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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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Immunogenicity of ffLuc-eGFP alters the response of tumors to chemotherapeutic agents in wildtype mice compared to GH mice.Labeled LLC tumors were inoculated subcutaneously into WT and GH c-Brd mice. When the average tumor size reached 125 mm3, each strain of mice was randomized into two groups to receive either control vehicle (Cremophor EL + saline) or paclitaxel. Tumor size was measured periodically. A and B, Tumor growth (fold-increase relative to day 1) in WT and GH c-Brd mice (mean ± SE). Paclitaxel treatment was inefficacious in GH mice (A), but delayed tumor growth in WT mice (*, P<0.05 in a two-tailed T-test) (B). Ctrl, control vehicle; Tx, paclitaxel treatment. C, Spleen size in each group (mean ± SE). Spleens in paclitaxel-treated WT c-Brd mice were marginally bigger than those in vehicle-treated c-Brd mice but significantly bigger than those in both groups of GH mice. No significant difference was found between the two GH mouse groups. D and E, Enlarged spleens in paclitaxel-treated WT mice correspond to higher CD8/CD4 ratios. Splenocytes were prepared from spleens harvested from mice from each treatment group. These were stained with anti-mouse CD4 or CD8 antibodies, and analyzed by flow cytometry and Cellometer to obtain the ratio of the CD8+ to CD4+ subpopulation (CD8/CD4) in WT and GH c-Brd host mice (mean ± SE) (D). E, Regressional analysis demonstrated a significant correlation between CD8/CD4 ratio and spleen size (P<0.01).
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pone-0109956-g004: Immunogenicity of ffLuc-eGFP alters the response of tumors to chemotherapeutic agents in wildtype mice compared to GH mice.Labeled LLC tumors were inoculated subcutaneously into WT and GH c-Brd mice. When the average tumor size reached 125 mm3, each strain of mice was randomized into two groups to receive either control vehicle (Cremophor EL + saline) or paclitaxel. Tumor size was measured periodically. A and B, Tumor growth (fold-increase relative to day 1) in WT and GH c-Brd mice (mean ± SE). Paclitaxel treatment was inefficacious in GH mice (A), but delayed tumor growth in WT mice (*, P<0.05 in a two-tailed T-test) (B). Ctrl, control vehicle; Tx, paclitaxel treatment. C, Spleen size in each group (mean ± SE). Spleens in paclitaxel-treated WT c-Brd mice were marginally bigger than those in vehicle-treated c-Brd mice but significantly bigger than those in both groups of GH mice. No significant difference was found between the two GH mouse groups. D and E, Enlarged spleens in paclitaxel-treated WT mice correspond to higher CD8/CD4 ratios. Splenocytes were prepared from spleens harvested from mice from each treatment group. These were stained with anti-mouse CD4 or CD8 antibodies, and analyzed by flow cytometry and Cellometer to obtain the ratio of the CD8+ to CD4+ subpopulation (CD8/CD4) in WT and GH c-Brd host mice (mean ± SE) (D). E, Regressional analysis demonstrated a significant correlation between CD8/CD4 ratio and spleen size (P<0.01).

Mentions: The advantages illustrated above suggest that GH mice would constitute a superior preclinical model for drug assessment. We have shown that chemotherapeutic paclitaxel has no significant effect on growth of subcutaneous LLC tumors in syngeneic C57BL/6 hosts, irrespective of doses ranging between 6.7–22 mg/kg, QDx5 (Fig. S6). In this study syngeneic GH and WT mice carrying subcutaneous ffLuc-eGFP-labeled LLC tumors were randomized to receive vehicle or paclitaxel at 7.5 mg/kg, QDx5, considered to be a dose mimicking human treatment [8], [29]. As with unlabeled LLC growing in WT mice, paclitaxel had no effect on tumors growing in GH mice (Fig. 4A); in contrast, growth of the ffLuc-eGFP-labeled tumor was significantly delayed in treated WT mice (Fig. 4B). Interestingly, the spleens of paclitaxel-treated WT mice were significantly larger relative to the other three groups (Fig. 4C), and exhibited enlarged, disrupted lymphatic follicles (Fig. S7). Accordingly, the CD8/CD4 ratio of splenocytes increased in paclitaxel-treated WT mice (Fig. 4D), correlating with spleen size in all groups (Fig. 4E). There was no difference in the growth or response to paclitaxel of unlabeled LLC cells growing in WT vs. GH mice (not shown). These data suggest that paclitaxel treatment could produce a false-positive preclinical outcome by inducing a cytotoxic T cell response against a xenobiotic tumor antigen, but only in WT mice that had not been pre-tolerized to that antigen. Taken more broadly, our results show that tumor antigens can significantly influence preclinical tumor response to chemotherapy.


