluation of growth and response kinetics for traceable tumors.

Show MeSH
Related in: MedlinePlus