"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)

Immunogenicity of ffLuc-eGFP alters the response of tumors to chemotherapeutic agents in wildtype mice compared to GH mice.Labeled LLC tumors were inoculated subcutaneously into WT and GH c-Brd mice. When the average tumor size reached 125 mm3, each strain of mice was randomized into two groups to receive either control vehicle (Cremophor EL + saline) or paclitaxel. Tumor size was measured periodically. A and B, Tumor growth (fold-increase relative to day 1) in WT and GH c-Brd mice (mean ± SE). Paclitaxel treatment was inefficacious in GH mice (A), but delayed tumor growth in WT mice (*, P<0.05 in a two-tailed T-test) (B). Ctrl, control vehicle; Tx, paclitaxel treatment. C, Spleen size in each group (mean ± SE). Spleens in paclitaxel-treated WT c-Brd mice were marginally bigger than those in vehicle-treated c-Brd mice but significantly bigger than those in both groups of GH mice. No significant difference was found between the two GH mouse groups. D and E, Enlarged spleens in paclitaxel-treated WT mice correspond to higher CD8/CD4 ratios. Splenocytes were prepared from spleens harvested from mice from each treatment group. These were stained with anti-mouse CD4 or CD8 antibodies, and analyzed by flow cytometry and Cellometer to obtain the ratio of the CD8+ to CD4+ subpopulation (CD8/CD4) in WT and GH c-Brd host mice (mean ± SE) (D). E, Regressional analysis demonstrated a significant correlation between CD8/CD4 ratio and spleen size (P<0.01).
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getmorefigures.php?uid=PMC4219677&req=5

pone-0109956-g004: Immunogenicity of ffLuc-eGFP alters the response of tumors to chemotherapeutic agents in wildtype mice compared to GH mice.Labeled LLC tumors were inoculated subcutaneously into WT and GH c-Brd mice. When the average tumor size reached 125 mm3, each strain of mice was randomized into two groups to receive either control vehicle (Cremophor EL + saline) or paclitaxel. Tumor size was measured periodically. A and B, Tumor growth (fold-increase relative to day 1) in WT and GH c-Brd mice (mean ± SE). Paclitaxel treatment was inefficacious in GH mice (A), but delayed tumor growth in WT mice (*, P<0.05 in a two-tailed T-test) (B). Ctrl, control vehicle; Tx, paclitaxel treatment. C, Spleen size in each group (mean ± SE). Spleens in paclitaxel-treated WT c-Brd mice were marginally bigger than those in vehicle-treated c-Brd mice but significantly bigger than those in both groups of GH mice. No significant difference was found between the two GH mouse groups. D and E, Enlarged spleens in paclitaxel-treated WT mice correspond to higher CD8/CD4 ratios. Splenocytes were prepared from spleens harvested from mice from each treatment group. These were stained with anti-mouse CD4 or CD8 antibodies, and analyzed by flow cytometry and Cellometer to obtain the ratio of the CD8+ to CD4+ subpopulation (CD8/CD4) in WT and GH c-Brd host mice (mean ± SE) (D). E, Regressional analysis demonstrated a significant correlation between CD8/CD4 ratio and spleen size (P<0.01).
Mentions: The advantages illustrated above suggest that GH mice would constitute a superior preclinical model for drug assessment. We have shown that chemotherapeutic paclitaxel has no significant effect on growth of subcutaneous LLC tumors in syngeneic C57BL/6 hosts, irrespective of doses ranging between 6.7–22 mg/kg, QDx5 (Fig. S6). In this study syngeneic GH and WT mice carrying subcutaneous ffLuc-eGFP-labeled LLC tumors were randomized to receive vehicle or paclitaxel at 7.5 mg/kg, QDx5, considered to be a dose mimicking human treatment [8], [29]. As with unlabeled LLC growing in WT mice, paclitaxel had no effect on tumors growing in GH mice (Fig. 4A); in contrast, growth of the ffLuc-eGFP-labeled tumor was significantly delayed in treated WT mice (Fig. 4B). Interestingly, the spleens of paclitaxel-treated WT mice were significantly larger relative to the other three groups (Fig. 4C), and exhibited enlarged, disrupted lymphatic follicles (Fig. S7). Accordingly, the CD8/CD4 ratio of splenocytes increased in paclitaxel-treated WT mice (Fig. 4D), correlating with spleen size in all groups (Fig. 4E). There was no difference in the growth or response to paclitaxel of unlabeled LLC cells growing in WT vs. GH mice (not shown). These data suggest that paclitaxel treatment could produce a false-positive preclinical outcome by inducing a cytotoxic T cell response against a xenobiotic tumor antigen, but only in WT mice that had not been pre-tolerized to that antigen. Taken more broadly, our results show that tumor antigens can significantly influence preclinical tumor response to chemotherapy.

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